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1 Graduate Institute of Veterinary Microbiology, College of Veterinary Medicine, National Chung Hsing University, Taichung 402, Taiwan ROC
2 Department of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung 912, Taiwan ROC
3 Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, Taichung 402, Taiwan ROC
Correspondence
Long Huw Lee
lhlee{at}mail.nchu.edu.tw
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Oligonucleotide primers used for RT-PCR in this study are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The virus encodes at least 13 primary translation products; eight of them are structural components and the other five are non-structural proteins (Varela & Benavente, 1994; Bodelon et al., 2001; Shmulevitz et al., 2002; Touris-Otero et al., 2004). Viruses have been isolated frequently from the gastrointestinal and respiratory tracts of chickens with a variety of disease conditions (Rosenberger, 2003). Among them, viral arthritis and pale bird syndrome are the most commonly recognized diseases in poultry.
It has been demonstrated that the expression and release of pro-inflammatory cytokines, such as interleukin-1 (IL-1) and type I interferons, is a primary antiviral response in virus-infected cells (Jacobs & Langland, 1996; Iordanov et al., 2001; Maggi et al., 2003). In mammals, IL-1, which exhibits a broad spectrum of activities, has been extensively studied. It is a low molecular mass protein produced by many different cell types, but with stimulated macrophages being the main producers (Dinarello, 2000). IL-1 is an important factor in the pathogenesis of many diseases and in the mediation of a host response to infections through inflammatory and immunological events (Dinarello, 2000). IL-1β is a member of the IL-1 family (Burger et al., 2006). The primary translation product of the IL-1β gene is converted into a mature protein of approximately 17 kDa by the IL-1β converting enzyme, a proteinase that belongs to the caspase family of proteases (Nicholson & Thomberry, 1997). It is not glycosylated in spite of a number of potential N-glycosylation sites being present in its protein sequence and is transported out of the cells, then enters the circulation.
The gene encoding chicken IL-1β (chIL-1β) has been cloned and characterized (Weining et al., 1998). Subsequently, many studies on the production of chIL-1β have been described. Increased levels of IL-1β mRNA have been described in the intestinal tissues and in the livers of birds infected with Salmonella enterica serovar Typhimurium (Withanage et al., 2004), in gut extracts of birds infected with Eimeria tenella or Eimeria maxima (Laurent et al., 2001) and in intestinal intraepithelical lymphocytes after Eimeria maxima infection (Hong et al., 2006). In addition, the potential role of IL-1β in viral infections of chicken has also been demonstrated. For example, large increases in IL-1β mRNA were seen in the brain tissues of chickens infected with Marek's disease virus (Jarosinski et al., 2005) or in chicken macrophages exposed to infectious bursal disease virus (Khatri et al., 2005). Moreover, enhanced IL-1β activity was detected in culture supernatant of macrophages from poults suffering from poult enteritis and mortality syndrome (PEMS) caused by a reovirus infection (Heggen et al., 2000). Increases of IL-1β mRNA expression level have also been detected in a chicken macrophage cell line treated with the PEMS-associated reovirus (Heggen-Peay et al., 2002). These results suggest that IL-1β is an important factor in controlling the pathogenesis of many diseases. Recently, IL-1β proteins of chicken, duck, goose, turkey and pigeon were shown to be structurally and functionally homologous, although the IL-1β-encoding region of pigeon clustered into a lineage distinct from those of the other four species in the phylogenetic analysis (Wu et al., 2007).
We performed experiments to characterize chIL-1β mRNA expression in macrophages in response to ARV. The results may provide insights into the role of chIL-1β in the pathogenesis of viral arthritis in ARV-infected birds.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The virus has a minimum titre of 4x106 TCID50 ml–1 as calculated by the Reed & Munch method (Reed & Munch, 1938).
Chicken macrophages were prepared from the blood of specific-pathogen-free (SPF) Leghorn birds as described previously (Klasing & Peng, 1987). Briefly, peripheral blood mononuclear cells (PBMCs) were separated from blood by centrifugation through Ficoll/Hypaque (Ficoll type 400; Sigma) and were removed, washed twice with normal saline and resuspended in RPMI 1640 culture medium. Cells were placed in six-well tissue culture plates and incubated for 24 h at 37 °C. Plates were then thoroughly washed with PBS three times to remove non-adherent cells. Adherent cells (macrophages) were scraped with a cell scraper, resuspended with the same medium, and incubated in 24-well tissue culture plates for an additional 24 h before use.
Animals.
All Leghorn SPF chickens used in this study were purchased from the Research Institute for Animal Health, Council of Agriculture, Taipei, Taiwan. Chickens at 4–8 weeks of age were routinely used for macrophage preparation.
Virus purification and infectious subviral particle (ISVP) generation.
Reoviral particles were extracted from CEF cells infected with ARV S1133 by Freon extraction, layered onto 20–40 % CsCl gradients and centrifuged at 35 000 r.p.m. (SW41, Beckman) for 18 h (Yin et al., 2000). Virion (1.36 g ml–1) and empty particle (EP) (1.30 g ml–1) bands were collected, pelleted and used for further studies. The procedures to determine concentrations of virions or EPs of ARV in purified preparations were described previously for mammalian reovirus (Smith et al., 1969; Connolly & Dermody, 2002). Concentrations of purified reovirus were estimated by the empirically determined relation: 5.42 optical density units at 260 nm are equivalent to 1 mg reovirions, which is equivalent to 1.13x1013 virion particles (Smith et al., 1969). Virion yields were usually between 0.8 and 2.1 mg virions per ml (9.00x1012–2.37x1013 virion particles ml–1). Concentrations of EPs were determined from the equivalence 1 mg viral protein ml–1=1.8x1013 particles ml–1 (Connolly & Dermody, 2002). EP yields were usually between 1.7 and 4.2 mg ml–1 (3.06x1013–7.56x1013 EPs ml–1). To generate ISVP, reovirus particles or EPs (5x1010) were digested in 100 µl chymotrypsin reaction buffer containing 200 µg chymotrypsin (Sigma) ml–1 at 37 °C for 90 min as described previously (Schnitzer et al., 1982). Digested particles were pelleted and analysed by SDS-PAGE. Gels were stained with Coomassie blue to confirm digestion of µB/µBC to form 
(Duncan, 1996) and the removal of 
B protein (Schnitzer et al., 1982; Duncan, 1996). The titres of purified virions and ISVPs were measured in CEF cells as described above. Titres were usually between 4x109 and 2x1010 TCID50 ml–1 of virions or between 5x105 and 5x106 TCID50 ml–1 of ISVPs.
Virus inactivation.
The purified virions at an m.o.i. of 10 TCID50 per cell were inactivated with 2.5 % binary ethylenimine (BEI) at 37 °C for 15 h (Larghi & Nebel, 1980). Inactivated virus preparations were blind-passaged three times on CEF cells to confirm inactivation.
Virus infection of macrophages.
To assess the steps in viral replication required to induce chIL-1β mRNA expression, purified virions or EPs and ISVPs and BEI-inactivated virions prepared as described above were used to treat macrophages. Macrophages (5x104) were treated with dsRNA+ virion or ISVP at an m.o.i. of 10 TCID50 per cell for 60 min at room temperature (22 °C) (Duncan, 1996), virus inoculum was removed and cells were washed three times with PBS at room temperature before adding pre-warmed culture medium with or without drug inhibitors. Cells were then incubated at 37 °C for the specified period of time before collection for subsequent assays. The procedures for the EP assay were those described for virion, except that cells were treated with 5x103 EPs or ISVP per cell.
Preparation of total RNA and RT-PCR.
Macrophages collected at specified times were washed twice with PBS after medium was removed and total RNA was then isolated using TRIzol reagent (Life Technologies) as specified by the manufacturer.
To detect chIL-1β mRNA (Weining et al., 1998) and chicken glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (Panabieres et al., 1984), purified total RNA was used as the template in RT-PCR. The sequences of primers used for this amplification are listed in Supplementary Table S1 (available in JGV Online). RT-PCR was carried out as described previously (Lee et al., 1994). After 25 cycles of amplification, PCR products were separated on 1.5 % agarose gels, stained with ethidium bromide and quantified by Image Quan Software (Molecular Dynamics) and IPLab gel program (Signal Analytic Corp.). GAPDH mRNA expression was included to indicate that the differences of gene expression were not due to differences in the concentration of total RNA templates. The results were expressed as the ratio of mean count of chIL-1β mRNA expression level to that of GAPDH mRNA expression level. The experiment was performed twice and each experiment was treated in triplicate.
Virus titration.
To detect virus yields in macrophages, cell cultures were subjected to three cycles of freezing and thawing. Supernatants were collected after low speed centrifugation and used for titration in CEF cells (Lee et al., 1992).
Statistical analysis.
Statistical analysis was performed using Student's t test and significance difference levels were determined based on the respective controls.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), when subjected to SDS-PAGE, showed the expected distribution of viral proteins (Fig. 1b, lane 1) (Varela & Benavente, 1994; Schnitzer et al., 1982). Fig. 1(a) shows that the stain penetrated into the EPs, which showed an irregular shape. Analysis of EPs on SDS-PAGE showed that in addition to some extra minor bands their major protein components were similar to those of virions (Fig. 1b, lane 3) (Schnitzer et al., 1982). Examination of viral genomic dsRNA indicated that virions (dsRNA+ particles) contained dsRNA and confirmed that the EPs (dsRNA–) lacked genomic dsRNA (Fig. 1c). In addition, the viral protein composition of dsRNA+ virions or dsRNA– particles treated with chymotrypsin was assessed by SDS-PAGE to determine if the generated viral particles were ISVPs. The results showed that although the protein band , the cleaved product of viral protein µB/µBC, was very faint, viral proteins µB/µBC and B were digested and completely disappeared (Fig. 1b, lanes 2 and 4), as previously described (Duncan, 1996; Su et al., 2006). These data and results of the infectivity tests of enzyme-treated dsRNA+ virions (titre: 5x105–5x106 ml–1) indicated that these particles were ISVPs.
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). The virus yield at 36 h post-inoculation (p.i.) in 20 mM NH4Cl-treated cells was 1x102 TCID50 ml–1, lower than that in cells treated with 2 mM NH4Cl (3x105 TCID50 ml–1), which was similar to that in cells without the NH4Cl treatment. Expression of chIL-1β mRNA was assessed by RT-PCR. Virus induced macrophages to express chIL-1β mRNA in the absence of or in the presence of 2 mM NH4Cl throughout the time course of the experiment. ChIL-1β mRNA expression was inhibited in the presence of 10 or 20 mM NH4Cl within 6 h p.i. Expression of chIL-1β mRNA was detected by 6 h p.i. and was maintained over the time course of the experiment, but the expression level was lower than that in the control untreated cultures or in samples treated with 2 mM NH4Cl (data not shown). A representative result of chIL-1β mRNA expression in dsRNA+ virion-treated cells in the presence of 10 mM NH4Cl is shown in Fig. 2(b), panel i and (c). Similar results were obtained from cells treated with the same amount of dsRNA+ virions and incubated in the presence of 200 µM E64, which inhibits cysteine-containing protease activity in the endolytic pathway (Barrett et al., 1982) (Fig. 2b, panel ii and c), or 100 nM bafilomycin A, which inhibits disassembly of avian and mammalian reoviruses (Seglen, 1983; Labrada et al., 2002) (Fig. 2b panel iii and c). Taken together, these results demonstrated that treatment of macrophages with NH4Cl, E64 or bafilomycin A blocked ARV-induced chIL-1β mRNA expression at an early stage during viral replication, suggesting that virus disassembly might be involved in the rapid induction of chIL-1β mRNA expression. However, NH4Cl might not completely block viral disassembly so that induction of chIL-1β mRNA expression occurred as infection proceeded. GAPDH mRNA expression in each sample for RT-PCR was included and showed that each sample contained a consistent amount of RNA for RT-PCR.
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), indicating that ISVPs generated within endosomes or absorbed to cell surface receptors were able to induce chIL-1β mRNA expression. To assess if ISVPs were able to bypass the blocks mediated by NH4Cl to the expression of chIL-1β mRNA induced by dsRNA+ virions, the ability of ISVPs to induce chIL-1β mRNA expression in the presence of 10 mM NH4Cl was tested. The results from RT-PCR showed that the expression level of chIL-1β mRNA induced by ISVPs in the presence of NH4Cl was slightly lower than that in the absence of NH4Cl in the early phase of induction, but the expression was maintained at a higher level than that induced by dsRNA+ virions in the early phase (Fig. 3). These data and the results obtained from Fig. 2(b and c)
provide additional evidence that the NH4Cl-mediated inhibition of chIL-1β mRNA expression during the early phase in viral replication might be due to the ability of the inhibitors to block viral disassembly to form ISVPs. These findings suggest that expression of chIL-1β mRNA was also elicited by steps subsequent to ISVP formation in viral replication.
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; Rankin et al., 1989). Each concentration of ribavirin tested inhibited virus replication (Fig. 4a). In the presence of ribavirin, the expression pattern of chIL-1β mRNA induced by dsRNA+ virions was different from that in the absence of ribavirin (Fig. 4b, panel iii and c). In the presence of ribavirin, chIL-1β mRNA was rapidly induced at 30 min p.i., with maximal levels reached by 2 h p.i. After 2 h p.i., a rapid decrease in chIL-1β mRNA was observed and it reached a background level by 6 h p.i. (Fig. 4b, panel i and c). The results suggested that ribavirin could only efficiently block the stable, sustained expression that occurred at and after 6 h p.i. Both early and late phases of expression patterns of chIL-1β mRNA in macrophages were abolished in the presence of both NH4Cl and ribavirin (Fig. 4b, panel ii and c), further suggesting that viral disassembly or viral RNA synthesis in viral replication was required for the rapid, transient or the stable, sustained expression of chIL-1β mRNA induced by ARV, respectively.
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, panel iv and c). BEI-inactivated virions only induced the rapid, transient level of chIL-1β mRNA expression in the early stage of induction, similar to the results obtained from cells treated with dsRNA+ virions in the presence of ribavirin (Fig. 4b, panel i and c). These results also suggested therefore that viral RNA synthesis during primary transcription or steps subsequent to primary transcription in viral replication was required for the induction of the stable, sustained expression of chIL-1β mRNA.
To determine if dsRNA+ ISVPs were able to bypass the blocks mediated by ribavirin to chIL-1β mRNA expression induced by virions, cells were treated with dsRNA+ virions or ISVPs in the absence or presence of 200 µM ribavirin. The results from RT-PCR are shown in Fig. 4(d). As expected, treatment with ribavirin blocked the stable, sustained levels of chIL-1β mRNA expression induced by dsRNA+ virions. In cells treated with ISVPs, ribavirin efficiently blocked mRNA expression of chIL-1β over the time course of the experiment and only the rapid, transient expression pattern was observed. These results provided additional evidence that the inhibition of stable, sustained levels of chIL-1β mRNA expression mediated by ribavirin was due to the ability of this inhibitor to block viral RNA synthesis. These findings indicated that chIL-1β mRNA expression in a stable, sustained manner induced by dsRNA+ virions was elicited by viral primary transcription or subsequent steps in viral replication.
Effects of cycloheximide (CH) on chIL-1β mRNA expression induced by ARV
To determine if protein translation was required for chIL-1β mRNA expression induced by ARV, cells were mock-treated or treated with dsRNA+ virions in the presence or absence of CH, which is reported to inhibit protein synthesis (Watanabe et al., 1967; Nonoyama et al., 1974; Lau et al., 1975), and expression of chIL-1β mRNA was assessed by RT-PCR at various times (Fig. 5). The presence of CH (4 µg ml–1) in media of mock-treated macrophages did not affect the viability of cells for at least 12 h of treatment. Expression of chIL-1β mRNA was not blocked in cells treated with virions in the presence or absence of CH. These results suggested that viral protein synthesis was not required for inducing expression of chIL-1β mRNA by ARV. In cells treated with both dsRNA+ virions and CH (Fig. 5), expression of chIL-1β mRNA at 3 h p.i. was increased by approximately 2.8-fold higher than that seen for cells treated solely with dsRNA+ virions, suggesting that a superinduction activity could be elicited by CH during the rapid, transient expression of chIL-1β mRNA in dsRNA+ virion-treated PBMCs.
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). Both dsRNA– particles and ISVPs induced only rapid, transient levels of chIL-1β mRNA expression within 6 h p.i., which was inhibited by NH4Cl (Fig. 6), similar but extremely weak in comparison to those induced by active dsRNA+ virions or by BEI-inactivated virions. These results indicated that dsRNA– particles lacking genomic dsRNA were not able to induce a stable, sustained chIL-1β mRNA expression that occurred at and after 6 h p.i., but induced a rapid, transient chIL-1β mRNA expression within 6 h p.i. These findings provide further support for the hypothesis that viral disassembly and viral RNA synthesis are indispensable for respective induction of the rapid, transient and the stable, non-transient expression of chIL-1β mRNA in macrophages induced by ARV.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In addition, the chIL-1β mRNA expression level induced by dsRNA+ ISVPs was slightly inhibited in the early phase of infection by NH4Cl; viral RNA synthesis steps, which occur after ISVP entry of cells, were also required for the induction of high level expression because only the rapid, transient expression of chIL-1β mRNA was induced by ISVPs, in the presence of ribavirin (Fig. 4d). Moreover, dsRNA– particles and dsRNA– ISVPs were able to induce the rapid, transient mRNA expression of chIL-1β that was inhibited by NH4Cl. These data demonstrated that virion disassembly was required to induce the rapid, transient expression of chIL-1β mRNA and that the rapid appearance of chIL-1β mRNA was a specific event of macrophages in response to exposure to ARV. This expression may be directly induced by protein products that remained attached to ISVPs after proteolytic cleavage. The most likely such candidates are the cleaved products of µBC, and ' (Duncan, 1996), and those reovirion proteins, such as C, which became exposed after viral disassembly [probably similar to the conformational changes in mammalian reovirus protein 1 during viral disassembly in acidic endosomes (Dryden et al., 1993)]. Our results also provided evidence to indicate that viral RNA synthesis was required for stable, sustained expression of chIL-1β mRNA. First, the ability of dsRNA+ virions and ISVPs to induce the stable, sustained chIL-1β mRNA expression was abolished in cells treated with the viral RNA synthesis inhibitor ribavirin. Second, dsRNA– particles and ISVPs or BEI-inactivated virions were only able to induce the rapid, transient expression of chIL-1β mRNA. These results indicated that viral disassembly mediated and viral RNA synthesis-mediated responses of ARV-induced cells with distinct expression patterns of chIL-1β mRNA and that each induction pattern could be operational when the other induction pattern was inhibited, suggesting that induction pathways involved in both early and late phases of expression pattern of chIL-1β mRNA were different.
Since inhibition of intracellular viral disassembly by NH4Cl, bafilomycin A or E64, or that of viral RNA synthesis by ribavirin should not prevent the extracellular attachment or internalization of the virus (Basak & Turner, 1992; Donelli et al., 1992; Greber et al., 1993), our data suggested that viral attachment and viral internalization might not be sufficient to induce chIL-1β mRNA expression because the early or late chIL-1β mRNA expression was abolished when viral disassembly or viral RNA synthesis were blocked, respectively. The possibility that receptor engagement by ARV might play a role in the induction of chIL-1β mRNA expression could not be excluded. However, because the exact nature or identity of cell receptors for ARV is unknown, this possibility is currently not possible to assess. In contrast to the requirement of both viral disassembly and RNA synthesis, viral protein synthesis was not required for inducing chIL-1β expression because CH, which inhibits protein synthesis (Watanabe et al., 1967; Lau et al., 1975), did not abolish this early expression of chIL-1β mRNA. Although these results did not exclude a contribution of post-translational events, such as viral assembly, these findings strongly suggest that viral protein synthesis and subsequent steps in ARV replication were not required to induce this early transient process.
In recent years, several reports have demonstrated that Toll-like receptors (TLRs) are key elements in recognition of microbial pathogens, an important prerequisite for the activation of immune mechanisms responsible for their elimination (Akira & Takeda, 2004). dsRNA or viral infection has been recognised to play a major role in the activation of antiviral responses in virally infected cells by the expression and release of proinflammatory cytokines (Jacobs & Langland, 1996; Maggi et al., 2003). dsRNA is detected by TLR3 (Alexopoulou et al., 2001) and by intracellular pattern recognition receptors (PRRs) belonging to the retinoic acid inducible gene-like helicase family and these molecules have been found to be important for effective viral cellular immunity (Tabeta et al., 2004; Andrejeva et al., 2004; Yoneyama et al., 2004). In this study, we presented evidence that ARV RNA synthesis was involved in inducing a stable, sustained level of chIL-1β mRNA expression, because the inhibitor ribavirin effectively blocked mRNA induction of chIL-1β. Our results also demonstrated that the effect of dsRNA– particles on chIL-1β mRNA induction was extremely weak, compared with active dsRNA+ virions or BEI-inactivated virions. These results seemed to suggest that the presence of dsRNA, even if transcriptionally inactive, is an important factor in this response. However, completely uncoated dsRNA genomes have not been detected in cells infected with dsRNA viruses such as reovirus (Nibert et al., 1990). During reovirus replication, viral dsRNA remained within the viral cores throughout the viral replication cycle, and progeny genome was only synthesized after assembly of positive-sense mRNA into immature subviral particles. These are thought to be general features of reoviruses and, thus, ARVs are expected to share similar replication features. It could be that small amounts of incorrectly uncoated or packaged genome might be present in infected cells and induce chIL-1β mRNA expression (Jacobs & Langland, 1996). Alternatively, intracellular double-stranded secondary structures formed on viral mRNA after transcription are possibly the molecules that interact with TLR3 or intracellular PRRs and result in the activation of chIL-1β mRNA expression (Kariko et al., 2004).
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ACKNOWLEDGEMENTS |
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Received 28 February 2007;
accepted 1 December 2007.
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) are indicated. (c) ARV genomic RNA isolated from virions or EPs was separated in 1.8 % agarose gels and stained with ethidium bromide. M represents DNA ladder (ProTech). L, M and S represent the large-, medium- and small-classes of ARV genome segments (Spandidos & Graham, 1976
