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1 Department of Biology, University of Victoria, Victoria, BC V8W 2Y2, Canada
2 The Hospital for Sick Children, Toronto, ON M5G 1X8, Canada
3 Canadian Forest Service – Atlantic Forestry Centre, PO Box 4000, Regent Street, Fredericton, NB E3B 5P7, Canada
4 Department of Biosystems Engineering, E2-376 EITC, University of Manitoba, Winnipeg, MB R3T 5V6, Canada
Correspondence
David B. Levin
levindb{at}cc.umanitoba.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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A supplementary table showing primers used in real-time qPCR and RT-PCR is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Lepidopteran baculovirus genes are transcribed in a regulated temporal cascade that is divided into immediate-early, early, late and very late temporal classes. The major distinctions between early and late transcription events are that early genes are transcribed by host-cell RNA polymerase II (Hoopes & Rohrmann, 1991
) prior to the onset of virus DNA (vDNA) replication. The earliest expressed genes, such as the immediate-early genes ie0/1, ie2 and pe38, act as trans-regulators for the transcription of later-expressed viral genes. Recent genome-sequence analyses (Afonso et al., 2001; Garcia-Maruniak et al., 2004; Lauzon et al., 2004; Duffy et al., 2006) have revealed that non-lepidopteran baculoviruses do not appear to encode orthologues of members of the lepidopteran immediate-early class of genes, suggesting that immediate-early genes encoded by non-lepidopteran baculoviruses are significantly divergent from those of lepidopteran baculoviruses, and/or that the mechanisms of early gene expression differ from those in the the lepidopteran baculovirus model.
In lepidopteran baculoviruses, late gene expression is divided into two temporally distinct stages: late and very late. Late genes are expressed following vDNA replication and are transcribed by a hetero-oligomeric RNA polymerase encoded by the baculovirus genes lef-4, lef-8, lef-9 and p47 (Guarino et al., 1998). The virus-encoded very late factor gene (vlf-1) is classified as a late gene. The precise function or functions of VLF-1 are unclear. It was initially implicated as the primary regulator of very late gene transcription, but is also believed to be involved in virus capsid maturation (Vanarsdall et al., 2006).
The effect of VLF-1 on very late gene transcription, however, is well established. Transient-expression studies suggested that a threshold level of VLF-1 expression promotes trans-activation of very late genes, such as polyhedrin (polh) and p10 (McLachlin & Miller, 1994; Yang & Miller, 1998, 1999). Deletion of the vlf-1 gene from the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) resulted in dramatically reduced p10 expression (Vanarsdall et al., 2004, 2006). Furthermore, VLF-1 transactivation of late genes has been shown to require a promoter element, known as the ‘burst sequence’, upstream of the target gene (Mistretta & Guarino, 2005).
By using real-time quantitative PCR (qPCR) and RT-PCR, we characterized the timing of DNA replication, as well as the timing and order of specific gene-transcription events in midgut cells following infection of Neodiprion abietis larvae by their natural baculovirus pathogen, N. abietis NPV (NeabNPV). This study will set a benchmark for future studies of hymenopteran baculoviruses by permitting categorization of novel genes based on their temporal expression profile within host tissues.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. This procedure involved thawing frozen (–20 °C) NeabNPV-infected larvae in 5 vols 0.3 % SDS solution followed by homogenization. Each sample was filtered twice through 1 mm2 plastic mesh, followed by resuspension of solid matter in 0.3 % SDS, rehomogenization and refiltration until the filtrate was clear. Filtrates were then pooled, filtered twice through eight layers of cheesecloth and centrifuged for 15 min at 9000 g. The supernatant was discarded and the NeabNPV OB pellet was resuspended in 0.3 % SDS. Centrifugation and resuspension were repeated until a clear supernatant was obtained. The NeabNPV OB pellet was then resuspended in water and concentrated to 109 OBs ml–1.
Larval inoculation.
Balsam fir (Abies balsamea) branches infested with N. abietis were collected from the Old Man's Pond region near Corner Brook (latitude 4 ° 57' N, longitude 5 ° 57' W) in Western Newfoundland, Canada, in July 2003. The larvae were maintained at 4 °C on balsam fir branches until they were harvested. The larvae were starved for 12–15 h at 20 °C prior to being inoculated. Head capsules of the larvae were measured according to the procedure described by Carroll (1962), and only second- and third-instar larvae were selected for experimentation. NeabNPV OBs were mixed with a 10 % pasteurized liquid honey solution, and N. abietis test larvae were allowed to imbibe a 1 µl drop of honey/water solution, containing an estimated 104 OBs (Moreau et al., 2005). Control larvae were inoculated by imbibing 1 µl of a 10 % pasteurized liquid honey solution with no OBs. All larvae were observed to ensure that the inocula were ingested; those that failed to imbibe the solution were discarded. After inoculation, the larvae were transferred immediately to and maintained on sprigs of fresh balsam fir foliage, which had been surface-cleansed with 2 % bleach for 30 min and rinsed with water.
Larvae used in this experiment were sacrificed at various time points ranging from 0.5 to 72 h post-inoculation (p.i.). Each sacrificed larva was washed for 60 s in 2 % bleach and rinsed for 60 s in DEPC-treated water. All larvae were dissected under sterilized DEPC-treated PBS solution (pH 7.4). The head, immediately posterior to the head capsule, and the hind end anterior to the last proleg were cut off by using a sharp scalpel. The larval gut was pulled out of the body into fresh sterile PBS by using fine forceps. The food bolus was removed by extraction of the peritrophic membrane and the dissected gut was stored in 1 ml RNALater (Ambion) at –20 °C.
Viral RNA and DNA isolation.
Each excised larval midgut was homogenized in 50 µl TRIzol reagent (Invitrogen) containing sand (ignited and baked for 4 h). The ground midgut suspension was collected after the sand had settled, and TRIzol reagent (Invitrogen) was added to a total volume of 1 ml. Debris was collected by centrifugation at 12 000 g for 10 min at 4 °C and the TRIzol supernatant was incubated for 10 min at 25 °C. Each sample was extracted with 0.2 vol. chloroform. Tubes were mixed for 15 s and incubated 2 min at 25 °C. The TRIzol solution was separated into its respective phases by centrifugation at 12 000 g for 15 min at 4 °C and the aqueous phase was collected. The interphase and organic phase were stored at –20 °C for further processing. RNA was precipitated from the solution by addition of 0.5 vol. isopropyl alcohol, incubated for 10 min at 25 °C, and centrifuged at 12 000 g for 10 min at 4 °C. The supernatant was removed and the pellet was washed once with 1 ml 75 % ethanol. The sample was mixed by vortexing and centrifuged at 7500 g for 5 min at 4 °C. The pellet was dried and resuspended in RNase-free water (Invitrogen) by incubation at 60 °C for 10 min. RNA samples were stored at –80 °C.
DNA was precipitated from the interphase and organic phase with 0.33 vol. 100 % ethanol and incubated for 3 min at 25 °C. Precipitate was collected by centrifugation at 2000 g for 5 min at 4 °C and removal of supernatant. The pellet was dried and resuspended in RNase-free water (Invitrogen) by incubation at 60 °C for 10 min. DNA samples were stored at –80 °C.
RNA RT-PCR.
The RNA samples were treated with 3 U DNase (Invitrogen) in a buffered solution (20 mM Tris/HCl, pH 8.4; 50 mM KCl; 2 mM MgCl2) for 15 min at 25 °C. The reaction was terminated by addition of EDTA to 2.5 mM and incubation at 65 °C for 10 min. First-strand cDNA synthesis was performed by incubating 50 ng DNase-treated total RNA with 200 U SuperScript II reverse transcriptase (Invitrogen) in 20 µl PCR buffer (50 mM KCl; 25 mM Tris/HCl, pH 8.3; 500 µM dNTPs; 10 µM dithiothreitol; 10 U RNase inhibitor) at 25 °C for 10 min. The reaction was then incubated at 42 °C for 50 min and inactivated by incubation at 70 °C for 15 min. The synthesized cDNA was stored at –20 °C.
The primers used to amplify the temporal standards are listed in Supplementary Table S1, available in JGV Online. The amplicons were designed to yield products 200–300 bp in length. A positive control (NeabNPV or host larva genomic DNA), a negative control (cDNA from uninfected larvae) and a host insect control (28S rRNA gene-specific primers) were included in each PCR set. The DNA templates were amplified by using gene-specific primers and Platinum Taq polymerase (Invitrogen), according to the manufacturer's protocol, with a temperature regime of 95 °C (9 min), followed by 45 cycles of 95 °C (30 s), 55 °C (60 s) and 72 °C (90 s). All products were resolved by gel electrophoresis in 2 % agarose and stained with SYBR green (Molecular Probes).
qPCR.
vDNA copy number was determined by real-time qPCR with primers specific to the polh gene (Supplementary Table S1, available in JGV Online). The viral copy number was then normalized against host-genome copy number by qPCR with primers specific to the host actin gene (Supplementary Table S1). The polh and actin genes were amplified by PCR and cloned into pGEM-T (Promega). Plasmid DNA concentrations were quantified by using an ND-1000 spectrophotometer (NanoDrop) and dilution standards were generated, ranging from 2.5x105 to 4x102 copies. DNA templates were amplified with an MX4000 thermocycler (Stratagene) using gene-specific primers (0.67 µM) and 20 µM dNTPs in MX4000 qPCR buffer (Stratagene), with Platinum Taq polymerase (Invitrogen). The mixture was heated at 95 °C for 9 min, followed by 40 cycles of 95 °C (15 s), 55 °C (30 s) and 72 °C (45 s). All products were confirmed by resolution by gel electrophoresis in 2 % agarose and staining with SYBR green (Molecular Probes).
For each standard dilution, four independent qPCRs were performed using polh- or actin-specific primers, and standard curves were generated. For each larval DNA extract, four independent qPCRs were performed using polh- and actin-specific primers. The mean vDNA copy numbers were determined and the number of polh amplicons was normalized against the host actin gene to derive the mean number of viral copies per mean host actin gene copy number. The reported number of viral copies represented the mean of two larvae at each time point. The rates of vDNA replication were calculated from the slopes of the lines joining adjacent time points as (m2–m1)/(t2–t1).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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shows the quantity of NeabNPV virus DNA, relative to host actin gene copy number, in infected larvae. Initially, the number of vDNA copies appeared to increase rapidly and, by 30 min p.i., we detected approximately 104 copies of NeabNPV per host actin gene copy. NeabNPV copy number increased by 217 % between 30 min and 1 h p.i., and by 258 % between 1 and 2 h p.i. Between 2 and 4 h p.i., however, NeabNPV DNA copy number increased by only 2.67 % h–1. Another dramatic increase in vDNA copies was observed between 4 and 12 h p.i., at a rate of 21.38 % h–1. This trend continued, to a lesser degree, between 12 and 24 h p.i., when the vDNA copy number continued to increase by 8.45 % h–1, but an increase of only 1.92 % h–1 was observed between 24 and 72 h p.i.
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). RT-PCR products representing transcripts of the RNA polymerase subunit lef-8 appeared as early as 1 h p.i., whilst RT-PCR products of the RNA polymerase subunit lef-9 were detected from 6 h p.i. (Fig. 2a). RT-PCR products representing transcripts of the late-expressed gp41 and p74 genes were detected from 6 h p.i. (Fig. 2a). A weak RT-PCR product for NeabNPV vlf-1 transcript was detected as early as 12 h p.i., but RT-PCR products representing polh transcripts were not observed until 24 h p.i. (Fig. 2b). Transcripts for all genes examined were observed as late as 72 h p.i.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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We acknowledge that there are several limitations to in vivo studies of baculovirus DNA replication and transcription. In our experiments, N. abietis larvae were exposed per os to 104 OBs in an inoculum of 1 µl. Whilst we observed the larvae imbibe the inocula, we do not know the number of OBs that were actually ingested by each larva, nor do we know the number of virions that were released from these OBs, the number of virions that penetrated midgut epithelial cells or the total number of midgut cells that were infected. We do know, however, that the NeabNPV DNA detected was located within midgut epithelial cells of the larvae. The food bolus within the digestive tract was removed and larvae were observed voiding their guts within the first hour of being placed on fresh balsam fir sprigs. Any vDNA detected was, therefore, most probably due to virions that had passed through the peritrophic membrane and intercalated within the midgut epithelial brush border or had already penetrated into the midgut cells.
Another consideration in in vivo experiments is that infection is likely to be asynchronous in nature. Whilst the entire inoculum in cell-culture systems can be assumed to have infected target cells in a synchronous manner, individual virions may infect their target cells in a temporally staggered manner in vivo. Although the asynchronous nature of in vivo infections may be considered a limitation of this study, it might be argued that the in vitro studies are ultimately intended to model an in vivo system and that asynchronous infection is an important characteristic of natural viral infections.
We have previously examined the potential for asynchronous infection in NeabNPV-inoculated larvae by light and electron microscopy (Whittome, 2006). At 5 h p.i., 55.8 % of nuclei in sections of NeabNPV-inoculated N. abietis larvae midgut epithelial cells were infected, as indicated by the presence of both nuclear hypertrophy and virogenic stroma, whilst the remaining 44.2 % of observed nuclei displayed no cytopathic effects. By 8 h p.i., approximately 61.0 % of the total observed nuclei displayed virus-induced cytopathic effects, and approximately half of these contained nucleocapsids (31.7 % of total nuclei observed). The remaining 29.3 % of the nuclei exhibiting cytopathic effects displayed only hypertrophy and virogenic stroma, while 39.0 % of the observed nuclei had no detectable cytopathic effects or virus-induced phenotypes that were distinguishable from sections of midgut epithelial cells from uninfected N. abietis larvae (Whittome, 2006). These data suggest that there is a primary wave of infection in N. abietis larvae midgut epithelial cells during the first 5 h, and that secondary infections of midgut cells may be detected by 8 h p.i. Although the asynchronous nature of the infection influences our interpretation of transcription events at later time points, the uniformity of cytopathic effects in the early phase of infection (up to 5 h p.i.) suggests that our interpretations of the timing of early gene-transcription events (as detected by RT-PCR) are valid.
DNA replication kinetics of baculoviruses were first characterized in Spodoptera frugiperda multicapsid NPV (SfMNPV)-infected S. frugiperda cells. In this system, virus replication was maximal between 8 and 20 h p.i. (Knudson & Tinsley, 1978; Liu & Bilimoria, 1990). In Autographa californica multicapsid NPV (AcMNPV)-infected S. frugiperda cells, DNA replication occurs at a constant rate from 6 to 20 h p.i. (Rosinski et al., 2002). Studies of recombinant AcMNPV infection within host larvae have shown that the timings of vDNA replication and transcription in vivo are very similar to those observed in vitro (Flipsen et al., 1995; Washburn et al., 1995), suggesting that physiological barriers may not act as a significant impediment to viral infection.
We observed approximately 104 copies of the NeabNPV genome per copy of the host actin gene within 0.5 h p.i. Such a large initial number of vDNA copies was not expected to result from an inoculum of only 104 OBs. Whilst in vitro experiments are typically performed so that the m.o.i. can be determined a priori, this was not the case for our study. Attempts to quantify the vDNA copies of the OB inoculum were hindered by inefficient and inconsistent DNA extraction. Also, antibodies to indicate the efficiency of viral infection were not available.
Sections of NeabNPV OBs observed by transmission electron microscopy, however, revealed a mean of approximately seven nucleocapsids per 70 nm section. The majority of virions within the OBs are singly enveloped, but virions with at least two nucleocapsids are also observed (Whittome, 2006). NeabNPV OBs are roughly spherical with a diameter of approximately 1 µm. Assuming a random distribution of nucleocapsids within the volume of an OB, we estimate that NeabNPV OBs could contain approximately 100 nucleocapsids (Whittome, 2006). There is also precedence that multiple genome copies may be contained within individual nucleocapsids (Rosinski et al., 2002). Detection of viral copy number by qPCR may also have detected deleted genotypes that may be co-occluded with wild-type virus. Thus, it is possible that the large number of NeabNPV genomes detected by 0.5 h p.i. may reflect a very large number of viral genomes contained within the NeabNPV OBs.
We caution that the quantification of vDNA copies upon initial infection may be hindered by the proximity of the polh ORF to a repetitive region, approximately 1 kb upstream. Although baculovirus DNA replication origins rely on viral DNA polymerase for genome replication (Vanarsdall et al., 2005), host DNA polymerase may be capable of DNA synthesis of proximal loci (Lu & Miller, 1995). This would artefactually amplify the number of polh copies detected by qPCR.
A rapid increase in vDNA copies was observed between 0.5 and 2 h p.i. It is most probable that the NeabNPV DNA detected at least at the first three time points (0.5, 1 and 2 h p.i.) represents DNA from virions released from ingested OBs. A much lower rate of copy-number increase was observed between 2 and 4 h p.i., followed by a more rapid rate of copy-number increase between 4 and 24 h p.i., again followed by a lower rate of increase between 24 and 72 h. These observations are similar to AcMNPV replication kinetics in vivo (Rosinski et al., 2002), except that NeabNPV copy number continued to increase up to 72 h p.i. It is important to note that we defined the number of viral genomes in relation to the number of host actin gene copies, as we were not able to confirm that every cell within the midgut was infected. In addition, the ploidy of midgut regenerative cells is known to vary among the developmental stages within an instar, ranging from 1n to 16n and having a predominance of tetraploid cells (Hakim et al., 2001).
Our data suggest that the timing of transcription of NeabNPV genes is remarkably similar to the temporal pattern of gene expression in the lepidopteran baculoviruses. NeabNPV genes appear to be expressed in temporally discrete stages where early genes are expressed from 1 to 2 h p.i., followed by vDNA replication between 4 and 6 h p.i. Late gene expression appears to coincide with later stages of vDNA replication, occurring from about 6 h p.i.
Early genes, such as lef-1, lef-2 and dnapol, are typically transcribed within 2–3 h p.i. in the lepidopteran baculoviruses (Passarelli & Miller, 1993a, b; Sriram & Gopinathan, 1998; Tomalski et al., 1988; Chaeychomsri et al., 1995; Liu & Carstens, 1995; Huang & Levin, 2001). Transcription of the NeabNPV dnapol, lef-1 and lef-2 genes was detected from 2 to 72 h p.i. Characterization of lef-8 and lef-9 gene expression has been limited and lef-9 expression does not appear to be consistent between baculovirus species. In AcMNPV, significant transcription of lef-9 was reported from 6 h p.i. (Guarino et al., 1998). The lef-9 transcript of Bombyx mori NPV (BmNPV), however, was detected at 12 h p.i., but not at 6 h p.i. (Acharya & Gopinathan, 2002). Transcription of lef-8 in BmNPV was reported as early as 12 h p.i., but earlier time points were not investigated (Acharya & Gopinathan, 2002). Our data indicate that lef-9 transcription was consistent with the pattern observed in AcMNPV, as it was first detected at 6 h p.i., whilst a lef-8 transcript was detected as early as 1 h p.i. (Fig. 2a
).
Late gene expression was coordinated with the transcription of lef-9, as gp41 and p74 were both transcribed from 6 h p.i. This is consistent with the roles of lef-8 and lef-9 gene products as components of the viral RNA polymerase, but it is not consistent with studies of lepidoteran baculoviruses in cell culture that suggest a lag between detection of the lef-8 and lef-9 transcripts and late gene transcripts, such as gp41 and p74. In AcMNPV, for example, the lef-9 transcript was detected at 6 h p.i. (Guarino et al., 1998), whereas in independent studies, gp41 and p74 expression was detected at 12 h p.i. (Whitford & Faulkner, 1992; Kuzio et al., 1989). In this study, lef-9 and late gene transcripts were all detected at 6 h p.i. without an apparent lag. One possible explanation is that lef-9 was expressed in the period between the 2 and 6 h p.i. time points and that the lag is less attenuated in NeabNPV than in AcMNPV. Another factor is that we used RT-PCR, which may be more sensitive to very low levels of transcription, undetectable by other methods such as primer extension and oligonucleotide hybridization.
The VLF-1 protein is believed to be the primary transactivator of very late genes such as polh and p10 in the lepidopteran baculoviruses (McLachlin & Miller, 1994; Yang & Miller, 1998, 1999), and transcription vlf-1 of very late viral genes is typically coordinated with the transcription of polh. Transcription of the NeabNPV vlf-1 gene was observed from 12 to 72 h p.i., whilst the polh transcript was not observed until 24 h p.i. One possible explanation for this is that vlf-1 must be expressed to a sufficient threshold before transactivating polh (Yang & Miller, 1998), and insufficient VLF-1 results in delayed expression of the polh gene expression in NeabNPV.
The practical constraints of studying baculovirus infection in vivo, such as low RNA extraction yields (50–500 ng per larva), required that we employ sensitive PCR-based analyses rather than the standard nucleotide-hybridization techniques. This constraint bears two major drawbacks. First, the PCR-based analysis should be considered a qualitative measure of transcription events. Hybridization experiments, on the other hand, are semiquantitative, as the signal is collinear to the number of copies of the target oligonucleotide. Second, PCR-based analyses do not distinguish between transcript variants, whereas hybridization experiments do. The high RT-PCR signal at 72 h p.i. for all transcripts examined could be derived from low levels of early gene transcription or asynchronous infection. Although there is no evidence whether NeabNPV can be transmitted from cell to cell within the midgut tissue, transcripts may be detected from secondary infection. Despite these drawbacks, however, RT-PCR is a very sensitive and effective tool for analysing virus infection processes in vivo.
In this study, we investigated the replication kinetics and temporal gene-expression pattern of NeabNPV in vivo. As the genes and gene-expression patterns of non-lepidopteran baculoviruses are largely unknown, this study provides the first model for DNA replication and gene expression of a non-lepidopteran baculovirus. Knowing the timing of both DNA replication and transcription of NeabNPV genes that are orthologous to well-characterized lepidopteran baculovirus genes has enabled us to define early and late events in the NeabNPV infection process, and will facilitate the classification of uncharacterized NeabNPV genes as early- or late-expressed.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 5 December 2006;
accepted 27 March 2007.
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  C. S. Guy, P. M. Mulrooney-Cousins, N. D. Churchill, and T. I. Michalak Intrahepatic Expression of Genes Affiliated with Innate and Adaptive Immune Responses Immediately after Invasion and during Acute Infection with Woodchuck Hepadnavirus J. Virol., September 1, 2008; 82(17): 8579 - 8591. [Abstract] [Full Text] [PDF]
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