- Volume 69, Issue 10, 1988
Volume 69, Issue 10, 1988
- Review Article
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Some Highlights of Animal Virus Research in 1987
More LessWe present here our review of highlights in animal virus research for 1987. This follows the format set by its predecessors (McGeoch et al., 1986, 1987a), particularly in that it is a personal and highly selective view of results published in 1987. Molecular virology has again been emphasized, reflecting the interests of the authors.
We start by describing current work on human immunodeficiency virus (HIV) and on papillomaviruses, two fields of particular urgency and application in modern virology. Topics relating to many viruses are then treated successively under the general categories of classification, growth, genome organization, genome replication, processes of transcription, protein modification and processing, and structural analyses. The account closes with an examination of the uses of oligopeptides as tools in recent research.
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- Animal
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Electron Microscopy of Human Immunodeficiency Virus
More LessSummaryTwo cell lines of human T lymphocytes (H9 and CEM) chronically infected with isolates of human or simian immunodeficiency viruses were examined by electron microscopy. Scanning electron microscopy of H9 cells showed characteristic morphological changes in the cells after infection with human T cell lymphotropic virus type III (HTLV-IIIB); these included loss of cell microvilli and their replacement by rounded surface protrusions. Virus particles were present over the whole cell surface, but were more numerous between, rather than on, surface protrusions. In contrast, CEM cells infected with three other isolates of the virus showed little change in morphology compared with uninfected cells; they typically had a single large aggregate of virus particles at the posterior end of the cell. Transmission electron microscopy of sections of infected cells showed budding virus in only a small proportion of cells although many had mature virus particles on their surface. The immature particles released by budding had a ring-like morphology in contrast to the mature virus with its characteristic elongated nucleoid. Surface spikes were rarely seen on sectioned virus particles, but were more obvious on the simian immunodeficiency virus than on human isolates. Negative staining of purified virus preparations showed that the peripheral dense material of immature particles had a striated structure. Surface spikes were not seen by negative staining on either immature or mature virus particles. A third type of smaller particle with surface spikes was found in all negatively stained preparations.
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Transforming Genes of Canine Adenovirus Type 2
More LessSummaryGenes of canine adenovirus type 2 (CAd2) involved in transformation were localized and their functions investigated. Cells transformed by CAd2 whole DNA or the BamHI A fragment (leftmost 47%) totally lacked contact inhibition and were serum-independent, anchorage-independent and tumorigenic in newborn rats. All the cells transformed by the XbaI D (0 to 15.2%) and the EcoRI C (0 to 11.3%) fragments were morphologically transformed, but were serum-dependent, anchorage-dependent and not tumorigenic in newborn rats after 150 and 190 days observation, respectively, when 5 × 106 cells per rat were injected. No transforming activities were detected by DNA fragments smaller than EcoRI C. By Northern blot hybridization, it was shown that a 1 kb mRNA was encoded in the 0 to 45% region of the genome, and 1.1 kb and 2 kb RNAs in the 4.5 to 11% region. It was therefore suggested that these gene products are required for morphological transformation, and other gene(s) or element(s), which have not been identified, may be involved in serum independence, anchorage independence and tumorigenicity of the transformed cells.
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Qualitative and Quantitative Differences in the Immune Response to Foot-and-Mouth Disease Virus Antigens and Synthetic Peptides
More LessSummaryIn cross-immunization studies using foot-and-mouth disease virus (FMDV) antigens and a synthetic peptide, from a region within virus coat protein VP1, it has been shown that intact virus will prime the immune system for intact virus, virus subunits and synthetic peptide but not for disrupted virus. In contrast, peptide will prime for a response to peptide and virus subunits but not to intact virus or disrupted virus. Furthermore, studies on antibody populations in anti-virus and anti-peptide antisera demonstrated clear differences in the nature of the antibody response to the two antigens. This result is reflected in protection studies carried out on animals immunized with virus particles or peptides where there is a clearer correlation between in vitro neutralization and protection in vivo following peptide immunization. Thus, it has been shown that there are major qualitative and quantitative differences in the immune response to the FMDV particle and synthetic peptide.
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Frequency Analysis of Simian Virus 40-specific Cytotoxic T Lymphocyte Precursors in the High Responder C57BL/6 Mouse Strain
More LessSummaryStudies in this laboratory have shown that long term simian virus 40 (SV40)-specific cytolytic T lymphocyte (CTL) cultures established from the spleens of high responder C57BL/6 (B6; H-2b) mice exhibit a preference for the selection of H-2Db-restricted CTL clones. In this study, we have investigated the basis for this selection. Limiting dilution cultures were established using responder cells from the popliteal lymph nodes and the spleens of B6 mice immunized subcutaneously in the hind footpads or via the intraperitoneal route, respectively, with syngeneic SV40-transformed cells expressing a full length (1 to 708 amino acid residues) SV40 large T antigen. The relative frequency of CTL precursors (CTLp) able to expand in vitro in the presence of SV40-transformed stimulator cells and interleukin 2 and exhibit lytic activity against H-2b cells expressing full length T antigen ranged from 1/1900 to 1/15000 in the popliteal lymph node and from 1/8000 to 1/55000 in the spleen. In these two experimental systems, CTLp restricted to H-2Kb were apparently present at higher frequency than H-2Db-restricted CTLp. Furthermore, CTLp recognizing determinants within the amino-terminal or carboxy-terminal halves of T antigen were generated in approximately equal numbers. The relative affinity of SV40-specific CTL, assessed by inhibition with anti-Lyt 2 monoclonal antibody, indicated that CTL restricted to H-2Db interacted with their target with greater affinity than CTL restricted to H-2Kb. These data suggest that the predominance of isolation of H-2Db-restricted CTL clones from long term in vitro cultures may be a function of the relative affinity of this population as a whole, rather than due to the immunodominance of this subpopulation during the in vivo response to SV40 T antigen.
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Humoral and Cytotoxic T Cell Immune Responses to Internal and External Structural Proteins of Simian Virus 5 Induced by Immunization with Solid Matrix-Antibody-Antigen Complexes
More LessSummaryMonoclonal antibodies (MAbs) were complexed to solid matrices and used to purify virus proteins from simian virus 5 (SV5)-infected tissue culture cells, ideally in such a way as to bind equimolar amounts of antigen to antibody. The resulting solid matrix-antibody-antigen (SMAA) complexes were then used as immunogens and successfully induced specific humoral and cytotoxic T cell responses. By attaching more than one MAb to the solid matrix and using such complexes to purify the respective proteins multivalent immunization was achieved. Analysis of the cytotoxic T cell response of immunized animals indicated that both surface and internal SV5 structural proteins can act as target antigens. Immunization with SMAA complexes, in the absence of adjuvant, induced higher levels of antibody than the antigen alone precipitated on alum. However, immunization with SMAA complexes resulted in relatively less antibody being produced to the antigenic determinant through which the protein is coupled, via antibody, to the SMAA complex compared with the amount of antibody produced against other antigenic determinants on that protein. The particulate solid matrix used to form the SMAA complexes in most of the experiments was a ‘fixed’ and killed suspension of the Cowan A strain of Staphylococcus aureus, although preliminary results indicated that Protein A-Sepharose could also be used as a solid matrix. Prior immunization with S. aureus alone did not reduce the level of the immune response to the appropriate antigen on subsequent immunization with S. aureus-antibody-antigen complexes. In fact on immunization of mice with these complexes the level of antibody produced to the S. aureus matrix itself was less when S. aureus-antibody-antigen complexes were used as immunogens than when S. aureus or S. aureus-antibody complexes were used. Furthermore, rabbits immunized with S. aureus-mouse MAb-antigen complexes showed a vigorous immune response to the antigen.
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Immunization with Solid Matrix-Antibody-Antigen Complexes Containing Surface or Internal Virus Structural Proteins Protects Mice from Infection with the Paramyxovirus, Simian Virus 5
More LessSummaryA mouse model system has been developed to examine the ability of purified virus proteins to protect mice from infection with the paramyxovirus simian virus 5. The system is based on the infection of mouse lungs by intranasal administration of infectious virus. The relative amounts of virus proteins and nucleic acid present within infected lungs were estimated either by Western blot analysis of disrupted lung tissues or by in situ hybridization studies using cryostat sections of infected lungs. During a normal time course of infection in non-immunized mice increasing amounts of virus protein and nucleic acid were detected in the lungs until 3 days post-infection (p.i.). Thereafter the amount of virus present within the lungs remained relatively constant until 7 days p.i. when there was a rapid decrease. Cytotoxic T cells, but not neutralizing antibody, could be detected at the time when the amount of virus within the lungs was decreasing. Prior immunization of mice with solid matrix-antibody-antigen (SMAA) complexes containing either surface or internal virus structural proteins reduced the amount of virus replication within infected lungs, the greatest degree of protection being observed when nucleoprotein or matrix protein was used to immunize the mice. There was no correlation between the degree of protection observed and the level of neutralizing antibody present in immunized animals; no neutralizing antibody was detected in mice immunized with internal virus proteins even at the time of sacrifice 5 days p.i. We have previously shown that immunization of mice with SMAA complexes containing either surface or internal virus structural proteins can induce cytotoxic T cells and thus conclude that the most likely explanation for the protection observed in immunized mice is through the induction of cytotoxic T cells.
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Difference in the Production of Human Interferon-α and -β in Mouse Cells
More LessSummaryHuman interferon-α1 and interferon-β genes with their flanking regions were introduced into mouse LMTK- cells. Although transfected cells contained the interferon genes with a similar copy number and produced a similar amount of interferon-specific mRNA, cells containing the human interferon-β gene secreted about 10 times more human interferon than cells transfected with the human interferon-α1 gene. When the coding region of the interferon-β gene was replaced by that of the interferon-α1 gene (hybrid interferon β/α gene), the human interferon production of transfected cells fell by approx. one order of magnitude. These results show that in the case of exogenous interferon genes a translational or post-translational mechanism might significantly affect the final level of human interferons, resulting in higher titres of interferon-β than of interferon-α.
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Interferon-independent Increases in Class I Major Histocompatibility Complex Antigen Expression Follow Flavivirus Infection
More LessSummaryInfection of tertiary-passaged mouse embryo fibroblasts by four flaviviruses, West Nile (WNV), Kunjin, Murray Valley encephalitis and Japanese B encephalitis, resulted in a six- to 10-fold increase in the expression of individual H-2K and H-2D class I major histocompatibility complex (MHC) antigens 16 to 48 h after infection. The mechanism(s) by which flaviviruses increased antigen expression has not been fully elucidated, but appears to be mediated partly independently of interferon-β (IFN-β) secretion, as anti-IFN-αβ antibodies partially inhibited the WNV-induced increase but totally prevented increases caused by the addition of (i) pure IFN-β, (ii) IFN-β-containing supernatants from WNV-infected mouse embryo fibroblasts (MEF), or (iii) polyinosinic-polycytidylic acid. Actinomycin D treatment of MEF, which inhibited mRNA synthesis by >90% as determined by [3H]uridine uptake, totally inhibited the increased MHC expression caused by WNV infection. Thus, the increase in class I MHC antigen expression following infection is dependent upon cellular RNA synthesis.
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Attachment of Influenza C Virus to Human Erythrocytes
More LessSummaryBinding experiments with radioactively labelled influenza C virions were carried out to investigate the interaction of the virus with human erythrocytes. The erythrocytes from any of 35 different individuals were found to contain influenza C virus-binding sites though their number was variable among the individuals and was much less than that on mouse, rat and chicken erythrocytes. Attachment of influenza C virus to human erythrocytes was inhibited completely by prior treatment of the virus with anti-HE monoclonal antibody having a strong haemagglutination inhibition activity. Pretreatment of erythrocytes with neuraminidase or the neuraminate-O-acetylesterase of influenza C virus resulted in a marked reduction in the level of virus binding. Thus it appears that human erythrocytes have a low level of O-acetylated sialic acid-containing glycoconjugates that can interact specifically with the HE glycoprotein of influenza C virus. Proteolytic digestion of erythrocytes with ficin, bromelain or V-8 protease inhibited virus binding almost completely, suggesting that the erythrocyte receptor for influenza C virus is a glycoprotein. In contrast to these enzymes, trypsin treatment of erythrocytes reduced virus binding by only about 50%, and α-chymotrypsin treatment did not inhibit at all. It was also found that treatment of erythrocytes with monoclonal antibody to the M or N blood group antigen greatly inhibited virus binding to the cells. These results, taken together, suggest that most influenza C virus receptors on human erythrocytes, if not all, reside on glycophorin A which is known to possess the M or N blood group activity.
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The L Protein of Vesicular Stomatitis Virus Modulates the Response of the Polyadenylic Acid Polymerase to S-Adenosylhomocysteine
More LessSummaryTsG16(I) is a temperature-sensitive (ts) mutant of vesicular stomatitis virus, Indiana serotype, which overproduces polyadenylic acid [poly(A)] in an in vitro transcription system due to a mutation in the L protein. Others have reported that l-S-adenosylhomocysteine (S-Ado-Hcy) causes wild-type (wt) virus to overproduce poly(A) in vitro. The possibility that tsG16(I) constitutively expresses a property induced by S-Ado-Hcy in the case of wt virus was found not to be so since polyadenylation by the mutant was still sensitive to S-Ado-Hcy. Indeed, S-Ado-Hcy caused tsG16(I) to overproduce poly(A) in vitro to a greater extent than its parental wt virus. The increase in polyadenylation observed in response to saturating levels of S-Ado-Hcy differed for tsG16(I), for its parental wt virus and for another wt strain. To characterize which viral protein modulated the polyadenylation response to S-Ado-Hcy, purified virions were fractionated and their phenotypes in homologous and heterologous reconstitution assays were examined. The results indicated that the viral L protein modulated the response in all three stocks of virus. These data provide further evidence to suggest that the L protein of vesicular stomatitis virus plays a role in polyadenylation of the viral mRNA.
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Minute Virus of Mice Non-structural Protein NS-1 Is Necessary and Sufficient for Trans-activation of the Viral P39 Promoter
More LessSummaryThe genome of the autonomous parvovirus minute virus of mice (MVM) is organized in two overlapping transcription units: the genes coding for the two non-structural proteins (NS-1 and NS-2) are transcribed from a promoter (P04) located at map unit 4, whereas the promoter controlling the capsid protein genes (P39) lies at map unit 39. We studied the effect of viral proteins on the activity of the P39 promoter in vivo. By site-directed mutagenesis we constructed clones encoding only one of the two NS proteins. The activity of the P39 promoter was measured in HeLa or EL-4 cells transfected with these clones, either by an RNase protection assay or by following the expression of a reporter gene, CAT (which codes for chloramphenicol acetyltransferase), placed under the control of this promoter. We found that the P39 promoter of strain MVMi is activated in trans by a viral gene product, and evidence to suggest that NS-1 is the only viral gene product responsible for this trans-activation. We also determined that the mechanism of trans-activation is very rapid, since all species of viral mRNAs appear together in non-synchronized infected EL-4 cells within a 2 h interval.
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Typing Hepatitis B Virus by Homology in Nucleotide Sequence: Comparison of Surface Antigen Subtypes
SummaryThe complete nucleotide sequences of the DNA of three hepatitis B virus (HBV) genomes of subtype adw, cloned from plasma samples of asymptomatic carriers living in the mainland and Okinawa Prefecture of Japan and Indonesia were determined. All three comprised 3215 bp and differed in sequence by only 3.9 to 5.6%. When these isolates were compared with the reported sequences of two HBV genomes of the same subtype derived from American carriers, however, the differences were greater (8.3 to 9.3%) to an extent comparable with the nucleotide divergence between an HBV genome of subtype adw and that of a heterotypic subtype, such as adr, ayw or ayr. A total of 18 HBV genomes of various subtypes, including the three described here, 10 reported previously and five unpublished ones, were classified into four groups based on an inter-group divergence in nucleotide sequence of 8% or greater: group A (two adw genomes), group B (four adw), group C (three adw, four adr and one ayr) and group D (four ayw). Thus, the nine genomes of HBV subtype adw were distributed into three groups with considerably different sequences. These results indicate that the four major antigenically defined subtypes of envelope polypeptide do not reflect true genotypic variation of HBV. The fact that d to y, as well as w to r, subtypic change can be induced by an A → G point mutation at nucleotides 365 and 479 in the S gene, respectively, supports this view.
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Molecular Analysis of the Pyrimidine Deoxyribonucleoside Kinase Gene of Wild-type and Acyclovir-resistant Strains of Varicella-Zoster Virus
SummaryThe pyrimidine deoxyribonucleoside kinase (dPK) genes from five wild-type and four acyclovir-resistant varicella-zoster virus (VZV) strains were studied. One of the acyclovir-resistant strains was isolated from a patient receiving chronic acyclovir therapy. Acyclovir-resistant strains expressed the 1.8 kb VZV dPK transcript but lacked dPK activity. To determine the basis for the lack of enzyme activity the dPK gene from each strain was cloned and its DNA sequence determined. The VZV dPK gene was found to be highly conserved among strains, with greater than 99% nucleotide and amino acid homology. Each acyclovir-resistant VZV strain differed from its wild-type parent in only a single amino acid. The dPK genes from the acyclovir-resistant strains contained either point mutations near the putative thymidine-binding site of the enzyme or ones that resulted in the premature termination of protein synthesis. Single point mutations were sufficient to render these strains dPK-negative and highly resistant to acyclovir. The molecular basis for acyclovir resistance at the dPK locus of VZV is similar to that previously noted to render herpes simplex viruses resistant to acyclovir.
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Mutational Analysis of the Herpes Simplex Virus Type 1 Trans-inducing Factor Vmw65
More LessSummaryThe herpes simplex virus type 1 (HSV-1) polypeptide Vmw65 is a structural component of the virus particle and is also responsible for trans-induction of immediate early (IE) transcription. Functional domains of this polypeptide were investigated by constructing a series of 10 plasmids each with a 12 bp insertion in the gene encoding Vmw65. Plasmids were analysed for their ability to stimulate IE transcription in short term transfection assays, and the altered Vmw65 polypeptides were assayed for the ability to form an IE-specific protein-DNA complex (IEC) in vitro. A direct correlation was observed between stimulation of transcription and formation of IEC, strongly suggesting that IEC is an important intermediate in transcription activation. Plasmids were also tested for their ability to rescue the temperature-sensitive mutation in the HSV-2 assembly mutant ts2203, since marker rescue analysis indicated that this mutation maps within the gene encoding Vmw65. Five plasmids failed to rescue ts2203, thereby defining regions of Vmw65 required for virus assembly. The results show that distinct domains exist in Vmw65 for activation of transcription and assembly of virus.
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Ribonucleotide Reductase Encoded by Herpes Simplex Virus Is a Determinant of the Pathogenicity of the Virus in Mice and a Valid Antiviral Target
SummaryThe role of the herpes simplex virus (HSV)-encoded ribonucleotide reductase (RR) in the pathogenicity of the virus has been examined by use of mutants with lesions in either the large or small subunit of the enzyme. The virulence of the mutants in mice was reduced by about 106-fold when compared with that of the parental virus (HSV type 1 strain 17), while the virulence of a revertant of one of the mutants was restored to within about 100-fold of that of the parent virus. These experiments demonstrate that activity of the HSV RR is essential for virus pathogenicity in mice and suggests that the enzyme is a valid target for specific antiviral compounds.
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Human Cytomegalovirus ICP22, the Product of the HWLF1 Reading Frame, Is an Early Nuclear Protein that Is Released from Cells
More LessSummaryWe employed a murine monoclonal antibody (CH41) and a λgt11 library of human cytomegalovirus (CMV) DNA fragments to map the gene for a viral protein, denoted infected cell protein (ICP) 22, to the HWLF1 open reading frame in the S component of the CMV genome (0.92 to 0.93 map units). By using antibody CH41 in immunofluorescence, immunoprecipitation and immunoblotting analyses, ICP22 was readily detected as a β (delayed early) gene product during viral growth. The cellular localization of this protein was found to be nuclear by immunofluorescence analysis; however, it partitioned with the cytoplasm when cells were fractionated with non-ionic detergents. Analysis of cell-free medium showed that a proportion of ICP22 was released from cells as a soluble protein at both early (24 h) and late (72 to 120 h) times in infection. The function of this protein which has such diverse characteristics remains unknown.
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The Fusion Glycoproteins of Human Respiratory Syncytial Virus of Subgroups A and B: Sequence Conservation Provides a Structural Basis for Antigenic Relatedness
More LessSummaryTwo major antigenic subgroups (A and B) have been described for human respiratory syncytial virus. The complete nucleotide sequence was determined for the fusion (F) mRNA of the subgroup B strain 18537 and the amino acid sequence of the F protein deduced, for comparison with the previously described sequences for the A2 strain of subgroup A. The F proteins (excluding the cleaved signal peptide) were 91% identical between the subgroups, consistent with the previously described high degree of antigenic relatedness. The greatest divergence occurred within the F2 subunit immediately preceding the cleavage activation site.
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Bluetongue Virus Genome Remains Stable throughout Prolonged Infection of Cattle
More LessSummaryInfection of three calves with a highly plaque-purified strain of bluetongue virus (BTV) resulted in prolonged infections, during which virus and neutralizing antibodies co-circulated in peripheral blood. Oligonucleotide fingerprint analyses of the original challenge virus and of the final virus isolate obtained from each calf demonstrated the BTV genome to remain stable throughout prolonged infection as no differences in fingerprint patterns were detected. Six neutralizing monoclonal antibodies (MAbs), and a polyclonal rabbit antiserum, were produced against the challenge virus. This panel of MAbs recognized at least two distinct neutralizing epitopes as demonstrated by immune precipitation. Neutralizing epitopes remained stable through the prolonged infections, as all MAbs and the polyclonal rabbit antiserum neutralized the challenge virus and the final calf isolates to equivalent titres. These results suggest that antigenic drift is not the mechanism by which BTV is able to persist in cattle in spite of a strong humoral immune response.
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Comparisons of the Pestivirus Bovine Viral Diarrhoea Virus with Members of the Flaviviridae
More LessSummaryThe molecular features of bovine viral diarrhoea virus (BVDV), a member of the Pestivirus genus currently classified in the Togaviridae, were examined for characteristics resembling those of the Flaviviridae family. Like flaviviruses, BVDV possesses a single-stranded RNA genome (approx. 4.3 × 106 M r) deficient in a 3′ poly(A) tract. This RNA has a single open reading frame spanning the length of the genome in the viral RNA sense (positive polarity), implying an expression strategy involving the processing of a precursor polyprotein. With the exception of several short but significant stretches of identical amino acids within two non-structural proteins, no extended regions of nucleotide or amino acid sequence homology between BVDV and representatives of three serological subgroups of mosquito-borne flaviviruses were noted. However, comparison of the organization of protein-coding domains along the genomes and the hydropathic profiles of amino acid sequences revealed pronounced similarities. It is proposed that Pestivirus, of which BVDV is the prototype member, should no longer be grouped in the Togaviridae family, but rather be considered a genus of non-arthropod-borne viruses within the Flaviviridae.
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