Journal of General Virology Publish Ahead of Print

Evolution, dispersal and replacement of American genotype dengue type 2 viruses in India (1956-2005): Selection pressure and molecular clock analyses [ANIMAL VIRUSES - POSITIVE-STRAND RNA]

Wed, 18 Nov 2009 09:01:18 PST

This study reports the phylogeny, selection pressure, genotype replacement and molecular clock analysis of many previously unstudied dengue type 2 virus (DENV-2) strains, isolated in India over a time span of almost fifty years (1956–2005). Analysis of complete envelope (E) gene sequences of thirty-seven strains of DENV-2 from India together with globally representative strains, revealed that the American genotype which predominantly circulated in India during the pre-1971 period, was then replaced by the Cosmopolitan genotype. Two previously unreported amino acid residues, one in the American (402I) and one in the Cosmopolitan (126K) genotypes, functionally known to be involved in the cellular tropism of the virus, were shown to be under positive selection pressure. The rates of nucleotide substitution estimated for DENV-2 was 6.5 x 10-4 substitutions/site/year which is comparable with earlier estimates. The time to the most recent common ancestor of the pre-1971 Indian strains and the American genotype was estimated to be between 1895 and 1932, which correlates with the historical record of traffic between India and South America, suggesting transportation of the virus from the Americas. Post-1971 Indian isolates formed a separate subclade within the cosmopolitan genotype. The estimated time to the most recent common ancestor of the Indian cosmopolitan strains was about 47 years with further estimates indicating the migration of DENV-2 from India to countries across the Indian ocean during 1955-1966. Overall, the present study increases our understanding of the events leading to the establishment and dispersal of the two genotypes in India.

Characterization of novel polyomaviruses from Bornean and Sumatran orangutans [ANIMAL VIRUSES - SMALL DNA]

Wed, 18 Nov 2009 09:01:18 PST

Serological screening of sera from orangutans demonstrated a high percentage of sera that cross-reacted with SV40 polyomavirus antigens. Analysis of archival DNA samples from seventy-one Bornean and eight Sumatran orangutans with a broad-spectrum PCR assay resulted in the detection of polyomavirus infections in eleven animals from both species. Sequence analysis of the amplicons revealed considerable differences between the polyomaviruses from Bornean and Sumatran orangutans. The genome from two polyomaviruses, one from each species, was therefore amplified and sequenced. Both viral genomes revealed a characteristic polyomavirus architecture, but lacked an obvious agnogene. Neighbor-joining analysis positioned the viruses in a larger cluster together with viruses from bats, bovines, rodents, and several primate polyomaviruses from chimpanzees, African green monkeys, squirrel monkeys, and the human Merkel cell polyomavirus.

Regulation of the Epstein-Barr virus Zp promoter in B lymphocytes during reactivation from latency [ANIMAL VIRUSES - LARGE DNA]

McDonald, C., Elgueta Karstegl, C., Kellam, P., Farrell, P. J — Wed, 18 Nov 2009 09:01:20 PST

Ten novel mutations were introduced into the Zp promoter to test the role of sequences outside the established transcription factor binding sites in EBV reactivation. Most of these had only small effects but mutations in the ZID site were shown to strongly reduce Zp activity at early times after induction by anti-Ig. The binding of MEF2 transcription factor to ZID was characterised in detail and linked functionally to Zp promoter activity. The presence of XBP-1s, the active form of XBP-1, after administration of anti-Ig to Akata BL cells is consistent with a role for this factor in reactivation of the EBV lytic cycle, although signalling through MEF2D was quantitatively much more significant in activation of Zp. Silencing of Zp during latency is thought to be primarily a consequence of a repressive chromatin structure on Zp and this aspect of Zp regulation can be observed in the Akata genome through protection of Zp from activation by BZLF1 in the absence of signalling from the BCR.

Lymphocryptovirus phylogeny and the origins of Epstein-Barr virus [ANIMAL VIRUSES - LARGE DNA]

Wed, 18 Nov 2009 09:01:20 PST

Specimens from wild and captive primates were collected, and searched for new members of the genus Lymphocryptovirus (subfamily Gammaherpesvirinae) utilising PCR for the DNA polymerase gene. Twenty-one new viruses were detected. Together with previous findings, more than 50 distinct lymphocryptoviruses (LCVs) are now known, with hosts from six primate families (Hominidae, Hylobatidae, Cercopithecidae, Atelidae, Cebidae, Pitheciidae). Further work extended genomic sequences for 25 LCVs to 3.4-7.4 kbp. Phylogenetic trees were constructed, based on alignments of protein sequences inferred from the LCV genomic data. The LCVs fell into three major clades: Clade A, comprising New World viruses; Clade B, containing both Old World monkey viruses and hominoid viruses including Epstein-Barr virus (EBV); and Clade C, containing other hominoid viruses. By comparison with the primate tree, it was proposed that major elements of the LCV tree represented synchronous evolution with host lineages, with the earliest node in both trees being separation of Old and New World lines, but that some virus lineages originated by interspecies transfer. From comparisons of branch lengths, it was inferred that evolutionary substitution in Clade B has proceeded more slowly than elsewhere in the LCV tree. It was estimated that in Clade B a subclade containing EBV, a gorilla virus and two chimpanzee viruses derived from an Old World monkey LCV line approximately twelve million years ago, and another subclade containing an orang utan virus and a gibbon virus derived from a macaque LCV line approximately 1.2 million years ago.

Characterization of Hepatitis C virus NS3 modifications in the context of replication [ANIMAL VIRUSES - POSITIVE-STRAND RNA]

Wed, 18 Nov 2009 09:01:22 PST

Post-translational modifications (PTMs) of viral proteins regulate various stages of infection. With only 10 proteins Hepatitis C virus can orchestrate its complete viral lifecycle. The HCV non-structural protein 3 has many functions. It has protease and helicase activity, interacts with several host cell proteins and plays a role in translation, replication and virus particle formation. Organization of all these functions is necessary and could be regulated by PTMs. We therefore searched for modifications of the NS3 protein in the subgenomic HCV replicon. When performing a tag-capture approach coupled with two-dimensional gel electrophoresis analyses, we observed that isolated His6-NS3 yielded multiple spots. Individual protein spots were in-gel digested and analyzed by mass spectrometry. Differences observed between the individual peptide mass fingerprints suggested the presence of modified peptides and allowed us to identify N-terminal acetylation and an adaptive mutation of NS3 (Q1067R). Further analysis of other NS3 variants revealed phosphorylation of NS3.

Wild-type measles virus infection of primary epithelial cells occurs via the basolateral surface without syncytium formation or release of infectious virus [ANIMAL VIRUSES - NEGATIVE-STRAND RNA]

Wed, 18 Nov 2009 09:01:21 PST

The lymphotropic and myelotropic nature of wild-type measles virus (wt-MV) is well recognised, with dendritic cells and lymphocytes expressing the MV receptor CD150 mediating systemic spread of the virus. Infection of respiratory epithelial cells has long been considered crucial for entry of MV into the body. However, the lack of detectable CD150 on these cells raises the issue of their importance in the pathogenesis of measles. Here we have utilised a combination of in vitro, ex vivo and in vivo model systems to characterise the susceptibility of epithelial cells to wt-MV of proven pathogenicity. Low numbers of MV-infected epithelial cells in close proximity to underlying infected lymphocytes or myeloid cells suggested infection via the basolateral side of the epithelium in the macaque model. In primary cultures of human bronchial epithelial cells foci of MV-infected cells were only observed following infection via the basolateral cell surface. The extent of infection in primary cells was enhanced both in vitro and in ex vivo cornea rim tissue by disrupting the integrity of the cells prior to the application of virus. This demonstrates that whilst epithelial cells may not be the primary target cells for wt-MV, areas of epithelium in which tight junctions are disrupted can become infected using high multiplicities of infection. The low numbers of MV infected epithelial cells observed in vivo in conjunction with the absence of infectious virus release from infected primary cell cultures suggests that epithelial cells have a peripheral role in MV transmission.

Immunodominant epitopes in nsp2 of porcine reproductive and respiratory syndrome virus are dispensable for replication but play an important role in modulation of host immune response [ANIMAL VIRUSES - POSITIVE-STRAND RNA]

Wed, 18 Nov 2009 09:01:22 PST

The nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus is the largest protein of the virus. Besides its crucial role in viral replication, recent studies indicated its involvement in modulating host immunity. In this study, each of the six identified immunodominant nsp2 B-cell epitopes (ES2 through ES7) was deleted using a Type I PRRSV cDNA infectious clone. Deletion of ES3, ES4, or ES7 allowed the generation of viable virus. In comparison to the parental virus, the ES3 mutant showed increased cytolytic activity and more vigorous growth kinetics, while ES4 and ES7 mutants displayed decreased cytolytic activity and slower growth kinetics in MARC-145 cells. These nsp2 mutants were further characterized in a nursery pig disease model. Results showed that ES4 and ES7 mutants exhibited attenuated phenotypes, whereas the ES3 mutant produced higher peak viral loads in pigs. The antibody response reached similar levels as measured by IDEXX ELISA after 21dpi, and slightly higher levels of average virus neutralizing titers were observed from pigs infected by ES4 and ES7 mutants. The expression of innate and T helper 1 cytokines were measured in peripheral blood mononuclear cells or virus infected macrophages. The results consistently showed that the IL-1β and TNF- expression levels were down-regulated in cells stimulated (or infected) with the ES3 mutant, as compared to parental virus and other nsp2 deletion mutants. These results suggest that certain regions in nsp2 are non-essential for PRRSV replication but may play an important role in modulation of host immunity in vivo.

Recombination and selection pressure in the ipomovirus Sweet potato mild mottle virus (Potyviridae) in wild species and cultivated sweetpotato in the centre of evolution in East Africa [PLANT VIRUSES - RNA]

Tugume, A., Mukasa, S., Kalkkinen, N., Valkonen, J. — Wed, 18 Nov 2009 09:01:20 PST

Sweet potato mild mottle virus (SPMMV) is the type member of genus Ipomovirus (family Potyviridae). SPMMV occurs in cultivated sweetpotatoes (Ipomoea batatas Lam.; Convolvulacea) in East Africa but its natural wild hosts are unknown. In this study, SPMMV was detected in 283 plants (9.8%) of the 2864 wild plants (family Convolvulacea) sampled from different agro-ecological zones of Uganda. The infected plants belonged to 21 species that were previously not known to be natural hosts of SPMMV. The size of SPMMV coat protein (CP) was determined by western blot analysis, N-terminal protein sequencing and peptide mass fingerprinting. Data implicated a proteolytic cleavage site VYVEPH/A at the NIb/CP junction, resulting in a CP of ~35 kDa. Nearly-complete sequences of 13 SPMMV isolates were characterized. Phylogenetic analysis of non-recombinant CP-encoding sequences placed five isolates from wild species sampled in the central zone of Uganda to a separate cluster. Recombination events were detected in the 5'- and 3'-proximal parts of the genome providing novel evidence of recombination in genus Ipomovirus. Thirteen amino acids in the N-terminus of the P1 protein were under positive selection, whereas purifying selection was implicated for HC-Pro, P3, 6K1 and CP encoding regions. These data supported by previous studies on ipomoviruses provide indications of an evolutionary process in which the P1 proteinase responds to needs of adaptation.

Deletion of the rat cytomegalovirus immediate early 1 gene results in a virus capable of establishing latency but with lower levels of acute virus replication and latency that compromise reactivation efficiency [ANIMAL VIRUSES - LARGE DNA]

Wed, 18 Nov 2009 09:01:19 PST

The IE1 and IE2 proteins encoded by the major immediate-early (MIE) transcription unit of cytomegaloviruses are thought to play key roles in the switch between latent and lytic cycle infection. Whilst IE2 is essential for triggering the lytic cycle, the exact roles of IE1 have not been resolved. An MIE-exon 4 deleted rat cytomegalovirus (IE1) failed to synthesize the IE1 protein and did not disperse promyelocytic leukemia bodies (PML) early post-infection, but it was still capable of normal replication in fibroblast cell culture. However, IE1 had diminished ability to infect salivary glands persistently in vivo and to reactivate from spleen explant cultures ex vivo. Quantitation of viral genomes in spleens of infected animals revealed a reduced amount of IE1 virus produced during acute infection, suggesting a role for IE1 as a regulator in establishing a chronic or persistent infection, rather than in more directly influencing the latency or reactivation processes.

Host range expansion of Spodoptera exigua nucleopolyhedrovirus to Agrotis segetum larvae when the midgut is bypassed [INSECT VIRUSES - DNA]

Jakubowska, A. K., Lynn, D. E., Herrero, S., Vlak, J. M., van Oers, M. M. — Wed, 18 Nov 2009 09:01:21 PST

Given the high similarity in genome content and organization between Spodoptera exigua nucleopolyhedrovirus (SeMNPV) and Agrotis segetum (Agse) NPV, as well as the high percentages of similarity found between their thirty core genes, the specificity of these nucleopolyhedroviruses was analysed for the insect species S. exigua and A. segetum. The LD50 for AgseNPV in L2 A. segetum larvae was 83 OBs/larva and the LT50 was 8.1 days. AgseNPV was orally infectious for S. exigua, although the LD50 was 10,000-fold higher than for SeMNPV. The SeMNPV virus was not infectious for A. segetum larvae when administered orally, but an infection was established by injection into the hemocoel. Bypassing the midgut entry by intrahemocoelic inoculations suggested that the midgut is the major barrier in A. segetum larvae for infection by SeMNPV. Delayed-early genes of SeMNPV are expressed in the midgut of A. segetum larvae after oral infections, showing that the virus is able to enter midgut cells. The possible mechanisms of A. segetum resistance to SeMNPV in per os infections are discussed.

Mouse vaccination with dendritic cells loaded with prion protein (PrP) peptides overcomes tolerance and delays scrapie [TSE AGENTS]

Wed, 18 Nov 2009 09:01:19 PST

Prion diseases are presumably caused by the accumulation in the brain of a pathological protein called prion protein scrapie which results from the transconformation of cellular PrP, a ubiquitous glycoprotein expressed in all mammals. Since all isoforms of PrP are perceived as self by the host immune system, a major problem in designing efficient immunoprophylaxis or immunotherapy is to overcome tolerance. The present study was aimed at seeing whether bone-marrow-derived dendritic cells loaded with peptides previously shown to be immunogenic in PrP-deficient mice, can overcome tolerance in PrP-proficient wild type mice and protect them against scrapie. Results show that peptide-loaded dendritic cells elicit in such mice both lymphokine release by T cells and antibody secretion against native cellular PrP. Repeated recalls with peptide-loaded dendritic cells reduces the attack rate of 139A scrapie inoculated i.p. and retards by 40 days disease duration. Most interestingly, survival time in individual mice appears to be correlated with the level of circulating antibody against native cellular PrP.

Transcriptomic analysis of intestinal genes following acquisition of Pea enation mosaic virus by the pea aphid Acyrthosiphon pisum [PLANT VIRUSES - RNA]

Wed, 18 Nov 2009 09:01:19 PST

Viruses in the Luteoviridae family are strictly transmitted by aphids in a non-propagative circulative and persistent mode. Virions ingested by aphids, successively cross the gut and the accessory salivary gland epithelia before being released, together with saliva, in the plant vasculature. Virions transport through aphid cells occurs by a transcytosis mechanism. We conducted a transcriptomic analysis of intestinal genes of the pea aphid Acyrthosiphon pisum following uptake of Pea enation mosaic virus-1 (a member of the Luteoviridae family). Among the 6776 transcripts analysed, 128 were significantly regulated (105 genes down-regulated and 23 up-regulated). 5% of the identified genes were involved in intracellular trafficking, endocytosis and signal transduction, three important steps in the internalization and transport of virions. The limited levels of down- (maximum of 3.45 fold) and up-regulation (maximum of 1.37 fold) suggest that the virus hijacks a constitutive endocytosis-exocytosis mechanism without heavily perturbing cell metabolism. Although limited to about 19% of the aphid genes, this work represents the first large scale analysis of aphid gene regulation following virus acquisition. A better knowledge of this virus-vector interaction will be possible only when tools representing the complete genomic capacity of the aphid become available.

Identification of protein-protein interactions of the ODV associated proteins of HearNPV [INSECT VIRUSES - DNA]

Peng, K., Wu, M., Deng, F., Song, J., Dong, C., Wang, H., Hu, Z. — Wed, 11 Nov 2009 09:01:04 PST

The purpose of this study is to identify protein-protein interactions among the components of the occlusion-derived virus (ODV) of Helicoverpa armigera nucleopolyhedrovirus (HearNPV), a group II Alphabaculovirus of the family Baculoviridae. To achieve this, 39 selected genes of potential ODV structural proteins were cloned and expressed in the Gal4 yeast two-hybrid (Y2H) system. The direct cross Y2H assays identified 22 interactions. These included 13 binary interactions that of HA9-ODV-EC43, ODV-E56-38K, ODV-E56-PIF3, LEF3-Helicase, LEF3-alkaline nuclease (AN), GP41-38K, GP41-HA90, 38K-PIF3, 38K-PIF2, VP80-HA100, ODV-E66-PIF3, ODV-E66-PIF2 and PIF3-PIF2, and 9 self-associations including that of IE1, HA44, LEF3, HA66, GP41, CG30, 38K, PIF3 and P24. Five of these interactions, the interactions of LEF3-Helicase and LEF3-AN, and the self-associations of IE1, LEF3 and 38K, had been previously reported in Autographa californica MNPV (AcMNPV). Since HA44 and HA100 are the two newly identified ODV proteins of group II viruses, their interactions were further confirmed. The self-association of HA44 was verified with His pull-down assay and the interaction of VP80-HA100 was confirmed by co-immunoprecipitation assay. A summary of so far reported protein-protein interactions of baculovirus including 68 interactions with 45 viral proteins and 5 host proteins is presented, which will facilitate our understanding of the molecular mechanisms of baculovirus infection.

Interferon beta plus gamma efficiently reduces acyclovir-resistant herpes simplex virus infection in mice in a T-cell-independent manner [ANIMAL VIRUSES - LARGE DNA]

Wed, 11 Nov 2009 09:01:03 PST

Acyclovir (ACV)-resistant herpes simplex virus type 1 (HSV-1) causes severe diseases in immunocompromised patients, so identification of new therapies is needed. Interferons (IFNs) are used to treat several other viral infections in the clinic, and IFN- and IFN-β are shown to cooperatively reduce wild-type HSV-1 replication in the corneas of immunocompetent mice. Because IFN- is shown to exert antiviral effect mostly through T cells, whether combined IFN treatment could still inhibit ACV-resistant HSV-1 replication, especially in imunocompromised hosts, is unknown. In the present study, we evaluated the efficacy of combined IFN treatment on ACV-resistant HSV-1 mutants. In vitro results showed that IFN-β synergized with IFN- to inhibit HSV-1 replication in both human and mouse cell lines. Some ACV-resistant mutants were actually hypersensitive to combined IFN treatment. In vivo results showed that topical treatment with a low dose of IFN-β plus IFN- (200 U each) on mouse corneas efficiently reduced the viral loads up to 4, 4, and 3 logs, respectively in the eyes, trigeminal ganglia, and brain stems of wild-type and also immunocompromised, nude mice infected or co-infected with ACV-resistant HSV-1 in a manner independent of T cells. Highly efficient reduction of HSV acute replication by combined IFN treatment led to a dramatic decrease in subsequent viral reactivation from neural tissues, trigeminal ganglia, brain stems, and spinal cords of latently infected mice. Thus, combination of IFN-β and IFN- could be a potential treatment for ACV-resistant HSV-1 in immunocompromised patients.

The feline calicivirus p32, p39 and p30 proteins localise to the endoplasmic reticulum to initiate replication complex formation [ANIMAL VIRUSES - POSITIVE-STRAND RNA]

Wed, 11 Nov 2009 09:01:04 PST

In common with other positive strand RNA viruses, replication of feline calicivirus (FCV) results in rearrangement of intracellular membranes and production of numerous membrane bound vesicular structures on which viral genome replication is thought to occur. In this study, bioinformatics approaches have identified three of the FCV non-structural proteins, namely p32, p39 and p30 as potential transmembrane proteins. These proteins were able to target cyan fluorescent protein (CFP) to membrane fractions where they behaved as integral membrane proteins. Immunofluorescence microscopy of these proteins expressed in cells showed co-localisation with ER-markers. Further electron microscopy analysis of cells co-expressing FCV p39 or p30 with a horseradish peroxidase protein containing the KDEL ER-retention motif demonstrated gross morphological changes to the ER. Similar reorganisation patterns, especially for those produced by p30, were observed in naturally infected CRFK cells. Together the data demonstrate that the p32, p39 and p30 proteins of FCV locate to the ER and lead to reorganisation of ER membranes. This suggests that they may play a role in the generation of FCV replication complexes and that the endoplasmic reticulum may represent the potential source of the membrane vesicles induced during FCV infection.

The evolutionary trajectory of turnip mosaic potyvirus populations adapting to a new host [PLANT VIRUSES - RNA]

Ohshima, K., Akaishi, S., Kajiyama, H., Koga, R., Gibbs, A. J. — Wed, 11 Nov 2009 09:01:02 PST

Little is known about how some plant viruses establish successful cross-species transmission, while others do not; the genetic basis for adaptation is largely unknown. We have investigated the genetic changes that occur using the progeny of an infectious clone, p35Tunos, derived from the Turnip mosaic virus (TuMV) UK 1 isolate, which is a Brassica host type, but rarely infects Raphanus systemically and then only asymptomatically. The genetic trajectory leading to viral adaptation was studied in a TuMV isolate passaged in Nicotiana benthamiana (parental), Brassica rapa old (susceptible) host and Raphanus sativus new (almost insusceptible) host. Almost complete consensus genomic sequences were obtained by RT-PCR of viral populations passaged up to 35 times; 59 full sequences of 578,200 nucleotides. There were significant differences in the nucleotide and encoded amino acid changes in the consensus genomes from old and new hosts. Furthermore, a 3264 nt region corresponding to nts 3222-6485 of the UK 1 genome was cloned, and 269 clones from 23 populations were sequenced; this region covered 33% of the genome and represented a total of 878,016 nucleotides. The results showed that the nucleotide diversity and the non-synonymous / synonymous ratio of the populations from the new host were higher than those from the old host. The analysis of molecular variance showed the significant differences among the populations from old and new hosts. To our knowledge, this is the first report comparing the evolutionary trajectory dynamics of plant virus populations in old and new hosts.

Structural homology between Bamboo mosaic virus and its satellite RNAs in the 5' untranslated region [PLANT VIRUSES - RNA]

Chen, S.-C., Desprez, A., Olsthoorn, R. — Wed, 11 Nov 2009 09:01:05 PST

A structural element was identified in the 5'-proximal sequence of the Bamboo mosaic virus (BaMV) RNA. Mutational analysis of the hairpin showed that disruptions of the secondary structure or substitutions of the loop sequences resulted in reduced accumulation of BaMV genomic RNA. Phylogenetic analysis further suggested the presence of structural homologues of this hairpin in all other potexviruses. In addition, remarkable structural homology was discovered between the BaMV hairpin and a stem-loop in the 5' untranslated region of satellite RNAs responsible for attenuation of BaMV in co-infected plants. The role of this homology in the helper-satellite interaction is discussed.

Sequences of complete human cytomegalovirus genomes from infected cell cultures and clinical specimens [ANIMAL VIRUSES - LARGE DNA]

Wed, 11 Nov 2009 09:01:04 PST

We have assessed two approaches to sequencing complete human cytomegalovirus (HCMV) genomes (236 kbp) in DNA extracted from infected cell cultures (strains 3157, HAN13, HAN20 and HAN38) or clinical specimens (strains JP and 3301). The first approach involved amplifying genomes from the DNA samples as overlapping PCR products, sequencing these by the Sanger method, acquiring reads from a capillary instrument, and assembling these using the Staden programs. The second approach involved generating sequence data from the DNA samples using an Illumina Genome Analyzer (IGA), processing the filtered reads by reference-independent (de novo) assembly, utilizing the resulting sequence to direct reference-dependent assembly of the same data, and finishing by limited PCR sequencing. Both approaches were successful. In particular, the investigation demonstrated the utility of IGA data for efficiently sequencing genomes from clinical samples containing as little as 3 % HCMV DNA. Analysis of the genome sequences obtained showed that each of the strains grown in cell culture was a mutant. Certain of the mutations were shared among strains from independent clinical sources, thus suggesting that they may have arisen in a common ancestor during natural infection. Moreover, one of the strains (JP) sequenced directly from a clinical specimen was mutated in two genes, one of which encodes a proposed immune evasion function, viral interleukin-10. These observations imply that HCMV mutants exist in human infections.

Molecular evolution of GBV-B hepatitis virus during acute resolving and persistent infections in experimentally infected tamarins [ANIMAL VIRUSES - POSITIVE-STRAND RNA]

Takikawa, S., Engle, R., Faulk, K., Emerson, S., Purcell, R. H., Bukh, J. — Wed, 11 Nov 2009 09:01:03 PST

GBV-B causes acute hepatitis in experimentally infected tamarins. We compared evolutionary features in acute resolving and persistent GBV-B infection. We detected no evidence of evolution in four animals with clearance during weeks 9-12, whereas three animals with clearance during weeks 13-26 had several substitutions in their polyprotein sequence. A single tamarin had long-term GBV-B viremia; analysis of virus recovered at weeks 2, 5, 12, 20, 26, 52 and 104 demonstrated that mutations accumulated over time. Overall, the amino acid substitution rate was 3.5 x 10-3 and 1.1 x 10-3 substitutions/site/year during weeks 1-52 and 53-104, respectively. Thus, there was a significant decrease in evolution over time, as found for hepatitis C virus. The rate of non-synonymous substitution per non-synonymous site compared with that of synonymous substitution per synonymous site decreased over time suggesting reduction of positive selective pressure. These data demonstrate that prolonged GBV-B infection is associated with viral evolution.

Analysis of latent viral gene expression in natural and experimental latency models of human cytomegalovirus and its correlation with histone modifications at a latent promoter [ANIMAL VIRUSES - LARGE DNA]

Reeves, M. B., Sinclair, J. H. — Wed, 11 Nov 2009 09:01:02 PST

Human cytomegalovirus (HCMV) is an opportunistic human pathogen that establishes a lifelong latent infection that can periodically reactivate which, if unchecked by a robust immune response, can result in severe disease in immuno-compromised patients. Reactivation of latent virus in myeloid progenitor cells is concomitant with cellular differentiation through regulation of the MIEP by chromatin remodelling. In this study, we have analysed the expression of the latent gene transcript UL81-82as (LUNA). LUNA is expressed in latently infected CD34+ cells and expression decreases as CD34+ cells differentiate to immature dendritic cells. Upon maturation (and HCMV reactivation) a second wave of transcription occurs consistent with expression during lytic infection also. Furthermore, we show that the LUNA promoter is associated with acetylated histones during HCMV latency in experimentally and naturally infected CD34+ cells thus suggesting that latent gene promoters are, like the MIEP, regulated by post-translational modifications of their associated histone proteins.

The classical swine fever virus Npro product is degraded by cellular proteasomes in a manner that does not require interaction with IRF-3. [ANIMAL VIRUSES - POSITIVE-STRAND RNA]

Seago, J., Goodbourn, S., Charleston, B. — Wed, 11 Nov 2009 09:01:02 PST

Classical swine fever is a notifiable disease of pigs. The causative agent, classical swine fever virus (CSFV), is highly contagious and causes mild to severe hemorrhagic disease depending on the virulence of the strain. The RNA genome of CSFV is translated as a single polyprotein that is processed to yield 12 proteins. Like other pestiviruses, the first protein to be translated is the N-terminal auto-protease termed Npro. A novel pestiviral protein with no known cellular homologues, Npro antagonises type I interferon induction by binding and targeting the transcription factor IRF-3 for ubiquitin-dependent proteasomal degradation. In this study, CSFV-infected PK-15 cells and stable cell lines were used to show Npro is itself an unstable protein that is targeted for proteasomal degradation in a ubiquitin dependent manner. In addition, Npro is not degraded as a direct consequence of its ability to interact with IRF-3 or target IRF-3 for proteasomal degradation.

Small intestine CD4+ cell reduction and enteropathy in SHIV-KS661-infected rhesus macaques in presence of low viral load [ANIMAL VIRUSES - RETROVIRUSES]

Wed, 04 Nov 2009 09:01:44 PST

HIV-1, SIV and SHIV infection generally leads to death of hosts accompanied by high viremia and profound CD4+ T cell depletion. SHIV-KS661-infected rhesus macaques with high viral load set-point (HVL) ultimately experience diarrhea and wasting at half to one year after infection. On the contrary, infected-macaques with low viral load set-point (LVL) usually live asymptomatically through the observation period, and have therefore been referred to as Asymptomatic LVL (Asym LVL) macaques. Interestingly, some LVL macaques exhibited diarrhea and wasting similar to symptoms of HVL macaques, and were named Symptomatic LVL (Sym LVL) macaques. Here, we tested the hypothesis that Sym LVL macaques have the same degree of intestinal abnormalities as HVL macaques. The proviral DNA loads in lymphoid tissues and intestine of Sym LVL and Asym LVL were comparable and all infected monkeys showed villous atrophy. Notably, the CD4+ cell frequencies of lymphoid tissues and intestine in Sym LVL macaques were remarkably lower than those in Asym LVL and uninfected macaques. Furthermore, Sym LVL and HVL macaques exhibited an increased number of activated macrophages. In conclusion, intestinal disorders including CD4+ cell reduction and abnormal immune activation can be observed in SHIV-KS661-infected macaques independent of viral replication levels.

Combinatorial chloroquine and ribavirin treatment does not prevent death in a hamster model of Nipah and Hendra virus infection [ANIMAL VIRUSES - NEGATIVE-STRAND RNA]

Freiberg, A. N, Worthy, M. N, Lee, B., Holbrook, M. R — Wed, 04 Nov 2009 09:01:44 PST

Hendra virus (HeV) and Nipah virus (NiV) are recently-emerged, closely related and highly pathogenic paramyxoviruses that cause severe disease, such as encephalitis, in animals and humans with fatality rates of up to 75%. Due to their high case fatality rate following human infection and because of the lack of effective vaccines or therapy they are classified as biosafety level 4 pathogens. A recent study reported that chloroquine, an anti-malarial drug, was effective in preventing NiV and HeV infection in cell culture experiments. In the present study we analyzed the antiviral efficacy of chloroquine, individually and in combination with ribavirin, in the treatment of NiV- and HeV-infection in in vivo experiments, using a golden hamster model. While we were able to confirm the strong antiviral activity of both drugs in inhibiting viral spread in vitro, they did not prove to be protective in the in vivo model. Ribavirin delayed death from viral disease in NiV-infected hamsters by approximately five days, but we did not observe any significant effect in HeV-infected hamsters. Chloroquine did not protect hamsters when administered either individually or in combination with ribavirin, the latter indicating lack of a favorable drug-drug interaction.

Hepatitis C virus RNA replication is regulated by Ras-Erk signalling [ANIMAL VIRUSES - POSITIVE-STRAND RNA]

Gretton, S., Hughes, M., Harris, M. — Wed, 04 Nov 2009 09:01:42 PST

The hepatitis C virus NS5A protein has been previously demonstrated to partially attenuate activation of the Ras-Erk signalling pathway, via a conserved class II polyproline motif located towards the C-terminus of the protein. However, the role of Ras-Erk signalling in the virus life cycle remains undetermined. To investigate this, levels of RNA replication were measured in genotype 1 and 2 transient luciferase subgenomic replicon systems in the context of either pharmacological or genetic (dominant negative) inhibition of MEK1, a kinase in the Ras-Erk signalling cascade. Incubation in the presence of two inhibitors (U0126 and PD184352), resulted in a decrease in the levels of RNA replication, conversely incubation with inhibitor PD98059 resulted in a modest increase in replication. The results obtained with PD98059 could not be explained by an off-target effect on Cox-2, stability of replicon RNA or stimulation of global translation levels, suggesting stimulation by a yet uncharacterised mechanism. To verify data obtained using pharmacological inhibitors the transient replicon RNA was co-electroporated with a dominant negative mutant of MEK1. This resulted in a reduction in replication, confirming data seen with U0126 and PD184352. Our data are consistent with the hypothesis that a low level of Ras-Erk signalling activity is required for RNA replication, however complete inhibition of Ras-Erk signalling is inhibitory. These results suggest that perturbation of this signalling pathway by NS5A may be a mechanism to regulate levels of genomic RNA replication.

Detection of a novel hepatitis E-like virus in faeces of wild rats using a nested broad-spectrum RT-PCR [ANIMAL VIRUSES - POSITIVE-STRAND RNA]

Johne, R., Plenge-Bonig, A., Hess, M., Ulrich, R. G., Reetz, J., Schielke, A. — Wed, 04 Nov 2009 09:01:43 PST

Hepatitis E is a rare human disease in developed countries. It is caused by hepatitis E virus (HEV), which is most likely zoonotically transmitted to humans from domestic pigs and wild boars. Multiple reports on the detection of HEV-specific antibodies in rats suggested the presence of an HEV-related agent; however, infectious virus or a viral genome could not be demonstrated so far. Here, a nested broad-spectrum RT-PCR protocol was developed capable of detecting different HEV types including those derived from wild boar and chicken. Screening of 30 faecal samples of wild Norway rats (Rattus norvegicus) from Hamburg (Germany) resulted in the detection of two sequences with similarities to human, mammalian and avian HEV. Virus particles with morphology reminiscent of HEV were demonstrated by immune electron microscopy in one of these samples and the virus was tentatively designated as rat HEV. Genome fragments with sizes of 4,019 and 1,545 nucleotides were amplified from two samples. Sequence comparison to human and avian strains revealed only 59.9% and 49.9% sequence identity, respectively. Similarly, the deduced amino acid sequence for the complete capsid protein had 56.2% and 42.9% identity with human and avian strains, respectively. Inoculation of the samples onto three different permanent rat liver cell lines did not result in detectable virus replication as assayed by RT-PCR with cells of the fifth virus passage. Further investigations are necessary to clarify the zoonotic potential of rat HEV and to assess its suitability to serve in a laboratory rat animal model for human hepatitis E.

Feline infectious peritonitis; insights into feline coronavirus pathobiogenesis and epidemiology based on genetic analysis of the viral 3c gene [ANIMAL VIRUSES - POSITIVE-STRAND RNA]

Chang, H.-W., de Groot, R. J., Egberink, H. F., Rottier, P. J.M. — Wed, 04 Nov 2009 09:01:41 PST

Feline infectious peritonitis (FIP) is a lethal systemic disease caused by FIP virus (FIPV), a virulent mutant of apathogenic feline enteric coronavirus (FECV). We analyzed the 3c gene - a proposed virulence marker - in 27 FECV- and 28 FIPV-infected cats. Our findings suggest that functional 3c protein expression is crucial for FECV replication in the gut but dispensable for systemic FIPV replication. While intact in all FECVs, the 3c gene was mutated in the majority (71.4%) but not in all FIPVs, implying that mutation in 3c is not the (single) cause of FIP. Most FIP cats had no detectable intestinal FCoV and had seemingly cleared the primary FECV infection. In those with detectable intestinal FCoV, the virus always had an intact 3c and seemed acquired by FECV superinfection. Apparently, 3c-inactivated viruses do not - or only poorly - replicate in the gut, explaining the rare incidence of FIP outbreaks.

A history estimate and evolutionary analysis of rabies virus variants in China [ANIMAL VIRUSES - NEGATIVE-STRAND RNA]

Wed, 04 Nov 2009 09:01:44 PST

To investigate the evolutionary dynamics of RABV in China, we collected and sequenced 55 isolates sampled from 14 provinces of China over the last 40 years and performed a coalescent-based analysis of the G gene. This revealed that RABV currently circulating in China comprised three main groups. Bayesian coalescent analysis estimated the date of the most recent common ancestor for the current RABV Chinese strains to be 1412 (with a 95% confidence interval of 1006-1736). The estimated mean substitution rate for the G gene sequences (3.961x10-4 substitutions per site, per year) was in accordance with previous reports for RABV.

1918 and 2009 H1N1 influenza are not pathogenic in birds [ANIMAL VIRUSES - NEGATIVE-STRAND RNA]

Wed, 04 Nov 2009 09:01:43 PST

The susceptibility of chickens to both 1918 and 2009 H1N1 influenza virus was evaluated. The intravenous pathogenicity index of 1918 and 2009 H1N1 viruses in chickens was 0. Chickens did not develop clinical signs following experimental inoculation simulating natural infection. No gross pathological changes were observed in any tissues from chickens between 2 and 18 days post infection (dpi) and viral RNA was not detected in mucosal secretions or tissues by real-time RT-PCR. Seroconversion was not detected in any of the chickens following inoculation with H1N1 2009 virus, whereas half the chickens developed influenza specific antibodies at 28 days post infection with 1918 influenza suggesting limited infection. Viral RNA was detected in mallard ducks following 1918 inoculation at 3 dpi in cloacal swabs but not in tissues by real-time RT-PCR and all ducks seroconverted by 28 dpi. Both 1918 and 2009 H1N1 influenza viruses behave as LPAI in gallinaceous poultry.

Production, characterization and in vitro testing of HBcAg-specific VHH-intrabodies [ANIMAL VIRUSES - SMALL DNA]

Wed, 04 Nov 2009 09:01:46 PST

Infections with the Hepatitis B virus represent a global health problem since these account for 350 million chronic infections worldwide that result in 500,000 to 700,000 deaths each year. Control of viral replication and HBV-related disease and mortality are of utmost importance. Because the currently available antiviral therapies all have major limitations, new strategies to treat chronic HBV infection are eagerly awaited. Six single-domain antibodies (VHHs) targeting the nucleocapsid protein of HBV (HBcAg) have been generated and three of these bound strongly to HBcAg of both subtype ayw and adw. These three VHHs were studied as intrabodies directed towards the nucleus or the cytoplasm of a hepatoma cell line that was co-transfected with HBV. A speckled staining of HBcAg was observed in the cytoplasm of cells transfected with nucleotropic VHH-intrabodies. Moreover, an increased intracellular accumulation of Hepatitis B e antigen (HBeAg) and a complete disappearance of intracellular HBcAg signal were observed with nuclear targeted HBcAg-specific VHHs. These results suggest that HBcAg-specific VHHs targeted to the nucleus affect HBcAg and HBeAg expression and trafficking in HBV transfected hepatocytes.

Time - the emerging dimension of plant virus studies [REVIEWS]

Gibbs, A., Fargette, D., Garcia-Arenal, F., Gibbs, M. — Wed, 04 Nov 2009 09:01:45 PST

Recent research has revealed that some plant viruses, like many animal viruses, have measurably evolving populations. Most of these viruses have single-stranded positive-sense RNA genomes, but a few have single-stranded DNA genomes. The studies show that extant populations of these viral species are only decades to centuries old, and the genera in which they are placed have diverged since agriculture was invented, and spread around the world during the Holocene. We suggest that this is not mere coincidence but evidence that the conditions generated by agriculture during this era have favoured particular viruses. There is also evidence, albeit less certain, that some plant viruses, including a few shown to have measurably evolving populations, have much more ancient origins. We discuss the possible reasons for this clear discordance between short-term and long-term evolutionary rate estimates, and how it might result from a large timescale dependence of the evolutionary rates. We also discuss briefly why it is useful to know the rates of evolution of plant viruses.

Molecular evidence of horizontal transmission of HCV within couples [ANIMAL VIRUSES - POSITIVE-STRAND RNA]

Wed, 04 Nov 2009 09:01:45 PST

HCV transmission has decreased with the adoption of universal blood donor screening and social policies to reduce the risk of infection in IDU, but it is still a worldwide health problem. Our objective was to evaluate the phylogenetic relationships among sequences from different HCV genomic regions from sexual partners of infected patients. Nine couples with a stable relationship and without other risk factors for HCV infection and 42 control patients were selected, so NS3 and NS5B regions were analyzed. The phylogenetic analysis showed that viruses from couples 3, 4, 6, 7 and 8 had a common origin, clustering in the same monophyletic group, with bootstrap values higher than 70. For the other couples, monophyletic groups were observed, however without bootstrap support. In conclusion, using two different viral genome regions, we observed a common source of infection from two members of five couples. These data strongly support HCV transmission within couples.

Detection of norovirus-, sapovirus- and rhesus enteric calicivirus-specific antibodies in captive juvenile macaques [ANIMAL VIRUSES - POSITIVE-STRAND RNA]

Farkas, T., Dufour, J., Jiang, X., Sestak, K. — Wed, 04 Nov 2009 09:01:41 PST

SUMMARYThe objective of this study was to determine the prevalence of anti-norovirus (NoV), sapovirus (SaV) and Tulane virus (TV) antibodies in rhesus macaques of the Tulane National Primate Research Center (TNPRC) and evaluate the antigenic relationship between these viruses. A high prevalence of NoV (51-61%) and SaV (50-56%) binding antibodies and TV (69%) neutralizing antibodies were detected. Serum samples obtained during a human NoV outbreak and a multivalent anti-NoV hyperimmune serum were not able to neutralize TV infectivity. Conversely, low levels of cross-reactivity between the prototype TV and NoVs, but not between the TV and SaVs were detected by ELISA. These data indicate the preservation of some cross-reactive B-cell epitopes between the rhesus and human CVs. The high prevalence of human and rhesus CV-specific serum antibodies suggests the frequent exposure of colony macaques to enteric CVs including the possibility of CV transmission between human and NHP hosts.

Activation of transcription factor Nrf2 by hepatitis C virus induces cell survival pathway [ANIMAL VIRUSES - POSITIVE-STRAND RNA]

Burdette, D., Olivarez, M., Waris, G. — Wed, 04 Nov 2009 09:01:40 PST

Oxidative stress has been implicated in various human diseases including in the pathogenesis of hepatitis C virus (HCV). Previous studies have shown the induction of oxidative stress in cultured cells expressing HCV genes. The transcription factor, Nrf2 is known to be activated in response to oxidative stress, but the mechanism of its activation is not clearly understood. In this study, we first determined the induction of Nrf2 and then investigated the mechanism of Nrf2 activation in human hepatoma cells infected with HCV (JFH-1). Our results showed the induction and nuclear translocation of Nrf2 in a time-dependent manner. The HCV-mediated activation of Nrf2 was abrogated in the presence of an antioxidant, PDTC (pyrrolidine dithiocarbamate) and Ca2+ chelator, BAPTA-AM (1, 2-bis (aminophenoxy)ethane N,N,N,N-tetraacetic acid-tetra (acetoxymethyl) ester), which suggest a role of both reactive oxygen species (ROS) and Ca2+ signaling in Nrf2 activation process. Using the inhibitors of cellular kinases, we further show that HCV-mediated phosphorylation/activation of Nrf2 is mediated by mitogen-activated protein (MAP) kinases, p38 MAPK, and janus kinase (JNK). We also observed the enhanced phosphorylation of Akt and its downstream substrate Bad in HCV-infected cells. Furthermore, using a siRNA approach our results suggest a potential role of HCV-mediated Nrf2 activation in the survival of HCV-infected cells, a condition favorable for liver oncogenesis. Taken together, these results provide an insight into the mechanisms by which HCV induces intracellular events relevant to chronic HCV infection.

Levels of soluble ST2 in serum associate with severity of dengue due to TNF-{alpha} stimulation [ANIMAL VIRUSES - POSITIVE-STRAND RNA]

Wed, 04 Nov 2009 09:01:42 PST

The interleukin-1 receptor-like-1 protein (IL1RL-1), also known as ST2, has been previously shown to regulate T cell function and is produced by T cells and endothelial cells. It was recently reported to be elevated in mild dengue patients during acute disease. The ST2 gene encodes several splice products: L (long), V (short) and s (soluble). A cohort of 38 patients with dengue hemorrhagic fever (DHF) and mild dengue fever (DF) were evaluated using sST2 ELISA. The RNA expression of ST2 was evaluated by real time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) using patients’ peripheral mononucleated blood cells (PMBCs) and in vitro using human umbilical vein endothelial cells (HUVECs) exposed to sera from dengue patients. DHF patients had higher levels of serum sST2, tumour necrosis factor alpha (TNF-, interleukin-8 and interleukin-10 compared to DF patients and normal healthy control individuals. However, viremia was indistinguishable between mild and severe cases. No changes in ST2 mRNA expression in PBMCs were found in these two groups of dengue patients. In vitro, sST2 was elevated in HUVECs treated with patients' sera. Neutralisation of TNF- in patient sera by pre-treatment with a TNF antibody inhibited the up-regulation of sST2 expression in HUVECs. These results implicate serum TNF- in the modulation of expression of sST2 in an in vitro system and sST2 could be associated with severity of disease. Further studies to determine if sST2 levels are predictive of the severe form of the disease as its role in immune regulation are warranted.

Fast broad-range disinfection of bacteria, fungi, viruses and prions [TSE AGENTS]

Beekes, M., Lemmer, K., Thomzig, A., Joncic, M., Tintelnot, K., Mielke, M. — Wed, 28 Oct 2009 10:01:32 PDT

Effective disinfectants are of key importance for the safe handling and reprocessing of surgical instruments. We tested whether new formulations containing SDS, NaOH and 1-propanol (n-propanol) are simultaneously active against a broad range of pathogens including bacteria, fungi, non-enveloped viruses and prions. Inactivation and disinfection were examined in suspension and on carriers, respectively, using coagulated blood or brain homogenate as organic soil. Coomassie blue staining was used to assess whether formulations did undesirably fix proteins to rough surfaces. A mixture of 0.2% SDS and 0.3% NaOH in 20% n-propanol achieved potent decontamination of steel carriers contaminated with PrPTSE, the biochemical marker for prion infectivity, from 263K scrapie hamsters, or patients with sporadic or variant Creutzfeldt-Jakob disease. 263K scrapie infectivity on carriers was decreased by ≥5.5 log10 units [logs]. Furthermore, the formulation effectively inactivated poliovirus, hepatitis A virus and caliciviruses (including murine norovirus) in suspension tests. It also yielded significant titre reductions of bacteria (E. faecium, M. avium; >6 logs), fungi (spores of Aspergillus niger; >5 logs) or poliovirus (≥4 logs) embedded in coagulated blood on carriers. The formulation was not found to fix proteins more than was observed with water as cleaning reagent. SDS, NaOH and n-propanol can synergistically achieve fast broad-range disinfection.

Roles of the ERK MAP Kinase in the Regulation of Proinflammatory and Apoptotic Responses in Chicken Macrophages Infected with H9N2 Avian Influenza Virus [ANIMAL VIRUSES - NEGATIVE-STRAND RNA]

Xing, Z., Cardona, C. J., Anunciacion, J., Adams, S., Dao, N. — Wed, 28 Oct 2009 10:01:33 PDT

The mitogen-activated protein kinase (MAPK) family are responsible for important signaling pathways, which regulate cell activation, differentiation, apoptosis, and immune responses. Studies have shown that influenza virus infection activates MAPK family members in mammals. While ERK1/2 is important for virus replication, activation of p38 controls the expression of RANTES, IL-8, and TNF-. In this study we report that avian influenza virus (AIV) activates ERK, p38 and JNK kinases in avian species. In chicken macrophages, while ERK was required for H9N2 AIV replication, ERK regulated proinflammatory cytokines IL-1b, IL-6 and IL-8, which is distinct from what has been previously reported in mammalian cells. Moreover, ERK alone suppressed TNF- and FasL and inhibited TNF family-mediated extrinsic apoptosis in H9N2-infected chicken macrophages. Taken together, these findings suggest that ERK signaling may uniquely play important roles in avian host responses to AIV infection.

The Inhibition of PI3K-Akt Pathway Enhances Gamma-2 Herpesvirus Lytic Replication and Facilitates Reactivation from Latency [ANIMAL VIRUSES - LARGE DNA]

Wed, 28 Oct 2009 10:01:33 PDT

Cellular signaling pathways are critical in regulating the balance between latency and lytic replication of herpesviruses. Here we investigated the effect of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway on replication of two gamma-2 herpesviruses, murine gammaherpesvirus-68 (MHV-68) and human herpesvirus-8/Kaposi’s sarcoma associated herpesvirus (HHV-8/KSHV). We found that de novo infection of MHV-68 induced PI3K dependent Akt activation and the lytic replication of MHV-68 was enhanced by inhibiting the PI3K-Akt pathway with both chemical inhibitors and RNAi technology. Inhibiting the activity of Akt using Akt inhibitor VIII also facilitated the reactivation of KSHV from latency. Both lytic replication and latency depend on the activity of viral transactivator RTA and we further show that the activity of RTA is increased by reducing Akt1 expression. The data suggest that the PI3K-Akt pathway suppresses the activity of RTA and thereby contributes to the maintenance of viral latency and promote tumorigenesis.

Evidence for similarity assisted recombination and predicted stem-loop structure determinant in potato virus X RNA recombination [PLANT VIRUSES - RNA]

Draghici, H.-K., Varrelmann, M. — Wed, 28 Oct 2009 10:01:32 PDT

Virus RNA recombination, one of the main factors for genetic variability and evolution, is thought to be based on different mechanisms. Here, the recently described in vivo potato virus X (PVX) recombination assay (Draghici & Varrelmann, 2009), was applied to characterize structural parameters of recombination. The assay uses an Agrobacterium mediated expression system incorporating a PVX green fluorescent protein (GFP) labeled full-length clone. The clone contains a partial coat protein (CP) deletion that is defective in cell to cell movement, together with a functional CP + 3''-ntr transcript in Nicotiana benthamiana leaf tissue. The structural parameters assessed were the length of sequence overlap, the distance between mutations and the degree of sequence similarity. The effect on the observed frequency of reconstitution, and the composition of the recombination products were characterized. Application of four different type X intact PVX CP-genes with variable composition allowed the estimation of the junction sites of precise homologous recombination. Although one template switch would have been sufficient for functional reconstitution, between one to seven template switches were observed. Use of PVX GFP mutants with CP deletions of variable length resulted in a linear decrease of the reconstitution frequency. The critical length for homologous recombination observed was 20 to 50 nt. Reduction of the reconstitution frequency was obtained when a phylogeneticly distant PVX type Bi CP-gene was used. Finally, the prediction of CP and 3''-ntr RNA secondary structure demonstrated that recombination junction sites were mainly located in regions of stem-loop structures, allowing the categorization of the recombination observed as similarity assisted.

Molecular epidemiology and genetic diversity of Hepatitis B virus (HBV) genotype E in an Afro-Colombian isolated community [ANIMAL VIRUSES - SMALL DNA]

Wed, 21 Oct 2009 09:01:07 PDT

Hepatitis B virus (HBV) infection is a significant public health concern with 350 millions chronic carriers worldwide. Eight HBV genotypes (A-H) have been described so far. Genotype E (HBV/E) is largely distributed in West Africa, and has rarely been found in other continents, except for few cases in individuals with African background. In this study we characterized HBV genotypes in Quibdó, Colombia by partial S/P genes sequencing, and found for the first time HBV/E circulating in 9 Afro-descendent Colombian patients, which had no recent contact with Africa. The HBV/E presence in this community as a monophyletic group suggests that it was a result of a recent introduction by some Afro-descendant contact or, alternatively, the virus came with slaves brought to Colombia . By using sequences with sampling date we estimated the substitution rate about 3.2 x 10-4 s/s/y, which resulted in a time of the most recent common ancestor (TMRCA) of 29 years. In parallel we also estimated the TMRCA for HBV/E by using two previously estimated substitution rates (7.7 x 10-4 and 1.5 x 10-5 s/s/y). The TMRCA was around 35 years under the higher rate, and 1500 years under the slower rate. In sum, this work reported for the first time the presence of an exclusively African HBV genotype circulating in South America. We also discuss about the time of the entry of this virus in America based on different substitution rates estimated for HBV.

The Early Host Innate Immune Response to Duck Hepatitis B Virus Infection [ANIMAL VIRUSES - SMALL DNA]

Tohidi-Esfahani, R., Vickery, K., Cossart, Y. — Wed, 21 Oct 2009 09:01:11 PDT

The early phase after hepatitis B virus infection could play a crucial role in clearance and/or persistence of the virus, particularly in neonates. This work compared the early phase of duck hepatitis B virus infection in day old (D1) and 28 day old (D28) ducks to determine whether differences in viral or host innate immune response can be related to the difference in outcome. In the first phase almost immediately after inoculation virus was taken up by components of the reticulo-endothelial systems, particularly liver-specific macrophages, Kupffer cells. Very early after infection the induction of interferon alpha by infected hepatocytes occurs and was rapidly reinforced by recruitment of effector lymphocytes which directly or indirectly cause apoptosis eliminating infected hepatocytes as was seen in mature birds. In addition, a lack of lymphocytic infiltration of the liver was found in D1 ducks which supports the suggestion that the innate immune network is less effective in D1 ducks. Taken together these results suggest that failure of the co-ordinated innate immune response rather than a defect in induced anti-viral cell mediated immunity may be the key factor which makes baby ducks vulnerable to persistence of hepadnavirus infection.

ISG15, a ubiquitin-like interferon stimulated gene, promotes Hepatitis C Virus production in vitro: Implications for chronic infection and response to treatment [ANIMAL VIRUSES - POSITIVE-STRAND RNA]

Chen, L., Sun, J., Meng, L., Heathcote, J., Edwards, A., McGilvray, I. — Wed, 21 Oct 2009 09:01:08 PDT

Background & Aims: Up-regulation of interferon stimulated genes (ISGs), including interferon stimulated gene 15 (ISG15) and other members of the ISG15 pathway, in pre-treatment liver tissue of Hepatitis C virus (HCV) chronically-infected patients is associated with subsequent treatment failure (pegylated interferon-/ribavirin, pegIFN/rib). Here, we study the effect of ISG15 on HCV production in vitro. Methods: The levels of ISG15 and of its conjugation to target proteins (ISGylation) were increased by plasmid transfection, or ISGylation was inhibited by siRNA directed against the E1 activating enzyme Ube1L in Huh7.5 cells. Cells were infected with HCV J6/JFH1 virus, and HCV RNA and viral titers determined. Results: Levels of both HCV RNA and virus increased when levels of ISG15 and ISGylation were increased, and decreased when ISGylation was inhibited. The effects of ISGylation on HCV are independent of upstream IFN signaling: IFN-induced ISG expression is not altered by Ube1L knockdown. The effect is also not likely secondary to a cytokine effect: treatment of cells with purified ISG15 does not inhibit HCV production. Conclusions: Although ISG15 has antiviral activity against most viruses, ISG15 promotes HCV production. HCV might exploit ISG15 as a host immune evasion mechanism, and this may in part explain how increased expression of ISGs, especially ISG15, correlates with subsequent interferon-based treatment failure.

Viral host range factors C7 or K1 are essential for modified vaccinia virus Ankara late gene expression in human and murine cells, irrespective of their capacity to inhibit PKR-mediated eIF2{alpha} phosphorylation [ANIMAL VIRUSES - LARGE DNA]

Wed, 21 Oct 2009 09:01:07 PDT

Vaccinia virus (VACV) infection induces phosphorylation of eukaryotic translation initiation factor 2 (eIF2), which inhibits cellular and viral protein synthesis. In turn, VACV evolved the capacity to antagonise this anti-viral response by expressing the viral host range proteins K3 and E3. Here, we reveal that the host range genes K1L and C7L also prevent eIF2 phosphorylation in modified VACV Ankara (MVA) infection of several human and murine cell lines. Moreover, C7L-deleted MVA (MVA-C7L) lacked late gene expression, which could be rescued by the function of either host range factor K1 or C7. We demonstrate that viral gene expression was blocked after viral DNA replication, and that it was independent of apoptosis induction. Furthermore, we found that eIF2 phosphorylation in MVA-C7L infected cells is mediated by protein kinase R (PKR) as shown in murine embryonic fibroblasts (MEF) lacking PKR function, and demonstrated that this is not due to reduced E3L gene expression. The block of eIF2 phosphorylation by C7 could be complemented by K1 in cells infected with MVA-C7L encoding a re-inserted K1L gene (MVA-C7L-K1L). Importantly, our data illustrate that eIF2 phosphorylation by PKR is not responsible for the block of late viral gene expression. This suggests that other mechanisms targeted by C7 and K1 are essential for completing the MVA gene expression cycle and likely also for VACV replication in a diverse set of cell types.

Identification of a dominant endoplasmic reticulum retention signal in yellow fever virus pre-membrane protein [ANIMAL VIRUSES - POSITIVE-STRAND RNA]

Ciczora, Y., Callens, N., Seron, K., Rouille, Y., Dubuisson, J. — Wed, 21 Oct 2009 09:01:11 PDT

Yellow fever virus encodes two envelope proteins, pre-membrane (prM) and envelope (E), that accumulate in the endoplasmic reticulum (ER). The C-termini of prM and E form two anti-parallel transmembrane alpha-helices that contain ER retention signals. To further understand the ER retention of prME heterodimer, we characterized the subcellular localization of chimeric proteins made of a reporter protein fused to the transmembrane segments of yellow fever virus envelope proteins. We showed that at least three of the transmembrane segments of prME heterodimer are ER retention signals. Interestingly, increasing the length of these alpha-helices led to the export of the chimeric proteins out of the ER. Furthermore, adding a di-acidic export signal at the C-terminus of the first transmembrane segment of the E protein also induced export to the cell surface. However, adding this export signal at the C-terminus of the first transmembrane segment of E in the context of prME did not change the subcellular localization of prME heterodimer, suggesting the presence of a stronger ER retention signal outside of the first transmembrane segment of E. Importantly, the di-acidic export motif added to the C-terminus of the first transmembrane segment of the prM protein was not sufficient to export a chimeric protein out of the ER, indicating that this sequence is a dominant ER retention signal. Together, these data indicate that a combination of several signals of different strengths contributes to the ER retention of yellow fever virus envelope protein heterodimer.

The hepatitis C virus NS5A protein interacts with {beta}-catenin and stimulates its transcriptional activity in a PI3K-dependent fashion [ANIMAL VIRUSES - POSITIVE-STRAND RNA]

Milward, A., Mankouri, J., Harris, M. — Wed, 21 Oct 2009 09:01:07 PDT

Hepatitis C virus (HCV) infection is increasingly associated with the development of hepatocellular carcinoma (HCC). HCV is not thought to be directly oncogenic but, by modulating a range of cellular functions, may predispose patients to the development of liver tumours. However, the molecular mechanisms by which HCV infection might contribute to HCC remain to be characterised. In this regard, we previously showed that the HCV NS5A protein bound to the p85 regulatory subunit of phosphoinositide-3 kinase (PI3K), thereby stimulating the activity of the p110 catalytic subunit of the enzyme. One of the downstream consequences of this was the stabilisation of the proto-oncogene, β-catenin, with a concomitant stimulation of its transcriptional activity. Here, we further analyse the mechanism by which NS5A mediates activation of β-catenin. Although our previous data were consistent with a role for the PI3K downstream effector kinases, Akt and GSK-3β, in NS5A-mediated activation of β-catenin, we demonstrate here that it is in fact independent of either of these kinases. Truncation analysis revealed that both the N- and C-termini of NS5A are required for full activation of β-catenin. Furthermore, we demonstrate that NS5A, either alone or in complex with p85, is able to bind directly to β-catenin, again both N- and C-termini contribute to this interaction. We propose that NS5A activates β-catenin via a novel mechanism that involves a direct interaction between the two proteins and is augmented by PI3K activity. This may contribute to the association between chronic HCV infection and the development of HCC.

The genome of cyprinid herpesvirus 3 encodes 40 proteins incorporated in mature virions [ANIMAL VIRUSES - LARGE DNA]

Wed, 21 Oct 2009 09:01:10 PDT

Koi herpesvirus, also known as cyprinid herpesvirus 3 (CyHV-3), is the etiological agent of an emerging and mortal disease in common and koi carp. CyHV-3 virions present the characteristic morphology of other members of the order Herpesvirales, being composed of an envelope, a capsid containing the genome, and a tegument. In the present study, we identified CyHV-3 structural proteins and the corresponding encoding genes using liquid chromatography tandem mass spectrometry based proteomic approaches. In addition, exponentially modified protein abundance index (emPAI) analyses were used to estimate the relative abundance of identified proteins in CyHV-3 virions. These analyses resulted in the identification of 40 structural proteins that were classified based on bioinformatic analyses as capsid (3), envelope (13), tegument (2) and unclassified (22) structural proteins. Finally, a search for host proteins in purified CyHV-3 virions indicated the potential incorporation of up to 18 distinct cellular proteins. The identification of the proteins incorporated in CyHV-3 virions and the determination of the viral genes encoding these proteins are key milestones for further fundamental and applied research on this virus.

Parvovirus PARV4 visualisation and detection [ANIMAL VIRUSES - SMALL DNA]

Tuke, P. W, Parry, R. P, Appleton, H. — Wed, 21 Oct 2009 09:01:05 PDT

The parvovirus PARV4 is the most recently described member of the Parvoviridae which has a human host. To investigate the prevalence of PARV4 in the blood a quantitative TaqMan PCR (Q-PCR) was developed and plasma, sera or whole blood from a variety of population groups were examined. Eight samples were positive for PARV4, one at high copy number. The high titre positive plasma had an approximate viral load of 5 x 10⁸ genome equivalents per ml. Two human sera identified as PARV4 antibody positive by indirect immunofluorescence were used in immune electron microscopy (IEM) to try and visualise native PARV4 virus within the high titre human plasma. PARV4 virus particles were observed using one of these two sera. To our knowledge this is the first time that native PARV4 virus has been visualised.

Characterization of a Rare Natural Intertypic Type 2/Type 3 Penta-Recombinant Vaccine-Derived Poliovirus Isolated from a Child with Acute Flaccid Paralysis [ANIMAL VIRUSES - POSITIVE-STRAND RNA]

Wed, 21 Oct 2009 09:01:06 PDT

A type 2 vaccine-derived poliovirus (VDPV) (Strain CHN1025) with a 1.1% (10/903) difference from Sabin strain in the VP1 coding region was isolated from a child with poliomyelitis caused by a poliovirus variant infection. The patient was from Shandong province of China and developed acute flaccid paralysis in 1997. The child was infected with a rare and complicated penta-recombinant poliovirus with the uncommon genomic recombinant organization S2/S3/S1/S3/S1/S3. At least 5 successive rounds of recombination occurred in the VP1 capsid coding region and in the 2C, 3C (twice), and 3Dpol noncapsid coding regions during virus evolution, respectively. Strain CHN1025 had most of the characteristics of the type 2 vaccine strain; it had Sabin-specific epitopes, suggesting that the virus was antigenically undistinguishable from the Sabin 2 reference strain. Typical mutations in the 5'-UTR and VP1 associated with reversion to neurovirulence for Sabin 2 poliovirus were found, and the virus showed moderate neurovirulence in transgenic mice. A few nucleotide substitutions were located in the donor sequences, and two donor sequences contained no nucleotide substitutions, suggesting that these sequences were relatively new. The appearance of these mutations within approximately 192 days of at least five 5 successive rounds of recombination events derived from a single ancestral infection illustrates the rapid emergence of new recombinants among VDPVs. This is the first report on the isolation of a type 2/type 3 poliovirus capsid recombinant with one of the 5 crossover sites located in the VP1 coding region.

Differential Binding Patterns to Host Cells Associated with Particles of Several Human Alpha Papillomavirus Types [ANIMAL VIRUSES - SMALL DNA]

Broutian, T. R., Brendle, S. A., Christensen, N. D. — Wed, 21 Oct 2009 09:01:04 PDT

The focus of this research was to compare the binding profiles of HPV11, 16, 18 and 45 VLPs to HaCaT cells and to the extracellular matrix (ECM) secreted by these cells. All four HPV types tested bind to a component(s) of the ECM. HPV11 VLP binding is blocked when the ECM is pretreated with an anti-Laminin 5 (LN5) Pab. A series of treatments utilizing heparins and heparinase reveals that HPV18 VLP is dependent on heparan sulfates (HS) for binding to cells and ECM. HPV16 and 45 VLP binding is dependent on HS for binding to HaCaT cells and on both HS and LN5 for binding to ECM. These studies emphasize the need to study the binding characteristics of different HPV types before applying universal binding principles to all papillomaviruses.

The VP35 Protein of Ebola Virus Impairs Dendritic Cell Maturation Induced by Virus and Lipopolysaccharide [ANIMAL VIRUSES - NEGATIVE-STRAND RNA]

Wed, 14 Oct 2009 09:01:34 PDT

Ebola virus causes rapidly progressive hemorrhagic fever, which is associated with severe immuosuppression. In infected dendritic cells (DC), Ebola virus replicates efficiently and inhibits DC maturation without inducing cytokine expression, leading to impaired T cell proliferation. However, the underlying mechanism remains unclear. In this study, we report that Ebola virus VP35 impairs the maturation of mouse dendritic cells. When expressed in mouse immature DCs, Ebola virus VP35 prevents virus-stimulated expression of CD40, CD80, CD86, and MHC class II. Further, it suppresses the induction of cytokines such as IL-6, IL-12, TNF-, and interferon /β. Notably, Ebola VP35 attenuates the ability of DCs to stimulate the activation of CD4+T cells. Addition of type I interferon to mouse DCs only partially reverses the inhibitory effects of VP35. Moreover, VP35 perturbs mouse DC functions induced by lipopolysaccharide, an agonist of Toll-like receptor 4. Deletion of the amino terminus abolished its activity whereas a mutation in the RNA binding motif has no effect. Our work highlights a critical role of VP35 in viral interference of DC function with resultant deficiency in T cell function, which may contribute to the profound virulence of Ebola virus infection.

Vps4 and the ESCRT-III complex are required for the release of infectious hepatitis C virus particles [ANIMAL VIRUSES - POSITIVE-STRAND RNA]

Corless, L., Crump, C., Griffin, S. D. C., Harris, M. — Wed, 14 Oct 2009 09:01:30 PDT

The mechanisms by which infectious hepatitis C virus (HCV) particles are assembled and released from infected cells remain poorly characterised. In this regard, many other enveloped viruses, notably HIV-1, have been shown to utilise the host vacuolar protein sorting machinery (also known as the endosomal sorting complex required for transport or ESCRT) to traffic through the cell and effect the membrane rearrangements required for the formation of enveloped particles. We postulated that this might also apply to HCV. To test this hypothesis we established a method of conditional virus-like particle assembly involving trans-complementation of an envelope-deleted JFH-1 genome using plasmid transfection. This system reliably produced virus particles that were infectious and could be easily enumerated by focus-forming assay in Huh7 cells. Following co-transfection with plasmids expressing various dominant-negative forms of either components of the ESCRT-III complex, or Vps4 (the AAA ATPase that recycles the ESCRT complexes), a reduction in particle production was seen. No significant effect was observed after co-transfection of dominant-negative ESCRT-I or Alix, an ESCRT associated protein. Dominant-negative Vps4 or ESCRT-III components had no effect on either virus genome replication, or the accumulation of intracellular infectious particles. These data were confirmed using cell culture infectious HCV and we conclude, therefore, that HCV requires late components of the ESCRT pathway for release of infectious virus particles.

The capsid protein of Cowpea chlorotic mottle virus is a determinant for vector transmission by a beetle [PLANT VIRUSES - RNA]

Mello, A. F. S., Clark, A. J., Perry, K. L. — Wed, 14 Oct 2009 09:01:31 PDT

Cowpea chlorotic mottle virus (CCMV) is a bromovirus transmitted by species of chrysomelid beetles, including the spotted cucumber beetle, Diabrotica undecimpunctata. An experimental system was set up to identify the viral determinant(s) of the beetle transmission of CCMV. Nicotiana clevelandii was selected as an experimental plant host because it supports the replication and accumulation of both CCMV and a second member of the genus Bromoviridae, Cucumber mosaic virus (CMV). Using a reverse genetic system for CMV, a complementary DNA copy of the CCMV capsid protein gene was substituted for that of the CMV capsid protein gene. The resulting 'CMV-hybrid' consisted of wild-type CMV RNA 1, RNA 2, and a chimeric CMV RNA 3 expressing the CCMV structural protein. The CMV-hybrid replicated and formed virions in N. clevelandii; in electron micrographs the hybrid virus was indistinguishable from CCMV. In beetle feeding assays, both CCMV and the CMV-hybrid were transmitted by D. undecimpunctata, while beetle transmission of CMV was not observed. Conversely, only CMV was observed to be transmitted by the aphid Myzus persicae. Surprisingly, the CMV-hybrid was transmitted more efficiently than the parental CCMV, and a virus-induced alteration in beetle feeding behavior is proposed to account for the difference. These results indicate that the CCMV capsid protein is a viral determinant for beetle vector transmission.

Mechanisms of control of acute Friend virus infection by CD4+ helper T cells and their functional impairment by regulatory T cells [ANIMAL VIRUSES - RETROVIRUSES]

Nair, S. R, Zelinskyy, G., Schimmer, S., Gerlach, N., Kassiotis, G., Dittmer, U. — Wed, 14 Oct 2009 09:01:34 PDT

The role of Cytotoxic CD8+ T cells (CD8+ CTLs) is well defined in retroviral immunity but the role of CD4+ T helper (Th) cells is poorly understood. The Friend retrovirus (FV) murine infection model is a good model to study immune responses in retroviral infections and hence was used to characterize the role of Th cells during acute infection. In vivo depletion of Th cells in acutely infected mice demonstrated that Th cells were vital in controlling viral spread and onset of erythroleukemia and for the maintenance of FV-specific CD8+ T-cell and neutralizing antibody responses. Kinetic analysis of FV-specific Th cell responses using class-II tetramers showed that the magnitude of the Th cell response correlated with the level of resistance to FV-induced leukemia in different mouse strains. FV-specific CD4+ T-cell receptor (TCR) beta transgenic (CD4+ TCR beta-tg) T-cells were adoptively transferred into mice infected for different time periods (1 wpi, 2 wpi, 3 wpi) to investigate the direct anti-viral effect of CD4+ T cells in FV infection. Results indicated that FV-specific CD4+ TCR beta-tg T cells were functionally active until 2 wpi, including their ability to produce IFN-g and reduce viral loads. However, the donor cells lost their anti-viral activity starting from 3 wpi. Interestingly, in vivo depletion of regulatory T-cells (Tregs) at this time point restored IFN-g production by transferred CD4+ T cells. Current study reveals that Th cells were critical for recovery from acute Friend retroviral infection but were functionally impaired during the late phase of acute infection due to induced Tregs.

Detection of a novel reassortant epizootic hemorrhagic disease virus in the United States containing RNA segments derived from both exotic and endemic serotypes [ANIMAL VIRUSES - DOUBLE-STRAND RNA]

Wed, 14 Oct 2009 09:01:33 PDT

Epizootic hemorrhagic disease virus (EHDV) is a Culicoides-transmitted orbivirus that infects domestic and wild ruminants and is provisionally distributed throughout Africa, North America, Australia, East Asia, and the Middle East. Historically, of the seven proposed serotypes of EHDV, only EHDV-1 and EHDV-2 have been reported from North America. In 2006, EHDV isolates were recovered from moribund or dead white-tailed deer (Odocoileus virginianus) in Indiana and Illinois that could not be identified as either EHDV-1 or EHDV-2 by virus neutralization tests or by serotype-specific RT-PCR. Additional serological and genetic testing identified the isolates as EHDV-6, a serotype that, although originally described from Australia, has recently been recognized as an emerging pathogen of cattle in Morocco, Algeria, and Turkey. In 2007 and 2008, EHDV-6 was isolated again from white-tailed deer; this time in Missouri, Kansas, and Texas, suggesting that the virus is capable of overwintering and that it may become, or already is, endemic in a geographically widespread region of the United States. Genetic characterization of the virus indicates that it is a reassortant, such that the outer capsid proteins determining serotype specificity (VP2 and VP5) are derived from exotic EHDV-6, while the remaining structural and nonstructural proteins were apparently obtained from indigenous EHDV-2 (Alberta).

The antibody dependent enhancement (ADE) of dengue virus infection in U937 cells requires cholesterol rich membrane microdomains [ANIMAL VIRUSES - POSITIVE-STRAND RNA]

Wed, 14 Oct 2009 09:01:33 PDT

Dengue virus is the causative agent of dengue fever and the more severe forms of the infection known as dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS). Secondary infections with a serotype different from the primary infection are considered a risk factor for the development of DHF/DSS. One explanation for the increased risk of DHF/DSS development after heterologous secondary infections is the antibody dependent enhancement (ADE) hypothesis. This hypothesis postulates that pre-existing non-neutralizing antibodies will form immune complexes with the new serotype-infecting virus that in turn, will have enhanced capacity to infect macrophages and other Fc receptor (FcvR) bearing cells. Despite the evidence supporting the ADE hypothesis, the molecular mechanisms of ADE are not fully understood. In this work, we present evidence which indicates that intact lipid rafts are required for the ADE infection of U937 cells with dengue virus. Flow cytometry analysis to measure percentage of infected cells, showed that treatment of differentiated U937 cells with nystatin (30µg/ml), filipin (10µg/ml) or β-methyl cyclodextrin (30mM) reduces significantly (p< 0.05) the ADE of dengue 4 virus infection in vitro without any effect in viability and in the number of FcvR bearing cells. Later cholesterol replenishment by supplementing treated cell cultures with bovine fetal serum for 24 hrs, reestablished lipid raft integrity and reversed the alteration of the ADE in vitro (p< 0.05). Our results suggest that ADE of U937 infection by dengue virus requires the presence of cholesterol and cholesterol rich membrane microdomains.

Characterization of the Functional Requirements of West Nile Virus Membrane Fusion [ANIMAL VIRUSES - POSITIVE-STRAND RNA]

Moesker, B., Rodenhuis-Zybert, I. A., Meijerhof, T., Wilschut, J., Smit, J. M. — Wed, 14 Oct 2009 09:01:32 PDT

Flaviviruses infect their host cells by a membrane fusion reaction. In this study, we performed a functional analysis of the membrane fusion properties of West Nile virus (WNV) with liposomal target membranes. Membrane fusion was monitored continuously using a lipid mixing assay involving the fluorophore pyrene. Fusion of WNV with liposomes occurred on the time scale of seconds and was strictly dependent on mildly-acidic pH. Optimal fusion kinetics were observed at pH 6.3, the threshold for fusion being pH 6.9. Preincubation of the virus alone at pH 6.3 resulted in a rapid loss of fusion capacity. WNV fusion activity is strongly promoted by the presence of cholesterol in the target membrane. Furthermore, we provide direct evidence that cleavage of prM to M is a requirement for fusion activity of WNV.

0rganization of Influenza A virus envelope at neutral and low pH [ANIMAL VIRUSES - NEGATIVE-STRAND RNA]

Wed, 14 Oct 2009 09:01:35 PDT

Fusion of the Influenza A H1N1 virus envelope with endosomal membrane at low pH allows the intracellular delivery of the viral genome playing an essential role in the infection process. Low pH induces an irreversible modification of the virus envelope which has so far resisted to 3D structural analysis, partly due to virus pleiomorphy. Here we show that atomic force microscopy (AFM) in physiological buffer can image structural details of the virus envelope both at neutral pH and after a low pH treatment. At low and intermediate magnification, AFM of control virions confirms both the pleiomorphy and the existence of zones devoid of glycoprotein spikes at the virus surface established by electron microscopy. At higher magnification, the unique vertical resolution of the AFM in 3D topography demonstrates the lateral heterogeneity in spikes distribution and strongly suggests that, at least locally, they can be organized in an irregular honeycomb pattern. The surface honeycomb pattern was more easily detected due to an increase in spikes height following low pH treatment at low temperature which likely prevented disruption of the organization. This enhanced contrast associated with low pH treatment emphasizes differences in the glycoprotein distribution between virions. It is concluded that, in association with EM approaches, AFM can help to establish correlation between surface structure and Influenza virus infectivity/pathogenicity.

Hepatitis B virus X protein overcomes the growth-inhibitory potential of retinoic acid by down-regulating retinoic acid receptor-{beta}2 expression via DNA methylation [ANIMAL VIRUSES - SMALL DNA]

Jung, J. K., Park, S.-H., Jang, K. L. — Wed, 14 Oct 2009 09:01:36 PDT

Aberrant promoter methylation of retinoic acid receptor-β₂ (RAR--β₂) is frequently detected in hepatitis B virus (HBV)-positive hepatocellular carcinoma (HCC); however, the mechanism and its biological significance are unknown. Here, we report that HBx, the principal oncogene product of HBV, induces promoter hypermethylation of RAR-β₂ via up-regulation of DNA methyltransferases 1 and 3a, resulting in down-regulation of its expression in human HCC cells. In addition, HBx abolished the potentials of retinoic acid (RA) to down-regulate levels of G1-checkpoint regulators including p16, p21, and p27, resulting in activation of E2F1 in the presence of RA. As a consequence, HBx-expressing cells compared to the control cells were less susceptible to the RA-induced cell growth inhibition. These effects almost completely disappeared when levels of RAR-β₂ in HBx-expressing cells were restored by treatment with a universal DNA methylation inhibitor, 5'Aza-2'dC. Considering that RAR-β₂ is a major executor of the anti-tumor potential of RA, its epigenetic down-regulation by HBx should be an important step during HBV-mediated tumorigenesis.

Strain-specific proteolytic processing of the prion protein in prion diseases of ruminants transmitted in ovine transgenic mice [TSE AGENTS]

NICOT, S., BARON, T. — Wed, 14 Oct 2009 09:01:32 PDT

The cerebral prion protein (PrP) isolated in the absence of proteinase K digestion, from ruminants prion sources transmitted to ovine transgenic mice, was studied by Western blot. A C2 PrP fragment, showing strain-specific cleavages similar to those observed after proteinase K or thermolysin digestion, accumulated in the brain. "CH1641-like" scrapie was characterized by the unique accumulation of a more C-terminally cleaved PrP fragment (CTF14). A similar, protease-resistant, PrP product was observed after proteinase K or thermolysin digestion. Whereas classical BSE appeared highly resistant to thermolysin digestion, CH1641 and "CH1641-like" natural isolates did not show any remarkable feature regarding resistance to thermolysin. Thus the molecular strain-specific features in the brain of TSE-infected mice essentially reflect the PrP proteolytic processing occurring in vivo.

Mapping of QTL affecting classical scrapie incubation time in a population comprising several generations of scrapie infected sheep [TSE AGENTS]

Wed, 14 Oct 2009 09:01:31 PDT

Although susceptibility to scrapie is largely controlled by the PrP gene, the role of other genes that affect scrapie resistance in sheep is now confirmed. Following the detection of quantitative trait loci (QTL) on chromosomes 6 and 18 in a half-sib family with an ARQ-VRQ susceptible PrP genotype, the whole pedigree of a naturally infected flock was investigated to confirm these QTL regions in different PrP genotypes. The present study has allowed us to confirm the QTL on the chromosome 18, and to demonstrate QTL effects in several PrP genotypes.

Functions of Tat: the versatile protein of human immunodeficiency virus type 1 [REVIEWS]

Romani, B., Engelbrecht, S., Glashoff, R. H. — Wed, 07 Oct 2009 09:01:14 PDT

Human immunodeficiency virus type 1 (HIV-1) Tat is a multifunctional protein that contributes to several pathological symptoms of HIV-1 infection as well as playing a critical role in virus replication. Tat is a robust transactivating protein that induces a variety of effects by altering the expression levels of cellular and viral genes. The functions of Tat are therefore primarily related to its role in modulation of gene expression. In this review the functions of HIV-1 Tat that have been well documented, as well as a number of novel functions that have been proposed for this protein, are discussed. Since some of the functions of Tat vary in different cell types in a concentration dependent manner and because Tat sometimes exerts the same activity through different pathways, study of this protein has at times yielded conflicting and controversial results. Due to its pivotal role in viral replication and in disease pathogenesis, Tat and the cellular pathways targeted by Tat are potential targets for new anti-HIV drugs.

Effects of HPV-16 E5 deletion mutants on epithelial morphology: functional characterization of each transmembrane domain [ANIMAL VIRUSES - SMALL DNA]

Wed, 07 Oct 2009 09:01:15 PDT

Human papillomavirus type 16 (HPV-16) is the cause of cervical cancer. The HPV genome encodes three transforming proteins, E5, E6 and E7. E6 and E7 are the main transforming proteins of HPV while the role of E5 is still poorly understood. Using three dimensional organotypic raft cultures we show that HaCaT human keratinocytes expressing HPV-16 E5 form a very perturbed epithelium, with simultaneous hyperkeratinisation of some cells and defective differentiation of other cells. The basal layer is disturbed and many cells invade the collagen matrix. Many cells among the differentiated layers show characteristics of basal cells: progression through the cell cycle, expression of cytokeratin 14, lack of cytokeratin 1 and production of matrix metalloproteases (MMP). Using deletion mutants which encompass the three hydrophobic domains of E5, we have assigned the ability to invade the matrix to the first hydrophobic domain, and the capacity to induce MMP9 to the C-terminal four amino acids. We also show that invasion and production of MMP9 can be dissociated, as mutants still capable of invasion do not produce MMP9 and vice versa.

Cell density-dependent increase in the level of protease-resistant PrP in prion-infected Neuro2a mouse neuroblastoma cells [TSE AGENTS]

Nakamitsu, S., Kurokawa, A., Yamasaki, T., Uryu, M., Hasebe, R., Horiuchi, M. — Wed, 07 Oct 2009 09:01:15 PDT

Cells persistently infected with prions continuously produce protease-resistant prion protein (PrP-res). Here we show that the PrP-res level in prion-infected Neuro2a (N2a) neuroblastoma cells decreased to 50 % of the initial level over the first 48 h and then recovered by 96 h after seeding. The level of cellular prion protein (PrPC) also appeared to fluctuate, but did not influence the fluctuation of the PrP-res level. Prion-infected N2a cells, co-cultured with a higher number of prion-unsusceptible cells had twice as much PrP-res than those cultured without unsusceptible cells, suggesting that cell density influences the fluctuation of PrP-res levels. A direct cell-to-cell contact between cells, rather than soluble factors, was involved in the cell density-dependent increase in the PrP-res level. The cholesterol content, which is known to influence PrP-res formation, also changed depending on cell density. Our data suggest that alterations in cellular microenvironments controlled by cell density influence PrP-res formation.

Genetic relatedness of H6 subtype avian influenza viruses isolated from wild birds and domestic ducks in Korea and their pathogenicity in animals [ANIMAL VIRUSES - NEGATIVE-STRAND RNA]

Wed, 07 Oct 2009 09:01:13 PDT

We report the genetic characterization of the H6 avian influenza (AI) viruses isolated from domestic ducks and wild birds in Korea between April 2008 and April 2009. A phylogenetic analysis showed that the H6N1 viruses of wild birds and domestic ducks were the same genotype (K-1) and were similar to the H6N1 virus isolated from a live poultry market in 2003, as six of the eight gene segments of those viruses had a common source. However, the H6N2 viruses of domestic poultry were separated into four genotypes (K-2a, K-2b, K-2c and K-2d) by at least a triple reassortment between influenza viruses of low pathogenicity from Korean poultry (H9N2 and H3N2) and viruses from aquatic birds. In an experimental infection of animals, certain H6 AI viruses replicated well in chickens and mice without pre-adaptation, indicating that the H6 virus pathogenicity has the potential to be altered due to multiple reassortments, and these reassortments could result in an interspecies transmission into mammals

Differential activation profiles of Crimean-Congo hemorrhagic fever virus versus Dugbe virus infected antigen presenting cells [ANIMAL VIRUSES - NEGATIVE-STRAND RNA]

Wed, 07 Oct 2009 09:01:12 PDT

Crimean-Congo hemorrhagic fever virus (CCHFV) is a highly pathogenic tick-borne virus member of the Bunyaviridae family and the Nairovirus genus. To better elucidate the pathogenesis of CCHFV, we analyzed the host innate immune response induced in antigen presenting cells (APC) infected in vitro by CCHFV. Monocyte-derived dendritic cells (DCs) and macrophages (MPs) both were shown to be permissive to CCHFV and to replicate the virus as monitored by genomic and anti-genomic strand quantification. Virus replication was however controlled, corroborating an efficient IFN- induced response. The up-regulation of CD-83, CD-86 indicated that CCHFV induced a partial maturation of DCs which were shown also to activate the secretion of IL-6 and IL-8 but no TNF- . On the other hand, in MPs, CCHFV infection elicited a high Il-6 and TNF- response and moderate chemokine response. Nevertheless, when we compared the response of these APCs after infection with Dugbe virus (DUGV), a mild pathogenic virus genetically close to CCHFV, we found that, in spite of some similarities, DUGV induced a higher cytokine/chemokine response in MPs. These results suggest that CCHFV was able to selectively inhibit the activation of the inflammatory mediators in the in vitro infection and that these differences could be relevant in pathogenesis.

Regulation of Marburg virus budding by Nedd4.1; a different WW domain of Nedd4.1 is critical for the binding to Marburg and Ebola virus VP40 [ANIMAL VIRUSES - NEGATIVE-STRAND RNA]

Urata, S., Yasuda, J. — Wed, 07 Oct 2009 09:01:13 PDT

The VP40 matrix protein of Marburg virus (MARV) has been shown to be the driving force behind MARV budding, a process in which the PPPY L-domain motif of VP40 plays a critical role. Here, we report that Vps4B and Nedd4.1 play critical roles in MARV VP40-mediated budding. We showed that unidentified activities of the Nedd4.1 HECT domain along with the E3 ubiquitin ligase activity may be required for MARV budding. Moreover, we showed that the first WW domain of Nedd4.1, WW1, is critical for binding to MARV VP40, indicating that MARV VP40 and Ebola virus VP40 are recognized by a different WW domain of Nedd4.1. This is the first report showing that the viral L-domains containing PPxY have specificities for binding to WW domains. Our findings provide new insights into MARV budding, which may contribute to the development of novel anti-MARV therapeutic strategies.

Thogoto virus ML protein is a potent inhibitor of the IRF7 transcription factor [ANIMAL VIRUSES - NEGATIVE-STRAND RNA]

Buettner, N., Vogt, C., Martinez-Sobrido, L., Weber, F., Waibler, Z., Kochs, G. — Wed, 07 Oct 2009 09:01:12 PDT

The tick-transmitted orthomyxovirus Thogoto virus (THOV) encodes the ML protein acting as a viral suppressor of the host interferon (IFN) system. Here, we describe that type I IFN is strongly induced in primary mouse embryo fibroblasts as well as plasmacytoid dendritic cells upon infection with a THOV mutant lacking the ML gene. However, wild-type THOV encoding ML suppresses induction of IFN by preventing the activation of members of the IFN regulatory factor (IRF) family. We found that reporter gene expression dependent on IRF3 and IRF7 was strongly inhibited by ML. Further experiments revealed that ML interacts with IRF7 and prevents dimerization of the transcription factor and its association with the coactivator TRAF6. Interestingly, another IRF7 activation step, nuclear translocation, is not affected by ML. Our data elucidate ML protein as a virulence factor with an IRF-specific IFN-antagonistic spectrum.

Towards an Understanding of the Migration of Crimean-Congo Hemorrhagic Fever Virus [ANIMAL VIRUSES - NEGATIVE-STRAND RNA]

Mild, M., Simon, M., Albert, J., Mirazimi, A. — Wed, 07 Oct 2009 09:01:14 PDT

Crimean-Congo Hemorrhagic Fever (CCHF) is a lethal disease caused by CCHF Virus (CCHFV). It is one of the most widespread medically significant tick-borne pathogen, with a distribution that coincides well with the geographic occurrence of its tick vector, Hyalomma marginatum marginatum. Sporadic outbreaks of CCHF have previously been recognized in Asia, Africa, Middle East and Europe but in the 21st century, outbreaks have become more frequent in former Yugoslavia, Turkey and Iran. It has been suggested that CCHFV is a migrating pathogen but it is not clear to what extent. We have for the first time, analyzed the worldwide migration pattern of CCHFV. Our results showed that Turkey may be a donor in Europe, both towards east and west, while the United Arab Emirates acted as a donor in the Middle East and China was found to be the origin for genotype 2. Finally, we showed that migration of CCHFV was unrestricted between Iran and Pakistan. Considering the distribution and coincidence of the tick vector with CCHFV and CCHF, and the fact that the viral tick vector is present in Western Europe, future outbreaks may extend to include hitherto naive areas, suggesting that increased surveillance and geographic mapping of this lethal pathogen is needed.

The biological significance of amino acid substitutions in hepatitis B surface antigen (HBsAg) for glycosylation, secretion, antigenicity and immunogenicity of HBsAg and hepatitis B virus replication [ANIMAL VIRUSES - SMALL DNA]

Wed, 07 Oct 2009 09:01:15 PDT

Amino acid (aa) substitutions of hepatitis B surface antigen (HBsAg) may affect the antigenicity and immunogenicity of HBsAg, leading to immune escape and diagnostic failure. The aa positions 122 and 160 are known as determinants for HBsAg subtypes d/y and w/r, respectively. The substitution K122I has been shown to strongly affect HBsAg antigenicity. In this study, we investigated the significance of naturally occurring aa substitutions K122I, T123N, A159G, and K160N. Both T123N and K160N substitutions resulted in additional N-glycosylated forms of HBsAg, while the other mutations produced more glycosylated HBsAg as compared to the wild type (wt). Detection of HBsAg by ELISA and immunofluorescence staining indicated that variant HBsAg (vtHBsAg) with K122I was not recognized by HBsAg immunoassays while vtHBsAg with T123N, A159G, K160N and A159G/K160N had reduced antigenicity. DNA immunization in BALB/c mice revealed that wtHBsAg and vtHBsAg with T123N and K160N are able to induce antibodies to HBsAg (anti-HBs), whereas K122I and A159G greatly impair the ability of HBsAg to trigger anti-HBs responses. The cellular immune response to the HBsAg aa 29-38 epitope was enhanced by K160N substitution. Using replication competent clones of hepatitis B virus, T123N and A159G substitutions were shown to strongly reduce virion assembly. The aa substitution K160N appeared to compensate for the negative effect of A159G on virion production. These results reveal complex effects of aa substitutions on biochemical properties of HBsAg, on antigenicity and immunogenicity, and on the replication of the hepatitis B virus.

Expression of the NS3 protease of cytopathogenic BVDV results in the induction of apoptosis but does not block activation of the interferon beta promoter [ANIMAL VIRUSES - POSITIVE-STRAND RNA]

Wed, 30 Sep 2009 09:01:03 PDT

The Pestivirus, Bovine Viral Diarrhoea Virus (BVDV), can exist as two biotypes, cytopathogenic (cp) and non-cytopathogenic (ncp). The cp differs from ncp by the continual expression of free non-structural protein 3 (NS3). Cp BVDV infection of cultured cells induces apoptosis, whereas ncp BVDV infection has been reported to block the induction of IFN-β. To investigate the viral mechanisms underlying these effects NS3 or NS2-3 proteins of ncp and cp BVDV biotypes, together with the cognate NS3 cofactor NS4A, were expressed in cells and their effect upon apoptosis and induction of IFN-β was investigated. Expression of NS3/4A resulted in increased activity of caspase 9 and caspase 3, indicating the induction of the intrinsic apoptosis pathway. Mutational analysis revealed that a protease inactive NS3/4A was unable to induce apoptosis suggesting that the NS3 protease activity is required for initiation of apoptosis during cp BVDV infection. The ability of NS2-3 to modulate activation of the IFN-β promoter was also investigated. These studies confirmed that, unlike the related viruses HCV and GBV-B, the BVDV proteases are unable to inhibit TLR3 and RIG-I dependent activation of the IFNβ promoter. These data suggest that BVDV NS3/4A is responsible for regulating the levels of cellular apoptosis and provide new insights regarding the viral elements associated with cp biotype pathogenesis.

Selective modification of rice (Oryza sativa) gene expression by rice stripe virus infection [PLANT VIRUSES - RNA]

Wed, 30 Sep 2009 09:01:02 PDT

Rice stripe disease, caused by rice stripe virus (RSV), is one of the major virus diseases in East Asia. Rice plants infected with RSV usually show symptoms such as chlorosis, weakness, necrosis in newly emerged leaves, and stunting. To reveal rice cellular systems influenced by RSV infection, temporal changes in the transcriptome of RSV-infected plants were monitored by a customized rice oligoarray system. The transcriptome changes in RSV-infected plants indicated that protein synthesis machineries and energy production in the mitochondrion were activated by RSV infection, while energy production in the chloroplast and synthesis of cell structure components were suppressed. The transcription of genes related to host defense systems under hormone signals and those for gene silencing were not activated at the early infection phase. Together with concurrent observation of virus concentration and symptom development, such transcriptome changes in RSV-infected plants suggest that different sets of various host genes are regulated depending on the development of disease symptoms and the accumulation of RSV.

The requirement of cellular DDX3 for Hepatitis C Virus replication is unrelated to its interaction with the viral core protein. [ANIMAL VIRUSES - POSITIVE-STRAND RNA]

Wed, 30 Sep 2009 09:01:03 PDT

The cellular DEAD-box protein DDX3 was recently shown to be essential for HCV replication. Prior to that, we had reported that HCV core binds to DDX3 in yeast-two hybrid and transient transfection assays. Here, we confirm by co-immunoprecipitation that this interaction occurs in cells replicating JFH1 virus. Consistent with this result, immunofluorescence staining of infected cells revealed a dramatic redistribution of cytoplasmic DDX3 by core protein to the virus assembly sites around lipid droplets. Given this close association of DDX3 with core and lipid droplets, and its involvement in virus replication, we investigated the importance of this host factor in the virus lifecycle. Mutagenesis studies located a single amino acid in the N-terminal domain of JFH-1 core that when changed to alanine significantly abrogated this interaction. Surprisingly, this mutation did not alter infectious virus production and RNA replication, indicating that the core-DDX3 interaction is dispensable in the HCV lifecycle. Consistent with previous studies, siRNA-led knockdown of DDX3 lowered virus production and RNA replication levels of both WT JFH1 and the mutant virus unable to bind DDX3. Thus, our study shows for the first time that the requirement of DDX3 for HCV replication is unrelated to its interaction with the viral core protein.

High Prevalence of Neutralizing Activity against Multiple Unrelated HIV-1 Subtype B Variants in Sera from HIV-1 Subtype B Infected Individuals: Evidence for Subtype Specific Rather than Strain Specific Neutralizing Activity [ANIMAL VIRUSES - RETROVIRUSES]

van Gils, M. J, Edo-Matas, D., Schweighardt, B., Wrin, T., Schuitemaker, H. — Wed, 30 Sep 2009 09:01:05 PDT

It is assumed that an effective HIV-1 vaccine should be capable of eliciting neutralizing antibodies. However, even the best antibodies known to date lack neutralizing ability against a significant proportion of primary HIV-1 variants and despite great efforts, still no immunogen is available that can elicit humoral immunity that can protect against infection or disease progression.We tested sera from 35 participants from the Amsterdam Cohort Studies on HIV-1 infection, who were all infected with HIV-1 subtype B and therapy naive at the time of sampling, for neutralizing activity against a panel of 23 tier 2-3 HIV-1 variants, with a minimum of 5 HIV-1 variants per subtype A, B, C and D. Strong cross-clade neutralizing activity was detected in sera from 7 individuals. Strikingly, sera from 22 out of 35 individuals (63%) neutralized 3 or more of the 6 tier 2-3 HIV-1 subtype B viruses in the panel. There was a strong correlation between neutralization titer and breadth in serum. Indeed, the IC50 of sera with strong cross-clade neutralizing activity was significantly higher than the IC50 of sera with cross-subtype B activity, which in turn had a higher IC50 than sera with the lowest neutralization breadth.These results imply that humoral immunity, at least in HIV-1 subtype B infected individuals, is often subtype-specific rather than strain-specific and that the breadth of neutralization is correlated with the titer of neutralizing activity in serum. Considering the difficulties in designing a vaccine that is capable of eliciting cross-clade neutralizing activity, subtype-specific vaccines may be explored as an interesting alternative.

Characterization of neutrophil extracellular traps in cats naturally-infected with the feline leukemia virus (FeLV). [ANIMAL VIRUSES - RETROVIRUSES]

Wed, 30 Sep 2009 09:01:01 PDT

Feline leukemia virus (FeLV), a common, naturally-occurring gammaretrovirus in domestic cats, is associated with degenerative diseases of the hematopoietic system, immunodeficiency, and neoplasia. FeLV infection causes an important suppression of neutrophil functions, leading to opportunistic infections. Recently, a new microbicidal mechanism named NETosis was described in human, bovine, and fish neutrophils, as well as in chicken heterophils. The purpose of the present study was to characterize NETosis in feline neutrophils as well as evaluate neutrophil functions in naturally-infected, FeLV symptomatic and asymptomatic cats through the phagocytosis process, NET release, and MPO activity. Our results showed that feline neutrophils stimulated with protozoa parasites release structures constituted by DNA and histones, which were characterized as NETs by immunofluorescence. Quantification of NETs after neutrophils stimulation showed a significant increase in NET release by neutrophils from FeLV(-) and FeLV(+) asymptomatic cats as compared to FeLV(+) symptomatic ones. Moreover, released NETs and myeloperoxidase (MPO) activity in unstimulated neutrophils of FeLV(+) symptomatic cats are higher than those found in unstimulated neutrophils from FeLV(-) and FeLV(+) symptomatic cats. We report here for the first time NET release by feline neutrophils, along with the fact that NET induction may be modulated by a viral infection. Our results point to the observation that the NET mechanism appears to be over activated in FeLV(+) cats and that this feature could be considered a progression disease marker in feline leukemia viral infection.

Characterization of microRNAs encoded by the Bovine herpesvirus 1 genome [ANIMAL VIRUSES - LARGE DNA]

Wed, 30 Sep 2009 09:01:02 PDT

Bovine herpesvirus 1 (BoHV-1) is a ubiquitous and important pathogen of cattle worldwide. This study reports the identification of ten microRNA (miRNA) genes, Bhv1-mir-1 to Bhv1-mir-10, encoded by the BoHV-1 genome that were processed into 12 detectable mature miRNAs as determined by ultrahigh throughput sequencing bioinformatics analyses of small RNA libraries, and expression studies. We found that four of the miRNA genes were present as two copies in BoHV-1 genome resulting in a total of 14 miRNA-encoding loci. Unique features of the BoHV-1 miRNAs include, evidence of bidirectional transcription and a close association of two miRNA genes with the origin of replication including one miRNA that is encoded within the origin of replication. The miRNA gene Bhv1-mir-5 was encoded on the opposite DNA strand to the latency associated transcript, potentially giving rise to the antisense transcripts originating from this locus. The association of herpesvirus miRNAs with latency appears to be a common feature in the alphaherpesviruses. Analyses of the genome the Bovine herpesvirus 5 for putative miRNA gene orthologues identified a high degree of evolutionary conservation for nine of the BoHV-1 miRNA genes. The possible roles for BoHV-1 miRNAs in the regulation of known BoHV-1 transcription units and the genetics of the BoHV-1 genotypes are also discussed.

Quantitative Analysis of Epstein-Barr Virus (EBV)-Related Gene Expression in Patients with Chronic Active EBV Infection [ANIMAL VIRUSES - LARGE DNA]

Wed, 30 Sep 2009 09:01:01 PDT

Chronic active Epstein-Barr virus (EBV) infection is a systemic EBV-positive lymphoproliferative disorder characterised by persistent or recurrent infectious mononucleosis-like symptoms in patients with no known immunodeficiency. The detailed pathogenesis of the disease is unknown, and no standard treatment regimen has been developed. EBV gene expression was analysed in peripheral blood samples collected from 24 patients with chronic active EBV infection. The expression levels of six latent and two lytic EBV genes were quantified by real-time reverse transcription (RT)-PCR. EBV-encoded small RNA (EBER) 1 and BamHI-A rightward transcripts (BARTs) were abundantly detected in all patients, and latent membrane protein (LMP) 2 was observed in most patients. EBV nuclear antigen (EBNA) 1 and LMP1 were detected less frequently and were expressed at lower levels. EBNA2 and the two lytic genes were not detected in any of the patients. The pattern of latent gene expression was determined to be latency type II. EBNA1 was detected more frequently and at higher levels in the in clinically active patients. Quantifying EBV gene expression is useful in clarifying the pathogenesis of chronic active EBV infection and may provide information regarding a patient's disease prognosis, as well as possible therapeutic interventions.

Biochemical characterization of the (nucleoside-2'O)-methyltransferase activity of dengue virus protein NS5 using purified capped RNA oligonucleotides 7MeGpppACn and GpppACn [ANIMAL VIRUSES - POSITIVE-STRAND RNA]

Selisko, B., Peyrane, F. F., Canard, B., Alvarez, K., Decroly, E. — Wed, 23 Sep 2009 09:01:30 PDT

The Flavivirus RNA genome contains a conserved cap-1 structure, ^7MeGpppA_2'OMeG, at the 5' end. Two mRNA cap methyltransferase (MTase) activites involved in the formation of the cap, the (guanine-N7) and the (nucleoside-2'O)-MTases, reside in a single domain of non-structural protein NS5 (NS5MTase). We report on the biochemical characterization of the 2'OMTase activity of NS5MTase of dengue virus (NS5MTase_DV) using purified short capped RNA substrates (^7MeGpppAC_n or GpppAC_n). NS5MTase_DV methylates both types of substrate exclusively at the 2'O-position. The efficiency of 2'O-methylation does not depend on the methylation of the N7-position. Using ^7MeGpppAC_n and GpppAC_n substrates of increasing chain lengths, we found that both NS5MTase_DV 2'O-activity and substrate binding increased before reaching a plateau at n=5. Thus the cap and 6 nucleotides might define the interface providing efficient binding of enzyme and substrate. K_m values for ^7MeGpppAC₅ and the co-substrate S-adenosyl-L-methionine (AdoMet) were determined (0.39 and 3.26 ;micro;M, respectively). As other reported AdoMet-dependent RNA and DNA MTases, the 2'OMTase activity of NS5MTaseDV shows a low turnover of 3.25 x 10^-4 s^-1. Finally, we set up an inhibition assay and tested GTP- and AdoMet analogs as putative inhibitors of NS5MTase_DV. We confirmed efficient inhibition by the reaction product S-adenosyl-homocysteine (AdoHcy) (IC₅₀ 0.34 µM) and sinefungin (IC₅₀ 0.63 µM) demonstrating that the assay is sufficiently sensitive to conduct inhibitor screening and characterization assays.

Neutralisation of feline immunodeficiency virus by antibodies targeting the V5 Loop of Env [ANIMAL VIRUSES - RETROVIRUSES]

Wed, 23 Sep 2009 09:01:27 PDT

Neutralising antibodies (NAbs) play a vital role in vaccine-induced protection against infection with feline immunodeficiency virus (FIV). However, little is known about the appropriate presentation of neutralisation epitopes in order to induce NAbs effectively; the majority of the antibodies that are induced are directed against non-neutralising epitopes. Here, we demonstrate that a subtype B strain of FIV, designated NG4, escapes autologous NAbs but may be rendered neutralisation-sensitive following the insertion of two amino acids, Lysine and Threonine, at positions 556-557 in the fifth hypervariable (V5) loop of the envelope glycoprotein (Env). Consistent with the contribution of this motif to virus neutralisation, an additional three subtype B strains retaining both residues at the same position were also neutralised by the NG4 serum and serum from an unrelated cat (TOT1) targeted the same sequence in V5. Moreover, when the V5-loop of subtype B isolate KNG2, an isolate that was moderately resistant to neutralisation by NG4 serum, was mutated to incorporate the K-T motif, the virus was rendered sensitive to neutralisation. These data suggest that even in a polyclonal sera derived from FIV infected cats following natural infection, the primary determinant of virus neutralising activity may be represented by a single, dominant epitope in V5.

HLA-C is necessary for optimal HIV-1 infection of human peripheral blood CD4 lymphocytes [ANIMAL VIRUSES - RETROVIRUSES]

Wed, 23 Sep 2009 09:01:28 PDT

The hypothesis that open conformers of HLA-C on target cells might directly exert an effect on HIV infectability was originally suggested by our group. We exploited the peculiar specificity of mAb L31 for HLA-C open conformers to show that normal levels of Env-driven fusion were restored in HLA-C transfectants of a MHC-deleted (fusion-incompetent) cell line. The physiological relevance of this finding is now confirmed by this report, where siRNA technology is employed to silence HLA-C expression in 11 healthy donors peripheral blood lymphocytes (PBL). HIV infectability (by IIIB, Bal and primary isolates) was significantly reduced (p = 0.016) in silenced cells, compared to cells that maintained HLA-C expression, in 10/11 PBL donors. Normal infectability was resumed together with HLA-C expression, when the effect of siRNA interference waned, after several days in culture. We have obtained other confirmations of the HLA-C effect in several assays employing HLA-C positive and negative cell lines, a number of HIV strains and also pseudoviruses. In particular, viruses pseudotyped with Env genes from HIV strains AC10 and QH0692 were assayed on siRNA silenced lymphocytes from three healthy donors: the differences in infection with pseudoviruses were even higher than those observed in infections with normal viruses.

Specific Hydrophobic residues in the {alpha}4 helix of {lambda}CII are crucial for maintaining its tetrameric structure and directing the lysogenic choice [PHAGE]

Parua, P. K., Datta, A. B., Parrack, P. — Wed, 23 Sep 2009 09:01:29 PDT

The CII protein of the temperate bacteriophage lambda is the decision making factor that determines the viral lytic/ lysogenic choice. It is a homotetrameric transcription activator that recognizes and binds specific direct repeat sequences TTGCN₆TTGC in the lambda genome. The quaternary structure of CII is held by a four-helix bundle. It is known that the tetrameric organization of CII is necessary for its activity, but the molecular mechanism behind this requirement is not known. By specific site-directed mutagenesis of hydrophobic residues in the 4 helix of CII that constitutes the four-helix bundle, we found that residues leu70, val74 and leu78 were crucial for maintaining the tetrameric structure of the protein. When any of these residues was substituted a by polar one, CII lost its activity and failed to promote lysogeny. This loss of activity was accompanied by the inability of CII to form tetramers, to bind DNA or to activate transcription.

Involvement of the Fc{gamma} receptor IIA cytoplasmic domain in antibody dependent enhancement of dengue virus infection [ANIMAL VIRUSES - POSITIVE-STRAND RNA]

Moi, M. L., Lim, C.-K., Takasaki, T., Kurane, I. — Wed, 23 Sep 2009 09:01:28 PDT

Subneutralizing concentrations of antibody to dengue virus enhance dengue virus infection of Fc receptor-expressing cells. This phenomenon, referred to as antibody dependent enhancement (ADE), has been hypothesized to be responsible for the severe form of dengue virus infection, including dengue hemorrhagic fever and dengue shock syndrome. To further analyze the mechanisms of ADE in vitro, we introduced a series of cytoplasmic mutants to human FcRIIA. We then expressed the mutated FcRIIA on COS-7 cells and examined whether these mutants could enhance dengue virus infection. Wild type FcRIIA enhanced dengue virus infection, consistent with previous reports using FcR-positive monocytes. Disruption of the immune tyrosine activation motif (ITAM) in the cytoplasmic domain of FcRIIA or removing the sequences between the two ITAM regions abrogated ADE. These findings suggest that the specific structure of the FcRIIA cytoplasmic domain is essential for the ability of FcRIIA to mediate ADE.

Capsid gene divergence in Rabbit Haemorrhagic Disease Virus [ANIMAL VIRUSES - POSITIVE-STRAND RNA]

kinnear, m. w., Linde, C. C — Wed, 23 Sep 2009 09:01:28 PDT

The emergence and rapid global spread of the Rabbit Haemorrhagic Disease Virus remains enigmatic despite two decades of study, largely due to the difficulties associated with modelling substitution processes of the RNA genome for phylogenetic inference. We used Maximum Likelihood and Bayesian methods to investigate rates of molecular evolution in the capsid gene, finding evidence of positive selection and of variable substitution rates between nucleotide sites and between lineages. The Maximum Likelihood and Bayesian analyses produced fully congruent topologies, however strong support for older nodes of the phylogeny was only obtained from the Bayesian analyses that utilised the additional information of collection dates for RHDV isolates spanning 22 years. These dates also allowed calibration of the RHDV phylogenetic tree in a calendar year timescale and estimation of dates for the most recent common ancestors of virulent and benign strains. These dates suggested the divergence of RHDV approximately 20 years prior to the first report of haemorrhagic disease in rabbits.

Genetic characterization of early isolates of Japanese encephalitis virus: Genotype II has been circulating since at least 1951 [ANIMAL VIRUSES - POSITIVE-STRAND RNA]

Schuh, A. J, Li, L., Tesh, R. B, Innis, B., Barrett, A. — Wed, 23 Sep 2009 09:01:29 PDT

Japanese encephalitis virus (JEV) consists of five genotypes (GI-V). Phylogenetic characterization of 16 JEV strains isolated from the USSR, Japan and Korea during the 1930s-1970s revealed that 15 strains fell into GIII confirming that GIII was the predominant genotype of JEV in Japan and Korea between 1935 (isolation of the prototype strain; a GIII virus) and the 1990s (when GI supplanted GIII). One of the Korean isolates fell into GII demonstrating that GII has been circulating for at least 19 years longer than previously thought. Formerly, GII was associated with endemic disease and this genotype had never been isolated north of Southern Thailand. Additionally, the northern border of GIII prevalence was extended from Japan to the USSR.

Vitronectin receptors, {alpha}V-integrins, are recognized by several non-RGD containing echoviruses in a continuous laboratory cell line and also in primary human Langerhans' islets and endothelial cells [ANIMAL VIRUSES - POSITIVE-STRAND RNA]

Ylipaasto, P., Eskelinen, M., Salmela, K., Hovi, T., Roivainen, M. — Wed, 23 Sep 2009 09:01:30 PDT

Previously published data suggest that the RGD recognizing integrin, Vβ3, known as the vitronectin receptor, acts as a cellular receptor for RGD containing enteroviruses, coxsackievirus A9 and echovirus 9, in several continuous cell lines as well as in primary human Langerhans' islets. Since this receptor is also capable of binding the ligands by non-RGD-dependent mechanism we investigated here whether vitronectin receptors, V-integrins, might act as a receptor for other echoviruses which do not have RGD motif. Blocking experiments with polyclonal anti-Vβ3 antibody showed that both primary human islets and a continuous laboratory cell line of green monkey kidney origin (GMK) are similarly protected from adverse effects of several non-RGD containing echovirus (E-7, 11, 25, 30, 3) infections. In contrast, the corresponding studies on primary human endothelial cells showed that the receptor works only for E-25, E-30, E-32 and CAV-9. The inhibitory effect of the antibody was not restricted to prototype strains of echoviruses since GMK cells infected with several field isolates of the corresponding serotypes were also protected from virus induced cytopathic effects. Colocalization of virus particles with the receptor molecules in both GMK and primary human endothelial cells was demonstrated by live cell stainings in confocal microscopy. Remarkably in spite of similar virus-receptor colocalization and comparable protective effect of the Vβ3 antibody, the entry pathways of the studied virus strains seemed to be divergent.

Glycoprotein G from pseudorabies virus binds to chemokines with high affinity and inhibits their function [ANIMAL VIRUSES - LARGE DNA]

Viejo-Borbolla, A., Munoz, A., Tabares, E., Alcami, A. — Wed, 23 Sep 2009 09:01:29 PDT

Summary.Pseudorabies virus (PRV) is the aetiological agent of Aujeszky's disease in swine. In other animals, except higher order primates, PRV infection is often fatal. The mechanisms of PRV pathogenesis and immune modulation are largely unknown. PRV codes for 11 glycoproteins. Among them, glycoprotein G (gG) is the most abundant PRV protein found in the supernatant of PRV-infected cell cultures. PRV gG has low amino acid sequence similarity with gG from other animal alphaherpesviruses and its function is unknown. gG from other animal alphaherpesviruses, with the exception of at least equine herpesvirus 4, binds to chemokines. We show here that PRV gG binds to the human chemokine CL1 and several CC and CXC human chemokines with high affinity. Chemokine-binding activity can be detected in the supernatants of PRV-infected cell cultures, and insertional inactivation of the gene encoding gG from the PRV genome results in loss of chemokine-binding activity. Binding of PRV gG to chemokines inhibits chemokine-mediated cell migration, suggesting a role for PRV gG in immune evasion.

Novel circular DNA viruses in stool samples of wild-living chimpanzees [ANIMAL VIRUSES - SMALL DNA]

Wed, 16 Sep 2009 09:01:05 PDT

Viral particles in stool from wild-living chimpanzees were analyzed using random PCR amplification and sequencing. Sequences encoding proteins distantly related to the replicase protein of single stranded circular DNA viruses were identified. Inverse PCR was used to amplify and sequence multiple small circular DNA viral genomes from the stool of 7 chimpanzees. The novel viral genomes were related in size and genome organization to vertebrate circoviruses and plant geminiviruses but with a different location for the stem loop structure involved in rolling circle DNA replication. The full replicase genes of these viruses were most closely related to those of the much smaller (~1kb) plant nanoviruses circular DNA chromosomes. Because the novel viruses have characteristics of both animal and plant viruses, we named them Chimpanzee Stool Associated Circular Viruses (ChiSCV). Further viral metagenomics of animal samples will greatly increase our knowledge of viral diversity and evolution.

Characterization of phylogenetically diverse astroviruses of marine mammals [ANIMAL VIRUSES - POSITIVE-STRAND RNA]

Rivera, R., Nollens, H. H., Venn-Watson, S., Gulland, F. M.D., Wellehan, J. F.X. — Wed, 16 Sep 2009 09:01:04 PDT

SummaryAstroviruses are small nonenveloped positive stranded RNA viruses. Previously studied mammalian astroviruses have been associated with diarrheal disease. Knowledge of astrovirus diversity is very limited, with only six astrovirus species from mammalian hosts officially recognized, and one human and some bat astroviruses recently described. We used consensus PCR techniques for initial identification of five astroviruses of marine mammals; three from California sea lions (Zalophus californianus), one from a Steller sea lion (Eumetopias jubatus), and one from a bottlenose dolphin (Tursiops truncatus). Bayesian and maximum likelihood phylogenetic analysis found that these viruses showed significant diversity at a level consistent with novel species. Astroviruses that we identified from marine mammals were found across the mamastrovirus tree and did not form a monophyletic group. Recombination analysis found that a relatively recent recombination event may have occurred between a human and a California sea lion astrovirus, suggesting that both lineages may have been capable of infecting the same host at one point. The diversity found amongst marine mammal astroviruses and their similarity to terrestrial astroviruses suggests that the marine environment plays an important role in astroviral ecology.

Comprehensive full length sequence analyses of human parechoviruses; diversity and recombination [ANIMAL VIRUSES - POSITIVE-STRAND RNA]

Wed, 16 Sep 2009 09:01:04 PDT

Human parechoviruses (HPeVs) are highly prevalent pathogens among very young children. Although originally classified into 2 serologically distinct types, HPeV1 and -2, recent analyses of variants collected worldwide have revealed the existence of 12 further types classified genetically by sequence comparisons of complete genome sequences or the capsid gene, VP1. To investigate the nature of HPeV evolution, its population dynamics and recombination breakpoints, we generated 18 full length genomic sequences of the most commonly circulating genotypes, HPeV1 and -3, collected over a time span of 14 years from the Netherlands. In total, we analyzed 35 sequences by inclusion of previously published full length sequences. Analysis of contemporary strains of HPeV1 and those most similar to the prototype strain (Harris), showed HPeV1 variants to fall into two genetically distinct clusters much more divergent from each other than observed within other HPeV types. Future classification criteria for HPeVs may require modification to accommodate the occurrence of variants with intermediate degrees of diversity within type.Recombination was frequently observed among HPeV1, -4, -5, and -6 but was much more restricted among HPeV3 strains. Favoured sites for recombination were found to flank the capsid region and further sites were found within the nonstructural region, P2. In contrast to other HPeV types, the majority of the HPeV3 sequences remained monophyletic across the genome, a possible reflection of its lower diversity and potentially more recent emergence than other HPeV types, or biological and/or epidemiological constraints that limit opportunities for co-infections with potential recombination partners.

Sequence diversity, population genetics and potential recombination events in Grapevine rupestris stem pitting-associated virus in Pacific Northwest Vineyards [PLANT VIRUSES - RNA]

Alabi, O. J, Martin, R. R, Naidu, R. A — Wed, 16 Sep 2009 09:01:04 PDT

Grapevine rupestris stem pitting-associated virus (GRSPaV; genus Foveavirus; family Flexiviridae) is present in many grape-growing regions of the world. A total of 84 full-length coat protein (CP) sequences and 57 sequences representing the helicase-encoding region (HR) of the RNA-dependent RNA polymerase were obtained from wine grape cultivars grown in the Pacific Northwest (PNW) of the United States and their molecular diversity compared with corresponding sequences previously reported from other grape-growing regions. In pairwise comparisons, the CP sequences from PNW showed identities ranging between 80 and 100% at the nucleotide (nt) level and the HR sequences showed identities between 79 and 100%. A global phylogenetic analysis of the CP and HR sequences revealed segregation of GRSPaV isolates into four major lineages with isolates from PNW distributed in all four lineages, indicating a lack of clustering by geographical origin. Scion cultivars grafted onto rootstock were found to contain mixtures of more genetic variants belonging to different lineages than own-rooted cultivars. Assessment of population genetic parameters found that the CP was the more variable than the HR region. The discordant gene phylogenies obtained for some CP and HR sequences and the identification of potential intergenic recombination events involving parents from different lineages provided strong evolutionary evidence for genetic diversity among GRSPaV isolates. These results underscore the highly variable nature of the virus with implications for grapevine health status and distribution of virus-tested planting materials. This study also contributes to an increased understanding of molecular population genetics of viruses infecting deciduous woody perennials.

Molecular evolutionary dynamics of Ross River virus and implications for vaccine efficacy [ANIMAL VIRUSES - POSITIVE-STRAND RNA]

Jones, A., Lowry, K., Aaskov, J. G., Holmes, E. C., Kitchen, A. — Wed, 16 Sep 2009 09:01:06 PDT

Ross River virus (RRV) is a mosquito-borne member of the genus Alphavirus that causes epidemic polyarthritis (EPA) in humans, costing the Australian health system at least US$10 million dollars annually. Recent progress in RRV vaccine development requires accurate assessments of RRV genetic diversity and evolution, particularly as they may affect the utility of future vaccination. In this study, we provide novel RRV genome sequences and investigate the evolutionary dynamics of RRV from time-structured E2 gene data sets. Our analysis indicates that although RRV evolves at similar rates to other alphaviruses (mean evolutionary rate of ~8 x 10-4 nucleotide substitutions per site per year), the relative genetic diversity of RRV has been continuously low through time, possibly as a result of purifying selection imposed by replication in a wide range of natural host and vector species. Together, these findings suggest that vaccination against RRV is unlikely to result in the rapid antigenic evolution that could compromise the future efficacy of current RRV vaccines.

A region at the left end of the fowl adenovirus 9 genome that is non-essential in vitro has consequences in vivo [ANIMAL VIRUSES - LARGE DNA]

Corredor, J. C., Nagy, E. — Wed, 16 Sep 2009 09:01:05 PDT

The regions at the left and right ends of fowl adenovirus (FAdV) genomes are not well characterized in comparison to those of human adenoviruses. Using a series of deletion mutants, we analysed a 2.4-kb region near the left end of the FAdV-9 genome (nucleotides 400-2782) that contains packaging signal motifs VI and VII and open reading frames (ORFs) 0, 1, 1A, 1B, 1C and 2. Viable viruses with specific deletions in this region had wild-type characteristics in vitro, as measured by cytopathic effect, plaque morphology, virus titres and growth kinetics. However, one mutant (FAdV-94), which lacked these ORFs and retained the packaging motifs, did not replicate at wild-type levels in vivo, as judged in infected eggs by virus titres in allantoic fluid and in infected chickens by antibody responses, virus titres in feces and virus genome copy numbers in tissues. These findings indicate that some of the ORFs in this region, although dispensable in vitro, are important for in vivo replication of FAdV-9.

Genetic backbone modulates phenotype of Hepatitis B Surface Antigen "mutants" [ANIMAL VIRUSES - SMALL DNA]

Beale, M. A, Ijaz, S., Tedder, R. S — Wed, 16 Sep 2009 09:01:03 PDT

Hepatitis B virus vaccine and diagnostic escape mutants are a growing concern. The principle target of detection, HBsAg, encoded by S, is completely overlapped by the reverse transcriptase encoding P. With the increased incidence of nucleos(t)ide analogue resistance altering P, the concurrent impact on S must be assessed. HBV DNA from fifty nine HBsAg-positive plasmas was sequenced across the polymerase/surface region and the amino acid sequence of HBsAg inferred. ELISA's were formatted containing individually bound monoclonal antibodies directed against three discrete epitopes on HBsAg. Similar point mutations occurring in different genotypes were shown to influence epitope conformation differently indicating that genetic backbone is a major factor in predicting phenotype. C-terminal changes associated with antiviral resistance were found to modulate epitope profiles of HBsAg. Treatment options which may promote drug resistance should be avoided both to protect antiviral treatment and also to prevent facilitation of vaccine and diagnostic escape mutants.

Single amino acid changes in the Turnip mosaic virus viral genome-linked protein (VPg) confer virulence towards A. thaliana mutants knocked-out for eukaryotic initiation factors eIF(iso)4E and eIF(iso)4G [PLANT VIRUSES - RNA]

Wed, 09 Sep 2009 09:00:58 PDT

Previous resistance analyses of A. thaliana mutants knocked-out for eukaryotic translation initiation factors showed that disruption of either At-eIF(iso)4E or both At-eIF(iso)4G1 and At-eIF(iso)4G2 genes resulted in resistance against the Turnip mosaic virus (TuMV). In this study, we selected TuMV virulent variants overcoming those resistances and showed that two independent mutations in the region coding for the viral genome-linked protein (VPg) were sufficient to restore TuMV virulence in At-eif(iso)4e and At-eif(iso)4g1xAt-eif(iso)4g2 KO plants. As VPg-eIF(iso)4E interaction was previously shown to be critical for TuMV infection, a systematic analysis of the interactions between A. thaliana eIF4Es and VPgs of virulent and avirulent TuMV was performed. Our results suggest that the virulent TuMV variants could use an eIF4F-independent pathway.

Genomics and evolution of Aedes-borne flaviviruses [ANIMAL VIRUSES - POSITIVE-STRAND RNA]

Wed, 09 Sep 2009 09:00:58 PDT

We analyzed the complete coding sequences of all recognized species of Aedes-borne flavivirus, including previously uncharacterized viruses within the yellow fever virus (YFV), Spondweni virus (SPOV) and dengue virus (DENV) groups. Two major phylogenetic lineages were revealed, one included the YFV and Entebbe bat virus groups, and the other included the DENV, SPOV and Culex-borne flavivirus groups. This analysis supported previous evidence that Culex-borne flaviviruses evolved from ancestral Aedes-borne viruses. However, the topology at the junction between these lineages remains complex and may be refined by the discovery of viruses related to Kedougou virus. Additionally, viral evolution was found to be associated with the appearance of new biological characteristics; mutations that may modify the envelope protein structure were identified for 7 viruses within the YFV group, and an expansion of host-vector range was identified in the two major evolutionary lineages, which in turn may facilitate the emergence of mosquito-borne flaviviruses.

Assembly and export determine the intracellular distribution of Hepatitis B Virus core protein subunits [ANIMAL VIRUSES - SMALL DNA]

Weigand, K., Knaust, A., Schaller, H. — Wed, 09 Sep 2009 09:00:57 PDT

Little is known about the parameters and factors which determine the intracellular distribution of the Hepatitis B Virus (HBV) core protein (HBc). In order to study HBc in living cells, HBc was GFP-tagged. Being assembly incompetent, the GFP-fusion protein distributed equally throughout the cell. Mutational inactivation of known serine phosphorylation sites within the C-terminal region led to predominant intranuclear localization. Phosphorylation of these targets, presumably by an SR domain protein kinase (SRPK), resulted in predominantly cytoplasmic localization, which suggests active cytoplasmic export or retention. The phosphoserine itself and not its negative charge appears essential for the underlying mechanism. In addition, the arginine-rich, protamine-like domain surrounding these phosphorylation sites does not function as the dominant nuclear localization signal (NLS), as had been previously assumed, because neither deleting nor altering these sequences led to a change in intracellular HBc subunit distribution. Restoring the capability of the fusion protein to form capsids by co-assembly with assembly-competent, sterically uncompromised HBc subunits provided a second assay which included the effects resulting from capsid formation. Assembly was found to be the dominant factor in the cytoplasmic retention of the GFP-HBc fusion protein. Furthermore, the stability of these empty capsids was influenced by the cell cycle inhibitor Nocodazole. Thus, the intracellular distribution of the HBV core protein is dominated by cytoplasmic assembly, which is supported by the active nuclear export of HBc subunits, and modulated during the cell cycle by the instability of capsids.