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1 Department of Arbovirology, Institute for Animal Health, Pirbright, Woking, Surrey GU24 0NF, UK
2 Agence Française de Sécurité Sanitaire des Aliments, 22 rue Pierre Curie, 94703 Maisons-Alfort Cedex 07, France
3 Unité de Virologie Moléculaire, Etablissement Français du Sang Alpes-Méditerranée, 149 Boulevard Baille, 13005 Marseille, France
Correspondence
Houssam Attoui
houssam.attoui{at}bbsrc.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession number for the complete sequence of Middelburg virus strain MIDV-857 determined in this study is EF536323.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Alphaviruses are important pathogens of livestock animals and humans worldwide. The alphavirus particle contains a single copy of the virus genome, which is a linear, positive-sense, single-stranded RNA. The genome length of those alphaviruses that have been sequenced ranges from 11 442 nt for Semliki Forest virus to 11 851 nt for Ross river virus (Pfeffer et al., 1998). The genome has a 5'-terminal cap and the 3' end has a poly(A) tract. The 5' two-thirds of the genome encodes the non-structural proteins nsP1 to nsP4, followed by the 26S junction region, which promotes transcription of the intracellular subgenomic 26S RNA. This latter mRNA contains the structural genes that encode the capsid (C), E3, E2, 6K and E1 proteins and represents the remaining 3' one-third of the genome (Pfeffer et al., 1998).
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; Weaver et al., 2005; Table 1).
Antigenic cross-reactivities between alphaviruses reflect sequence conservation in the C protein and E1 glycoprotein, whilst antibodies directed against the E2 protein are usually virus specific (Griffin, 2001).
Within a given antigenic serocomplex, viruses exhibit more than 57 % nucleic acid and 56 % amino acid identities. Between members of distinct serocomplexes, these values are more than 62 and 60 %, respectively. Middelburg virus (MIDV, the only member of the MIDV serocomplex) is the least divergent of all antigenic complexes, exhibiting more than 67 % nucleic acid and 69 % amino acid sequence identity when compared with other alphaviruses. In contrast, Trocara virus is the most divergent from all other alphaviruses, exhibiting only 57 % nucleic acid and 53 % amino acid identity (Powers et al., 2001).
The type species of the genus Alphavirus is Sindbis virus, which has a very wide distribution, with isolates from Europe, Asia (India, the Philippines and China), Australia and many parts of Africa. Viruses related to Sindbis virus have also been isolated from New Zealand (Whataroa virus) and South America (Aura virus) (Griffin, 2001). However, many of the alphaviruses that are transmitted by mosquitoes are geographically restricted in their distribution. These viruses circulate primarily between their mosquito vectors and small mammals or birds, whilst infection of larger mammals (such as humans or horses, which are regarded as dead-end hosts) can result in severe or fatal forms of disease (Griffin, 2001). More recently, three alphaviruses have been isolated from fish (salmon pancreas disease virus and rainbow trout sleeping disease virus, classified as a single species) and from the elephant seal louse Lepidophthirus macrorihini (classified as a separate species) (Weaver et al., 2005).
The pathogenic alphaviruses can be divided into those that cause a rash and arthritis (mainly the Old World alphaviruses) and those that cause encephalitis (New World alphaviruses), although some have not yet been shown to cause any disease. An example is MIDV, which has never been isolated from mammals or from marine animals. MIDV was originally isolated in South Africa during the summer of 1957. Two isolates of MIDV were reported from Aedes caballus (isolate AR749) and other Aedes mosquitoes (isolate AR747) (Kokernot et al., 1957). Partial sequence information is available only for isolate AR749.
We isolated a new strain of MIDV (isolate MIDV-857) from the spleen of a horse that had died in Zimbabwe in 1993 with severe clinical signs similar to those caused by African horsesickness virus (AHSV; genus Orbivirus, family Reoviridae) (Mertens, 1994). This paper reports the sequence analysis of the complete MIDV-857 genome, using full-length amplification of cDNA (FLAC; Maan et al., 2007) and the SMART methodology described previously (Attoui et al., 2000).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Virus isolation and virus propagation.
Fourteen paired samples of whole blood and spleen were obtained from the Veterinary Research Laboratory in Causeway, Zimbabwe, during 1993. Thirteen of these tested positive for African AHSV in diagnostic RT-PCR assays (using first-round and nested PCR primers targeting AHSV genome segment 7; Zientara et al., 1995). However, one spleen sample, which tested negative for AHSV by PCR, contained a virus that lysed BHK-21 cells 18 h after infection. The unidentified virus was subsequently plaque-purified three times on Vero cells using SeaPlaque agarose and plaques were identified by staining with trypan blue. The virus was further propagated for two passages in BHK-21 cells for biochemical and molecular biology studies. The unidentified virus was also screened by PCR for equine infectious anemia virus, equine arteritis virus and equine herpesviruses.
Determination of the nature of the virus.
Clarified supernatants from infected cell cultures were treated with organic solvents (Freon 113 and Vertrel XF) to see whether the virus contained lipids (i.e. was enveloped or non-enveloped). Briefly, 10 ml of the clarified supernatant was mixed with an equal volume of the solvent and shaken vigorously. The solution was spun at 2000 g for 10 min at 4 °C, the supernatant was recovered and its infectivity was assessed by virus titration on BHK-21 cells. Virus preparations treated with either solvent were subsequently used in lipofection assays using Fugene-6 reagent (Roche) as described by the manufacturer. Briefly, BHK-21 cells were grown to subconfluency in six-well plates. In a separate tube, 7 µl Fugene-6 was added to 100 µl of the treated culture supernatant and mixed by pipetting. The supernatant was removed from the wells of the culture plates and replaced with fresh serum-free EMEM. The transfection mixture was added to the wells and mixed by gently shaking the plates. The plates were incubated for 6 h at 37 °C and the supernatant was then replaced with EMEM containing 5 % FBS.
Similarly, other aliquots of the virus suspension were treated with 1 % sodium deoxycholate. Briefly, 1 ml of the clarified supernatant was treated with an equal volume of 2 % sodium deoxycholate (final concentration 1 %). After incubation for 1 h at room temperature (Auletta & Marlowe, 1968), the mixture was diluted with an equal volume of fresh serum-free EMEM and infectivity was assessed by titration on BHK-21 cells.
To determine whether the virus genome was DNA or RNA, virus replication in all three cell types was also assessed by measurement of virus titre in the presence of 1 or 5 µg actinomycin D ml–1.
Extraction of viral RNA, cDNA synthesis and cloning.
RNA was extracted from cell culture supernatants or cell pellets at 72 h post-inoculation using RNA Now (Biogentex). The supernatant was clarified by low-speed centrifugation at 2000 g for 10 min and then concentrated from 200 ml down to 1 ml by ultrafiltration (molecular mass cut-off of 5000 Da; Sartorius). Five fractions of 200 µl of the resuspended material were extracted using RNA Now, as described by the manufacturer. The extracted RNA was dissolved in a total volume of 50 µl. Fractions of 16 µl were then used in ligation reactions with the anchor primer RTC12-Spacer: 5'-AGGTCTCGTAGACCGTGCACC(12)TCCAGGTGCACGGTC-3' (the number 12 in parentheses denotes 12 CH2 residues constituting a spacer arm). Ligation was carried out using 20 U T4 RNA ligase (NE Biolabs) at 16 °C overnight. Unligated primer was subsequently removed by filtration through concentrators (molecular mass cut-off of 10 000 Da; Sartorius) and the RNA purified using an RNaid kit (Qbiogen).
The anchor primer molecule can fold, so that complementary 3' and 5' sequences base pair, forming a hairpin-like structure, thus preventing internal mispriming. This provides a reverse transcriptase priming site, which can be used to copy the RNA molecules after the anchor primer has been ligated to their 3' termini. The purified and ligated RNA of MIDV was reverse transcribed using Superscript II reverse transcriptase at 40 °C in the presence of the SMART II oligonucleotide (5'-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG-3') as described previously (Attoui et al., 2000). Superscript II reverse transcriptase, like other Moloney murine leukemia virus reverse transcriptases, possesses a terminal cytidine transferase activity, thus adding poly(C) at the 3' end of the cDNA (Chenchik et al., 1998; Attoui et al., 2000). The SMART II oligonucleotide has a poly(G) sequence at its 3' end that can hybridize to the poly(C) at the 3' end of the nascent cDNA, inducing template switching and therefore providing a new template for the reverse transcriptase to copy. This approach introduces two distinct target sequences at the 3' and 5' ends, respectively, of the first cDNA copy. The resulting cDNA was PCR amplified using the 5' PCR primer 5'-AAGCAGTGGTATCAACGCAGAGT-3', the PCR-Spacer primer 5'-GTCCAGGTGCACGGTCTACGAGACCT-3' and a TripleMaster PCR kit (Eppendorf).
For rapid sequencing, the resulting full-length amplicon was sonicated and the DNA ends repaired using a cloned Pfu DNA polymerase (Clontech) in presence of dNTPs. Briefly, the DNA, in 500 µl TE buffer [10 mM Tris/HCl (pH 7.5), 1 mM EDTA] was sonicated using a microtip sonication probe, at an output capacity of 20 %, for 1 min. The smaller DNA fragments in the sonicated product were removed by ultrafiltration using Vivaspin 500 concentrators (PES membrane, molecular mass cut-off of 300 kDa; Sartorius). Only products that were larger than 800 bp were retained. The concentrate was diluted in TE buffer (pH 8.0) and reconcentrated again in the same column until the volume reached 40 µl. Five microlitres of 10x Pfu DNA polymerase buffer (Clontech) was added and mixed with dNTPs (0.25 mM final concentration) and incubated at 72 °C for 30 min to repair the DNA ends. The reaction was cleaned using a MinElute Reaction Cleanup kit (Qiagen). The DNA was eluted from the column using 15 µl water preheated to 70 °C. Two microlitres of 10x Taq DNA polymerase buffer (Invitrogen) was added and mixed with 0.2 µl 100 mM dATP (final concentration 1 mM) and 1 U Taq DNA polymerase for end tailing of the DNA. The mixture was incubated at 72 °C for 30 min and the DNA was purified using a MinElute Reaction Cleanup kit. The DNA was eluted from the column using 8 µl water preheated to 70 °C. The purified products were ligated into a pGEM-T plasmid (Promega) and transformed into JM109 competent bacteria as described by the manufacturer. The resulting clones were screened by PCR using M13 universal primers. Clones with PCR inserts larger than 800 bp were sequenced using a dRhodamine sequencing kit (Applied Biosystems).
Two primers were designed from the sequence of the 3' untranslated region (UTR) of the of MIDV-857 genome to confirm the sequence of this region and to estimate the length of the poly(A) tract. These primers were Ds1 (5'-GTAGGCACTAGATATAGTAGAACGG-3', nt 11101–11125) and Ds2 (5'-GGTAGGCAAAGGCATCATTAATCATC-3', nt 11152–11177). These primers were used with the PCR-Spacer primer in PCR.
Sequence analysis.
The contig sequence of the various cDNA clones was assembled using CONTIGEXPRESS (Vector NTI advance 10, version 10.1.1). The sequence of cDNA was compared with viral sequences deposited in GenBank/EMBL/DDBJ using the NCBI's BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/).
These sequences were aligned using CLUSTAL W (version 1.83; Thompson et al., 1994). Phylogenetic analyses were performed using the neighbour-joining method (Saitou & Nei, 1987) implemented in the MEGA 3.1 software (Kumar et al., 2004). The p-distance model and the Kimura two-parameter or the Poisson correction model were used for tree building. The alpha shape parameter used for the gamma distribution analysis was calculated using the PAML package (Yang, 1997). The shape parameter measures how variable the rates are among sites. With a value of 
>1, most sites have rates around 1 (similar rates) and only a few sites have either very low or very high rates. When 
1 (there is a relatively large amount of rate variation) and most sites have very low rates, but there are evolutionary hot spots with higher rates.
Zuker's algorithm, as implemented in version 1.5 of the Vienna RNA Package RNAFOLD (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi), was used to draw the theoretical secondary structures of the 3' UTRs of the RNA genomes.
Detection of recombination events between RNAs of the alphaviruses was carried out using the recombination detection program RDP (Martin & Rybicki, 2000), which compiles, alongside RDP itself, other recombination analysis programs including CHIMAERA, GENECOV, SISCAN and MAXCHI.
The GenBank accession numbers of the sequences used in this study are provided in Table 1
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Assays in BHK-21 cells gave a virus titre in the supernatant from infected BHK-21 or Vero cell cultures of approximately 108 TCID50 ml–1 at 18 h post-infection. A growth curve showing a time course for the titre determination is shown in Fig. 1.
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Nature of the virus
MIDV-857 was isolated from a horse that died after showing severe clinical signs of disease. The cause of death was initially thought to be African horse sickness. However, AHSV-specific RT-PCR assays gave negative results with tissue samples (blood and spleen; data not shown). Agarose gel electrophoresis of the RNA extracted from infected cell cultures also failed to show a dsRNA-segment migration pattern typical of AHSV isolates, indicating the absence of this virus (or any other member of the family Reoviridae) (data not shown).
The sample also tested negative for equine infectious anemia virus, equine arteritis virus and equine herpesviruses.
Treatment of the infected tissue culture supernatants with organic solvents (Freon 113 or Vertrel XF) or deoxycholate abolished infectivity. Following lipofection, high levels of infection were observed, indicating that lipofection could restore virus infectivity. The lipofected virus lysed the cells and lysis was maintained for six subsequent passages (this was to confirm that new virus progeny was produced). Mock-lipofected cells were not lysed, indicating that lysis only occurred when the replication-competent virus capsid was present.
The presence of actinomycin D did not prevent virus replication in BHK-21, Vero or C6/36 cells (as determined by subsequent titration in BHK-21 cells), indicating that the virus had an RNA genome. Titres of 108 TCID50 (at 18 h post-infection) were consistently obtained, whether cells were treated with actinomycin D or not.
Cloning and sequencing of the MIDV-857 genome
The genome of isolate MIDV-857 was amplified as a single, full-length RT-PCR product, using a combination of a modified anchor primer (FLAC) and the SMART methodology (Fig. 2a, b). Cloning and sequencing of the genome showed it to be 11 674 nt, excluding a poly(A) tract that varied in length between approximately 180 and approximately 220 nt. The estimated length of the poly(A) tail is in agreement with the activity of eukaryotic poly(A) polymerase, which adds between 200 and 250 adenylate residues to the 3' end of RNA (Bienroth et al., 1993). The complete nucleotide sequence of the amplified cDNA was determined and has been deposited in GenBank under accession number EF536323.
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), indicating that the maximum length of the poly(A) tail was approximately 220 nt. The organization of the non-structural and structural polyproteins is shown in Fig. 2(d) (individual ‘cleaved-protein’ start and end sites are also shown).
Sequence comparisons
Sequence analysis of the genome of MIDV-857 and comparisons with the GenBank/EMBL/DDBJ sequence databases clearly identified it as an isolate of MIDV, an alphavirus that has previously been isolated from mosquitoes but has never been associated with disease in mammals. The sequence databases only contained partial sequences for the structural and non-structural protein genes (GenBank accession numbers given in Table 1) of MIDV (isolate MIDV-SAAR749). MIDV-857 and MIDV-SAAR749 were 98 % identical at the nucleotide level and 99.5 % identical at the amino acid level in the structural polyprotein, and 100 % identical in the partial sequence of nsP4 that was available from GenBank. Although the 5' UTR of MIDV-857 was identical to that of the earlier isolate (Ou et al., 1982), there were significant differences in the 3' UTR. The sequence alignment not only identified six base changes between the two strains in this region (T/C or A/G transitions), but also identified a 112 nt duplication in the MIDV-857 3' UTR (Fig. 3b). The downstream section of the repeated sequence had lost three nucleotides at two distinct positions and deletions were detected at two other positions in the 3' UTR of MIDV-857 compared with that of MIDV-SAAR749 (Fig. 3a). The insertion in the MIDV-857 sequence changed the predicted secondary structure of the 3' UTR as shown in Fig. 4 (RNAFOLD program).
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Phylogenetic comparison
Phylogenetic trees were built with the coding sequences of different alphaviruses. The values for the alpha shape parameter (used for the gamma distribution analysis) were calculated for different sequence sets using the PAML package and are provided in Table 2.
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) and for the non-structural amino acid sequences using the p-distance model (Fig. 5b). The topologies showed that Western equine encephalitis virus (WEEV) clustered with Eastern equine encephalitis virus (EEV), whilst MIDV was clearly separate from any of the other serocomplexes.
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), the non-structural gene nucleotide sequences (using either the p-distance or Kimura two-parameter model; data not shown) or the E1 amino acid sequences were compared (Fig. 6a, b). However, when the structural polyprotein sequences were used in conjunction with a Poisson correction (using the calculated shape parameter alpha), the topology of the tree (Fig. 5d) was identical to that obtained with non-structural amino acid sequences (Fig. 5b; MIDV clustered separately from the Semliki Forest serocomplex).
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). In our hands, bootscanning within the RDP program package identified potential recombination events involving the E1 region. These data indicated that MIDV was generated by recombination events involving members of the Semliki Forest serocomplex as sequence donors. This could explain the discrepancies between the trees obtained by analyses of structural and non-structural genes. The recombination region included nt 2430–2980 (551 nt) in MIDV-857 (P > 0.01), where two separate events were identified. The first event concerned nt 2430–2585 where the parental donors were Semliki Forest virus and Mayaro virus. The second event concerned nt 2850–2980 where again the parental donors were Semliki Forest virus and Mayaro virus. These events were identified by RDP and the parallel programs run within the RDP package including CHIMAERA, GENECOV and MAXCHI. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Field investigations showed that Wesselsbron virus (genus Flavivirus) was responsible for that outbreak. Pools of mosquitoes collected from the same region were screened for other infectious agents and two strains of MIDV were isolated. These viruses produced viraemia and pyrexia in experimentally inoculated sheep. The intracerebral inoculation of monkeys (Cercopithecus aethiops pygerythrus) failed to produce viraemia or disease. However, chicks were found to become viraemic after inoculation, without any clinical signs. The virus was also found to be lethal to newborn mice by either the intracerebral or intraperitoneal route. Mosquitoes fed with virus-contaminated brain-blood suspension transmitted the virus upon feeding on a naïve lamb. As a consequence, this lamb developed viraemia and pyrexia (Kokernot et al., 1957).
Following this first isolation from mosquitoes in South Africa, the virus has subsequently been isolated from Aedes mosquitoes on a number of occasions, especially in Senegal from Aedes cumminsi (Robin et al., 1969). Attempts to isolate the virus from mammalian or avian hosts were unsuccessful. Interestingly, haemagglutination inhibition antibodies to MIDV were identified in Romania from migratory wild birds (Antipa et al., 1984).
This study reports the first full-length sequence of MIDV-857 isolated from of a horse, which died showing severe clinical signs of disease. These signs were highly similar to those of African horse sickness (including a rise in body temperature, tachycardia, pulmonary affection and generalized oedema, particularly of the head and neck). The biological samples obtained from the horse tested negative for several horse pathogens, particularly AHSV, equine infectious anemia virus, equine arteritis virus and equine herpesviruses. These results prompted attempts to characterize and identify the virus that had been isolated in cell culture from the spleen sample. After plaque purification, the virus was found to be sensitive to organic solvent treatment, suggesting that it was enveloped. However, lipofection of the purified Vertrel XF-treated virus re-established infectivity, indicating the presence of ‘capsid’ that was replication competent (after cell entry) but incapable of cell attachment and penetration by itself. Virus replication was not inhibited by the use of actinomycin D, suggesting that the virus had an RNA genome.
The anchor primer/SMART approach that we developed permitted PCR amplification of the MIDV-857 genome as a single PCR product of 11 674 nt. The genome organization was typical of alphaviruses and contained a poly(A) tail, which varied in length between approximately 180 and approximately 220 adenylate residues. Poly(A) tails are added by a cellular poly(A) polymerase, which incorporates between 200 and 250 adenylate residues into a tail (Bienroth et al., 1993). This explains the variation in the MIDV poly(A) tail lengths, as individual encapsidated genomes would have tails of distinct lengths.
Comparison of the complete MIDV-857 genome sequence with the GenBank/EMBL/DDBJ databases identified it as an isolate of the species Middelburg virus, which forms a distinct antigenic complex within the genus Alphavirus. Phylogenetic comparisons of the MIDV-857 non-structural sequence with those of other alphaviruses identified MIDV as a member of a phylogenetic group on its own (which correlates with MIDV being a member of a separate serocomplex). However, when E1 amino acid sequences were used for neighbour-joining phylogenetic reconstruction (using p-distances or a Poisson correction), a major shift was observed in the tree, with MIDV clustering within the Semliki Forest serocomplex.
This inconsistency between the inter-relationships of established Alphavirus serocomplexes and the evolutionary similarities based on sequence analysis has led previous authors to suggest that MIDV may be a member of the Semliki Forest virus complex clade (Powers et al., 2001). However, it appears likely that a recombination event (within the E1 gene) between MIDV and other viruses of this serocomplex has occurred, leading to inconsistent phylogenetic results depending on the region of the alphavirus genome that is being compared. This hypothesis was subjected to analysis using bootscanning, implemented in the RDP program, which identified possible recombination events between MIDV and viruses of the Semliki Forest serocomplex. The mechanisms that resulted in the proposed recombination event are not known.
The process of genome replication for plus-sense RNA viruses begins with the generation of a full-length complementary copy of the genomic RNA (Hardy, 2006). The synthesis of this anti-genomic minus-strand RNA by the RNA-dependent RNA polymerase (RdRp) must start at the 3' end of the genome in order to produce a full-length copy of the virus genome. Alphaviruses possess a highly conserved 3' sequence element (3' CSE; approximately 19 nt), which immediately precedes the poly(A) tail (Pfeffer et al., 1998). Both the poly(A) tail and the 3' CSE are required for virus replication and, more specifically, for efficient minus-strand RNA synthesis (Hardy & Rice, 2005; Kuhn et al., 1991, 1992; Raju et al., 1999). An analysis of the 3' UTR of MIDV-857 and MIDV-SAAR749 identified an insertion of 112 nt within the 3' UTR of MIDV-857. This insertion represents a direct repetition of the sequence located immediately upstream of the insertion site, but does not modify the 3' CSE region and therefore is not thought to have any deleterious effect on replication (as confirmed by the high titres of virus produced in cell cultures).
It appears likely that the insertion sequence modifies the secondary structure of the 3' UTR, although the effects of these changes remain unclear. However, some features of the predicted secondary structure fold are maintained between the UTRs of MIDV-857 and MIDV-SAAR749, and there are similarities to Semliki Forest virus and O'nyong nyong virus of the SFV serocomplex (data not shown).
Direct repeats have been described previously in the 3' UTRs of the flaviviruses (Gritsun & Gould, 2007a, b). It was considered likely that the 3' UTR of the flaviviruses (and possibly the open reading frames) had evolved through multiple duplication of a single RNA domain, and that short direct repeats appeared to represent an evolutionary remnant of these domains. Inspection of the UTRs of various alphaviruses has revealed stretches of 18–102 bases that occur at least twice in the viral RNA (Pfeffer et al., 1998). A mechanism by which such duplications could occur has been proposed for the RdRp of a dsRNA virus, with a loop mechanism at the origin of the partial duplication (Matthijnssens et al., 2006).
In conclusion, the isolation of MIDV from a diseased horse provides an important first indication that MIDV can cause disease in equids. The methodology used in the RT-PCR amplification of MIDV RNA avoids false priming, making it particularly important for the determination of the full sequence of the 3' end of the RNA. Finally, the sequence analysis showed that WEEV is not the only recombinant virus within the genus Alphavirus, as MIDV also has the same status. The E1 gene of MIDV is probably a recombinant product from viruses of the Semliki Forest virus serocomplex.
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ACKNOWLEDGEMENTS |
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Received 11 April 2007;
accepted 2 July 2007.
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