Physical and genomic characteristics identify chicken proventricular necrosis virus (R11/3 virus) as a novel birnavirus.

Chicken proventricular necrosis virus (CPNV), isolate R11/3, previously was isolated from transmissible viral proventriculitis-affected chickens and was determined to be the likely etiology of this disease. CPNV was identified as a birnavirus on the basis of virion size and morphology (icosahedral, approximately 75 nm in diameter, nonenveloped); buoyant density in cesium chloride (1.32 g/ml); a genome comprising bisegmented, double-stranded RNA (approximately 3.8 and 3.4 kilobase pairs); and nucleotide sequence analyses. Nucleotide sequencing of CPNV RNA, segment B, identified a single large open reading frame that encodes a 903-amino acid protein. The 903-amino acid protein was identified as the putative VP1, the viral RNA-dependent RNA polymerase (RdRp), on the basis of sequence homologies with other birnavirus VP1 proteins. The CPNV VP1 possessed the unique permuted RdRp sequence motif arrangement characteristic of birnaviruses; however, phylogenetic analyses based on VP1 demonstrated that CPNV is deeply divergent from other birnaviruses.

Detection of chicken proventricular necrosis virus (R11/3 virus) in experimental and naturally occurring cases of transmissible viral proventriculitis with the use of a reverse transcriptase-PCR procedure.

A reverse-transcriptase-polymerase-chain-reaction (RT-PCR) procedure was evaluated for detection of chicken proventricular necrosis virus (CPNV) in transmissible viral proventriculitis (TVP) -affected chickens. The RT-PCR procedure was compared with indirect immunofluorescence (IFA) and virus isolation for detection of CPNV in experimentally infected chickens. Microscopic lesions characteristic of TVP were detected on days 5-35 postexposure (PE) in CPNV-infected chickens; CPNV was detected by RT-PCR on days 3-14 PE in freshly collected proventriculi, and on days 1-14 PE in formalin-fixed paraffin-embedded (FFPE) proventriculi. CPNV was detected in proventriculi of experimentally infected chickens by IFA on days 3-10 PE, and by virus isolation on days 1-14 PE. With IFA used as a reference, sensitivity of the RT-PCR procedure with freshly collected and FFPE proventriculi was 88% and 100%, respectively; specificity was 83% and 86%, respectively. Proventriculi (FFPE) obtained from suspect TVP cases (n=19) were evaluated for presence of CPNV by RT-PCR and microscopic lesions consistentwith TVP. CPNV was detected by RT-PCR in proventriculi from 8/11 TVP (+) cases (24/36 tissue sections). TVP (+) cases were defined by microscopic lesions characteristic of TVP; CPNV was not detected in proventriculi (0/8 cases, 0/32 tissue sections) in the absence of these lesions. The association between presence of TVP-characteristic microscopic lesions and presence of CPNV was highly significant (P = 0.0014). These findings indicate the utility of the RT-PCR procedure for detection of CPNV and provide additional evidence for an etiologic role for this virus in TVP.

Virulence determinants of equine infectious anemia virus.

Equine infectious anemia virus (EIAV) is a macrophage-tropic lentivirus that rapidly Induces disease in experimentally infected horses. Because EIAV infection and replication is centered on the monocyte/macrophage and has a pronounced acute disease stage, it is a useful model system for understanding the contribution of monocyte/macrophages to other lentivirus-induced diseases. Genetic mapping studies utilizing chimeric proviruses in which parental viruses are acutely virulent or avirulent have allowed the identification of important regions that influence acute virulence. U3 regions in the viral LTR, surface envelope (SU) protein and the accessory S2 gene strongly influence acute disease expression. While the chimeric proviruses provide insight into genes or genome regions that affect viral pathogenesis, it is then necessary to further dissect those regions to focus on specific virus-host mechanisms that lead to disease expression. The V6 region of the viral env protein is an example of one identified region that may interact with the ELR-1 receptor in an important way and we are currently identifying S2 protein motifs required for disease expression.

EIAV S2 enhances pro-inflammatory cytokine and chemokine response in infected macrophages.

Equine infectious anemia virus (EIAV) infection is distinctive in that it causes a rapid onset of clinical disease relative to other retroviruses. In order to understand the interaction dynamics between EIAV and the host immune response, we explored the effects of EIAV and its S2 protein in the regulation of the cytokine and chemokine response in macrophages. EIAV infection markedly altered the expression pattern of a variety of pro-inflammatory cytokines and chemokines monitored in the study. Comparative studies in the cytokine response between EIAV(17) and EIAV(17DeltaS2) infection revealed that S2 enhances the expression of IL-1alpha, IL-1beta, IL-8, MCP-2, MIP-1beta and IP-10. Moreover, S2 specifically induced the expression of the newly discovered cytokine, IL-34. Taken together, these results may help explain the effect of cytokine and chemokine dysregulation in EIAV pathogenesis and suggest a role of S2 in optimizing the host cell environment to promote viral dissemination and replication.

The S2 accessory gene of equine infectious anemia virus is essential for expression of disease in ponies.

Equine infectious anemia virus (EIAV) is a macrophage-tropic lentivirus that persistently infects horses and causes a disease that is characterized by periodic episodes of fever, thrombocytopenia, and viremia. EIAV encodes only four regulatory/accessory genes, (tat, rev, ttm, and S2) and is the least genetically complex of all known lentiviruses. We sought to determine the role of the EIAV S2 accessory gene of EIAV by introducing mutations that would prevent S2 expression on the p19/wenv17 infectious molecular clone. Virus derived from the p19/wenv17 molecular clone is highly virulent and routinely fatal when given in high doses (J. Virol. 72 (1998) 483). In contrast, an S2 deletion mutant on the p19/wenv17 background is unable to induce acute disease and plasma virus loads were reduced by 2.5 to 4.0 logs at 15 days post-infection. The S2 deleted virus failed to produce any detectable clinical signs during a 5-month observation period. These results demonstrate that S2 gene expression is essential for disease expression of EIAV.

Partial characterization of an adenovirus-like virus isolated from broiler chickens with transmissible viral proventriculitis.

Transmissible viral proventriculitis (TVP) was experimentally reproduced in specific-pathogen-free chickens using a homogenate of proventricular tissue obtained from TVP-affected commercial broiler chickens. Thin-section electron microscopy revealed intranuclear, approximately 70-nanometer (nm), adenovirus-like viruses (AdLV) within proventricular lesions. The AdLV, designated AdLV (R11/3), could not be propagated using various avian and mammalian cell cultures or by inoculation of embryonated chicken eggs by yolk, allantoic, or chorioallantoic membrane routes. However, AdLV (R11/3) was successfully propagated by amniotic inoculation of embryonated chicken eggs, with detection of the virus in proventriculi and intestinal contents of hatched 2-day-old chicks (8 days postinoculation). Virus propagation was evident in in ovo-inoculated chicks by (1) gross and microscopic lesions in proventriculi consistent with TVP, (2) immunohistochemical localization of AdLV (R11/3) antigens in proventricular epithelium, (3) thin-section electron microscopic detection of intranuclear, approximately 70-nm AdLVs within proventricular epithelium, and (4) negative-stain electron microscopic detection of extracellular, approximately 70-nm AdLVs in intestinal contents. Indirect immunofluorescence and polymerase chain reaction procedures that specifically recognize groups I, II, and III avian adenoviruses failed to recognize AdLV (R11/3). The findings suggest an etiologic role for AdLV (R11/3) in TVP and indicate that this virus is distinct from known avian adenoviruses.

Antigenic and genomic characterization of turkey enterovirus-like virus (North Carolina, 1988 isolate): identification of the virus as turkey astrovirus 2.

A small round virus (SRV) was isolated in 1988 from droppings of enteritis-affected turkeys in North Carolina and tentatively identified as an enterovirus on the basis of size (18-24 nm in diameter), intracytoplasmic morphogenesis, and a single-stranded RNA genome of approximately 7.5 kb. Additional characterization studies based on antigenic and genomic analyses were done to determine the relationship of this turkey enterovirus-like virus (TELV) to turkey astrovirus 2 (TAstV2), a recently characterized SRV of turkeys. Cross-immunofluorescence studies with TELV- and TAstV2-specific antisera indicated a close antigenic relationship between these viruses. TELV RNA was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) procedures with oligonucleotide primers specific for TAstV2 polymerase gene (open reading frame [ORF] 1b) and capsid protein gene (ORF 2). Subsequent sequence analyses of these TELV-derived RT-PCR products indicated a high degree of similarity with polymerase gene (98.8%) and capsid gene (96.9%) of TAstV2. These studies definitively identify TELV (North Carolina, 1988 isolate) as TAstV2.

Mechanism of feline immunodeficiency virus envelope glycoprotein-mediated fusion.

Feline immunodeficiency virus (FIV) shares remarkable homology to primate lentiviruses, human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). The process of lentiviral env glycoprotein-mediated fusion of membranes is essential for viral entry and syncytia formation. A detailed understanding of this phenomenon has helped identify new targets for antiviral drug development. Using a model based on syncytia formation between FIV env-expressing cells and a feline CD4+ T cell line we have studied the mechanism of FIV env-mediated fusion. Using this model we show that FIV env-mediated fusion mechanism and kinetics are similar to HIV env. Syncytia formation could be blocked by CXCR4 antagonist AMD3100, establishing the importance of this receptor in FIV gp120 binding. Interestingly, CXCR4 alone was not sufficient to allow fusion by a primary isolate of FIV, as env glycoprotein from FIV-NCSU(1) failed to induce syncytia in several feline cell lines expressing CXCR4. Syncytia formation could be inhibited at a post-CXCR4 binding step by synthetic peptide T1971, which inhibits interaction of heptad repeat regions of gp41 and formation of the hairpin structure. Finally, using site-directed mutagenesis, we also show that a conserved tryptophan-rich region in the membrane proximal ectodomain of gp41 is critical for fusion, possibly at steps post hairpin structure formation.

Influence of long terminal repeat and env on the virulence phenotype of equine infectious anemia virus.

The molecular clones pSPeiav19 and p19/wenv17 of equine infectious anemia virus (EIAV) differ in env and long terminal repeats (LTRs) and produce viruses (EIAV(19) and EIAV(17), respectively) of dramatically different virulence phenotypes. These constructs were used to generate a series of chimeric clones to test the individual contributions of LTR, surface (SU), and transmembrane (TM)/Rev regions to the disease potential of the highly virulent EIAV(17). The LTRs of EIAV(19) and EIAV(17) differ by 16 nucleotides in the transcriptional enhancer region. The two viruses differ by 30 amino acids in SU, by 17 amino acids in TM, and by 8 amino acids in Rev. Results from in vivo infections with chimeric clones indicate that both LTR and env of EIAV(17) are required for the development of severe acute disease. In the context of the EIAV(17) LTR, SU appears to have a greater impact on virulence than does TM. EIAV(17SU), containing only the TM/Rev region from the avirulent parent, induced acute disease in two animals, while a similar infectious dose of EIAV(17TM) (which derives SU from the avirulent parent) did not. Neither EIAV(17SU) nor EIAV(17TM) produced lethal disease when administered at infectious doses that were 6- to 30-fold higher than a lethal dose of the parental EIAV(17). All chimeric clones replicated in primary equine monocyte-derived macrophages, and there was no apparent correlation between macrophage tropism and virulence phenotype.