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Enterococcus cecorum (EC) is the dominant enteric commensal of adult chickens and contributes to the gut consortia of many avian and mammalian species. While EC infection is an uncommon zoonosis, like other enterococcal species it can cause life-threating nosocomial infection in people. In contrast to other enterococci which are considered opportunistic pathogens, emerging pathogenic strains of EC cause outbreaks of musculoskeletal disease in broiler chickens. Typical morbidity and mortality is comparable to other important infectious diseases of poultry. In molecular epidemiologic studies, pathogenic EC strains were found to be genetically clonal. These findings suggested acquisition of specific virulence determinants by pathogenic EC. To identify divergent genomic features and acquired virulence determinants in pathogenic EC; comparative genomic analysis was performed on genomes of 3 pathogenic and 3 commensal strains of EC. Pathogenic isolates had smaller genomes with a higher GC content, and they demonstrated large regions of synteny compared to commensal isolates. A molecular phylogenetic analysis demonstrated sequence divergence in pathogenic EC genomes. At a threshold of 98% identity, 414 predicted proteins were identified that were highly conserved in pathogenic EC but not in commensal EC. Among these, divergent CRISPR-cas defense loci were observed. In commensal EC, the type IIA arrangement typical for enterococci was present; however, pathogenic EC had a type IC locus, which is novel in enterococci but commonly observed in streptococci. Potential mediators of virulence identified in this analysis included a polysaccharide capsular locus similar to that recently described for E. faecium, an epa-like locus, and cell wall associated proteins which may bind host extracellular matrix. This analysis identified specific genomic regions, coding sequences, and predicted proteins which may be related to the divergent evolution and increased virulence of emerging pathogenic strains of EC.

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.

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.

The macrophage-tropic lentivirus, equine infectious anemia virus (EIAV), encodes the small auxiliary protein S2 from a short open reading frame that overlaps the amino terminus of env EIAV S2 is dispensable for virus replication in cultured cells but is required for disease production. S2 is approximately 7 kDa and has no overall amino acid sequence homology to other cellular or viral proteins. Therefore it is likely that S2 plays a role as an adaptor protein. To further investigate S2 function we performed a yeast-2-hybrid screen to identify cellular proteins that interact with EIAV S2. The screen identified two human cellular proteins, amplified in osteosarcoma (OS-9) and proteasome 26S ATPase subunit 3 (PSMC3) that interact with S2. The equine homologues of these proteins were cloned and their interactions with S2 confirmed using co-immunoprecipitation assays. We identified two OS-9 isoforms that interact with S2 and a third splice variant that does not, indicating a region of OS-9 apparently required for the S2 interaction. The roles of these cellular proteins during EIAV infection have not been determined., (Copyright 2010 Elsevier B.V. All rights reserved.)

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., (Copyright 2009 Elsevier Inc. All rights reserved.)

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.

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.

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.

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.

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.

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.

A transforming growth factor (TGF)-beta was isolated and cloned from hybrid striped bass (Morone saxatilis x M. chrysops) anterior kidney mononuclear cells. This isolate (Genbank accession number AF140363) contains an open reading frame of 1146 bases coding for a 382 amino acid protein most similar to rainbow trout TGF-beta (57.3 and 78.6% identity with precursor and active protein, respectively) and rat TGF-beta 1 (41.1 and 68.8% identity with precursor and active protein, respectively). Consensus primers were demonstrated to amplify specifically by polymerase chain reaction (PCR), a TGF-beta segment from 14 species of teleost fish comprising 10 taxonomic families in 7 orders. A reverse transcription quantitative competitive polymerase chain reaction (RT-qcPCR) assay was devised to measure TGF-beta mRNA expression in teleost fish. Higher levels of TGF-beta mRNA expression were detected in mononuclear cells of peripheral blood than from spleen or anterior kidney.

The nucleoprotein (NP) of Newcastle disease virus (NDV) was selected to study the relative importance of an internal structural protein in the avian immune response. The NP gene of the virulent, neurotropic NDV Texas GB (TGB) strain was cloned and sequenced. Nucleotide sequence data for the NP gene allowed comparison of the deduced amino acid sequences for the NP genes of NDV-TGB and the avirulent duck isolate NDV-D26. These comparisons demonstrated an 89% nucleotide sequence homology and a 97% homology between the deduced amino acid sequences. The NDV-TGB NP expressed in recombinant vaccinia virus (rVAC) was electrophoretically and immunologically identical to the wild-type NDV-TGB. Although inoculation of chickens with the recombinant vaccinia virus expressing the NDV NP gene elicited anti-NDV antibodies in higher titers than in birds inoculated with live LaSota NDV, this strong anti-NDV response did not protect against lethal challenge with NDV-TGB.

The 3' end of the turkey coronavirus (TCV) genome (1740 bases) including the nucleocapsid (N) gene and 3' untranslated region (UTR) were sequenced and compared with published sequences of other avian and mammalian coronaviruses. The deduced sequence of the TCV N protein was determined to be 409 amino acids with a molecular mass of approximately 45 kDa. The TCV N protein was identical in size and had greater than 90% amino acid identity with published N protein sequences of infectious bronchitis virus (IBV); less than 21% identity was observed with N proteins of bovine coronavirus and transmissible gastroenteritis virus. The 3' UTR showed some variation among the three TCV strains examined, with two TCV strains, Minnesota and Indiana, containing 153 base segments which are not present in the NC95 strain. Nucleotide sequence identity between the 3' UTRs of TCV and IBV was greater than 78%. Similarities in both size and sequence of TCV and IBV N proteins and 3' UTRs provide additional evidence that these avian coronaviruses are closely related.

Thrombocytopenia is a consistent finding and one of the earliest hematological abnormalities in horses acutely infected with equine infectious anemia virus (EIAV), a lentivirus closely related to human immunodeficiency virus. Multifactorial mechanisms, including immune-mediated platelet destruction and impaired platelet production, are implicated in the pathogenesis of EIAV-associated thrombocytopenia. This study was undertaken to investigate whether regenerative thrombopoiesis and platelet destruction occurred in ponies acutely infected with EIAV. Circulating large, immature platelets were increased in ponies acutely infected with EIAV late in the infection when platelet count was at a nadir. Morphometric analysis of bone marrow from acutely infected ponies revealed significant increased in megakaryocyte area and megakaryocyte nuclear area. A trend toward increased numbers of megakaryocytes was also observed. Platelets from acutely infected ponies had increased surface-bound fibrinogen and ultrastructural changes consistent with in vivo platelet activation. Platelets also had hypofunctional aggregation responses to three agonists in vitro. We conclude that thrombocytopenia in ponies acutely infected with EIAV is regenerative and suggest that bone marrow platelet production is not severely compromised in these ponies. Our findings reveal that in vivo platelet activation occurs in ponies acutely infected with EIAV, and as a result platelets are hypofunctional in vitro. Activation of platelets in vivo may cause platelet degranulation or formation of platelet aggregates, which would result in removal of these damages platelets from circulation. This may represent a form of nonimmune-mediated platelet destruction in ponies acutely infected with EIAV., (Copyright 1999 Academic Press.)

A reverse transcriptase, polymerase chain reaction (RT-PCR) procedure was used to amplify a segment of the genome of turkey coronavirus (TCV) spanning portions of the matrix and nucleocapsid (MN) protein genes (approximately 1.1 kb). The MN gene region of three epidemiologically distinct TCV strains (Minnesota, NC95, Indiana) was amplified, cloned into pUC19, and sequenced. TCV MN gene sequences were compared with published sequences of other avian and mammalian coronaviruses. A high degree of similarity (>90%) was observed between the nucleotide, matrix protein, and nucleocapsid protein sequences of TCV strains and published sequences of infectious bronchitis virus (IBV). The matrix and nucleocapsid protein sequences of TCV had limited homology (<30%) with MN sequences of mammalian coronaviruses. These results demonstrate a close genetic relationship between the avian coronaviruses, IBV and TCV.

Equine infectious anemia virus (EIAV), a macrophage-tropic lentivirus, causes persistent infections of horses. A number of biologic features, including the rapid development of acute disease, the episodic nature of chronic disease, the propensity for viral genetic variation, and the ability for many infected animals to eventually control virus replication, render EIAV a potentially useful model system for the testing of antiretroviral therapies and vaccine strategies. The utility of the EIAV system has been hampered by the lack of proviral clones that encode promptly pathogenic viral stocks. In this report, we describe the generation and characterization of two infectious molecular clones capable of causing acute clinical syndromes similar to those seen in natural infections. Virus derived from clone p19/wenv17 caused severe debilitating disease at 5 to 7 days postinfection; initial febrile episodes were fatal in two of three infected animals. Virus derived from a second clone, p19/wenv16, caused somewhat milder primary febrile episodes by 10 to 12 days postinfection in two of two infected animals. Virus derived from both clones caused persistent infections such that some animals exhibited chronic equine infectious anemia, characterized by multiple disease episodes. The two virulent clones differ in envelope and rev sequences.

The macrophage tropic lentivirus, equine infectious anemia virus (EIAV), encodes a dUTPase in the pol gene that is required for efficient replication in macrophages. Two naturally occurring variants of the enzyme were expressed as recombinant proteins in Escherichia coli; metal chelate affinity chromatography was used to purify histidine-tagged recombinant enzymes to greater than 80% homogeneity in a single chromatographic step. Biochemical and enzymatic analyses of these preparations suggest that this method yields dUTPase that is suitable for detailed mutational analysis. Specific activities of preparations ranged from 4 x 10(3) to 5 x 10(4) units/mg. Recombinant EIAV dUTPase was highly specific for dUTP with a Km in the range of 3 to 8 microM. The enzyme was sensitive to inhibition by dUDP with little inhibition by other nucleotides or the reaction products, dUMP and PPi. The subunit organization of recombinant EIAV dUTPase was probed by gel filtration, glycerol gradient centrifugation, and chemical cross-linking, and is a trimer. We have begun mutational analyses by targeting a conserved domain present at the carboxyl terminus of all dUTPases that shares high homology to the phosphate binding loops (P-loops) of a number of ATP- and GTP-binding phosphatases. The P-loop-like motif of dUTPases is glycine rich but lacks the invariant lysine found in authentic P-loops. Deletion of this motif leads to loss of dUTPase activity; a series of point mutations that have been shown to inactivate authentic P-loops also abolish EIAV dUTPase activity.

Equine infectious anemia virus (EIAV) is a lentivirus that replicates predominantly in mature tissue macrophages. Viral expression is strongly influenced by the state of differentiation of the host cell. While blood monocytes can be infected, viral transcription is limited until the cell differentiates into a mature macrophage. Activation of mature macrophages infected with EIAV might also alter viral expression, presumably through binding of cellular transcription factors to viral nucleic acid sequences within the long terminal repeat (LTR). Using DNA amplification techniques, we compared LTR sequences of U.S. field strains of EIAV to sequences of a laboratory adapted strain of the virus. All field strain sequences were more closely related to Wyoming strain than to the Malmquist laboratory adapted strain or a previously sequenced infectious molecular clone of EIAV. Primary equine monocyte-derived macrophage cultures were infected with virulent and avirulent strains of EIAV and the effects of macrophage stimulation on EIAV expression were determined. Stimulation of macrophages with phorbol ester activated the cells to secrete tumor necrosis factor alpha (TNF alpha). This activation signal also resulted in a significant downregulation of viral expression as determined by supernatant reverse transcriptase activity. This effect occurred independent of the virulence of the virus strain used or the nucleic acid sequence of the viral LTR. This may represent an adaptive response of EIAV to evade the host immune response and establish a persistent infection.

Equine infectious anemia virus is a lentivirus that replicates in mature tissue macrophages of horses. Ponies were infected with equine infectious anemia virus. During febrile episodes, proviral DNA was detectable, but viral mRNA was not detectable. As cultured blood monocytes from these ponies differentiated into macrophages, viral expression was upregulated. In situ hybridization confirmed that viral transcription occurred in mature macrophages.

The retrovirus equine infectious anemia virus (EIAV) encodes a dUTPase situated between reverse transcriptase and integrase. We have described the inability of EIAV with a 270-bp dUTPase deletion, delta DU EIAV, to replicate to wild-type (WT) levels in equine macrophages (D. S. Threadgill, W. K. Steagall, M. T. Flaherty, F. J. Fuller, S. T. Perry, K. E. Rushlow, S. F. J. LeGrice, and S. L. Payne, J. Virol. 67, 2592-2600, 1993). Here we describe the construction of a second dUTPase-deficient virus (DUD71E) containing a single amino acid substitution in dUTPase. delta DU and DUD71E replicate to 2% of WT levels in macrophages by 7 days postinfection, when WT EIAV is highly cytopathic. To identify the replication block(s), we analyzed DNA synthesis, integration, and transcription. DNA synthesis was normal in macrophages, with evidence of full-length viral DNA by 24 hr postinfection. The level of integrated delta DU and DUD71E DNA appeared to be decreased 2- to 3-fold compared to WT. Steady-state levels of full-length viral transcripts were decreased over 100-fold, indicating that replication of dUTPase-deficient EIAV is blocked between viral DNA synthesis and transcription. As dUTP hydrolysis normally plays a role in preventing incorporation of uracil into newly synthesized DNA, we investigated the possibility that dUTPase-deficient EIAV DNA contains uracil. In vitro assays showed that while WT virions do not utilize dUTP, dUTPase-deficient virus and recombinant RT synthesize uracil-containing DNA. The presence of uracil in viral DNA recovered from delta DU- and DUD71E-infected macrophages was also demonstrated. In macrophages, a virally encoded dUTPase may be necessary to prevent the incorporation of uracil into viral DNA.

The Rice strain of pseudorabies virus (PRV) was intranasally instilled in pigs that were seronegative to PRV. Cells were scraped or brushed from tonsillar surfaces biweekly until pigs were euthanatized at either 10 or 16 weeks after infection. The DNA extracted from tonsillar cells or parenchyma were subjected to polymerase chain reaction analysis, using either a single set of oligonucleotide primers or nested primers from the PRV gII glycoprotein gene. Pigs became seropositive to PRV by 3 weeks after infection. The virus was isolated from the trigeminal ganglia and tonsils of pigs that were euthanatized or died 1 to 2 weeks after infection, but not from pigs that were euthanatized 10 or 16 weeks after infection. The PRV gene products were consistently detected in trigeminal ganglia and tonsils of all pigs at 1, 10, and 16 weeks after infection, and sporadically in the nasal mucosa, lymph nodes, and lungs of pigs that were euthanatized or died during the first 2 weeks after infection. Cells collected biweekly from tonsillar surfaces were mostly nucleated, squamous epithelial cells with fewer lymphocytes and neutrophils. Polymerase chain reaction analysis of DNA extracted from these cells revealed PRV DNA in a large proportion of the samples when sufficient cells were collected to provide 1 microgram of extracted DNA for use in the reaction mixtures. A second group of pigs had PRV strain 4892 intranasally instilled. The virus was isolated from tonsillar swab specimens until 3 weeks after infection. Tonsillar brushing specimens were collected biweekly until 14 weeks after infection.(ABSTRACT TRUNCATED AT 250 WORDS)

An IgG1 mouse monoclonal antibody, designated 1.646, is described which recognizes a cytoplasmic antigen of equine mononuclear phagocytes. Indirect fluorescent antibody staining of peripheral blood leukocytes reveals a granular cytoplasmic staining, predominantly in adherent blood mononuclear cells. Indirect fluorescent antibody staining is positive for alveolar and peritoneal macrophages. In some horses, a few neutrophils are also stained. In equine tissue samples stained by immunohistochemistry, the distribution of positive cells is consistent with the distribution of tissue macrophages. The most intense and reliable staining occurs with splenic and lymph node macrophages. Hepatic Kupffer cells also stain with antibody 1.646, although the intensity of that staining is somewhat variable between horses. A granular pattern of staining typical of lipofuscin deposition is also seen in liver sections. There is also pale staining of some biliary and renal tubular epithelium. Equine erythrocytes, platelets and lymphocytes are not recognized by this antibody, and neither are monocyte/macrophages of human, canine or feline origin. Antibody 1.646 recognizes two proteins (150 and 30 kDa) of equine monocyte-derived macrophages when assayed by Western immunoblot. Because of the distribution of staining (tissue mononuclear phagocytes, lipofuscin-containing storage granules, biliary and renal tubular epithelium, and some neutrophils) we hypothesize that antibody 1.646 recognizes a cytoplasmic antigen that is closely associated with lysosomal membranes.

The putative dUTPase domain was deleted from the polymerase (pol) gene of equine infectious anemia virus (EIAV) to produce a recombinant delta DUpol Escherichia coli expression cassette and a delta DU proviral clone. Expression of the recombinant delta DUpol polyprotein yielded a properly processed and enzymatically active reverse transcriptase, as determined by immunoblot analysis and DNA polymerase activity gels. Transfection of delta DU provirus into feline (FEA) cells resulted in production of virus that replicated to wild-type levels in both FEA cells and fetal equine kidney cells. In contrast, the delta DU virus replicated poorly (less than 1% of wild-type levels) in primary equine macrophage cultures, as measured by reverse transcriptase assays. Preparations of delta DU virus contained negligible dUTPase activity, which confirms that virion-associated dUTPase is encoded in the pol gene region between the RNase H domain and integrase, as has been demonstrated previously for feline immunodeficiency virus (J. H. Elder, D. L. Lerner, C. S. Hasselkus-Light, D. J. Fontenot, E. Hunter, P. A. Luciw, R. C. Montelaro, and T. R. Phillips, J. Virol. 66:1791-1794, 1992). Our results suggest that virus-encoded dUTPase is dispensable for virus replication in dividing cells in vitro but may be required for efficient replication of EIAV in nondividing equine macrophages, the natural host cells for this virus.

In situ hybridization of tissues from two horses infected with the wild-type Wyoming strain of equine infectious anemia virus (EIAV) identified the liver, spleen, lymph nodes, kidney, lung, and adrenal gland as the primary host tissue sites for viral transcription during acute infection. Combined immunohistochemistry, with a monoclonal antibody recognizing a cytoplasmic antigen of equine mononuclear phagocytes, and in situ hybridization for viral RNA identified most infected cells as mature tissue macrophages. In contrast, in situ hybridization of adherent peripheral blood mononuclear cells collected from horses on various days during the first 2 weeks postinfection with the Wyoming strain of EIAV failed to detect any viral RNA in these cells. For the two horses described here, serum reverse transcriptase activity correlated directly with the degree of replication detected in tissue macrophages on the day of sacrifice. These results suggest that unlike other lentivirus infections in which mature tissue macrophages accumulate cytoplasmic viral RNA to a high level but fail to produce infectious virions, mature tissue macrophages are the likely primary source of the high titer of viremia present during acute infection with EIAV. No significant posttranscriptional block of viral replication in tissue macrophages appears to occur with EIAV.

The complete nucleotide sequence of the sixth largest segment of ssRNA (RNA-6) of the tick-borne orthomyxo-like Dhori/India/1313/61 virus was determined by using cloned cDNA derived from infected cell mRNA and dideoxynucleotide sequencing of viral RNA. RNA-6 contains 962 nucleotides and is predicted to encode a protein of 270 amino acids with an M(r) of 30,498 in its first open reading frame (ORF). This protein is likely to represent the viral membrane (M1) protein, based on its predicted M(r) of 29,000 (estimated by PAGE), its relatively high abundance in infected cells and amino acid composition analysis. In addition, a second ORF was found which overlaps the M1 protein gene sequence by 327 nucleotides. This additional reading frame, in the +3 frame, potentially can encode a protein of 141 amino acids. However, S1 nuclease analysis of RNA-6 mRNA from infected cells indicated that there was only a single abundant RNA species corresponding in size to the full-length genomic RNA (0.96 kb). Further studies are needed to determine whether expression of the second ORF occurs and how that expression might arise.

An adult horse infected with a virulent, cell culture-adapted strain of equine infectious anemia virus (EIAV) developed cyclical thrombocytopenia in which the nadir of platelet counts coincided with peak febrile responses. In order to investigate the mechanism of thrombocytopenia during acute febrile episodes, four adult horses were experimentally infected with the wild-type Wyoming strain of EIAV. Platelet counts decreased from baseline as rectal temperature increased. Serum reverse transcriptase activity increased above background levels in all horses, coincident with increase in rectal temperature. All horses developed an EIAV-specific immune response detectable by Western immunoblot by postinfection day 10. Increases in platelet-associated immunoglobulins G and M were detectable by direct fluorescent-antibody test and flow cytometric assay. Viral replication in bone marrow megakaryocytes was not detectable by in situ hybridization. Results suggest an immune-mediated mechanism of thrombocytopenia in horses infected with EIAV. Despite an inability to identify virion particles in association with platelet-bound antibody, the cyclical nature of the thrombocytopenia and the occurrence of a marked cell-free viremia concomitant with fever and thrombocytopenia suggest immune complex deposition on platelets. We propose that clearance of virus and antibody-coated platelets from the peripheral circulation by hepatic Kupffer cells and splenic macrophages may target infectious virus particles, in the form of immune complexes, to host cells most permissive for in vivo viral replication.

The complete nucleotide sequence of the second largest RNA segment of Dhori/India/1313/61 virus was determined and the deduced amino acid sequence was compared with the polymerase (P) proteins of influenza A, B, and C viruses. RNA segment 2 (2224 nucleotides) of Dhori virus contains a single long open reading frame that can encode a 716-amino acid polypeptide (81.3 kDa). The predicted polypeptide shares between 27 and 31% sequence identities with the PB1 polypeptides of influenza A, B, and C viruses. Among the regions most highly conserved are the sequences around the Asp-Asp motif common to many RNA polymerases. In spite of the high level of sequence identity between the Dhori RNA segment 2 gene product and the influenza A, B, and C virus PB1 proteins the amino acid composition of the Dhori protein indicates an acidic charge feature at pH 7.0 in contrast to the basic nature of the PB1 proteins of the influenza viruses. We suggest that the Dhori PB1-like protein be designated the P alpha protein of this virus.

The complete nucleotide sequence of the fourth largest segment of single-stranded RNA of the tick-borne, orthomyxo-like Dhori/Indian/1313/61 virus was determined by using cloned cDNA derived from infected cell mRNA. The fourth RNA contains 1586 nucleotides and can code for a protein of 521 amino acids with a molecular weight of 58,675 Da. The predicted polypeptide possesses an amino-terminal hydrophobic region that may function as a signal sequence to initiate translocation across the endoplasmic reticulum membrane and a carboxyl-terminal hydrophobic region that could serve as a stop transfer sequence for anchoring this protein in the membrane. The envelope protein of Dhori virus does not display significant amino acid sequence homology with any of the envelope proteins (hemagglutinin or neuraminidase) of the influenza virus family members (or any other virus group) suggesting that the Dhori envelope protein is unique and may at least in part account for its divergent biological properties with other orthomyxoviruses.

Infection 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.

We have shown that a single defective-interfering (DI) particle of early (5th) passage Sindbis virus induces maximal amounts of interferon in an 'aged' primary chick embryo cell. The capacity of such DI particles to induce interferon is inactivated by small amounts of u.v. radiation (i/e dose = 232 ergs/mm2). The I/e dose for inactivation of the interferon-inducing capacity of infectious virus particles is 399 ergs/mm2 and for infectivity is 101 ergs/mm2. Pre-treatment with interferon blocks formation of interferon in response to either DI or infectious virus particles. Our results suggest that Sindbis virus genes must be expressed to form the interferon inducer, which is presumably a molecule of double-stranded (ds)RNA. We postulate that for interferon induction, the genomic RNA which codes for genes G and A must be translated into products whose concerted action produces a dsRNA molecule upon synthesis of a segment of RNA complementary to the genome. The RNA from early passage DI particles is sufficiently large (25S, 1.6 x 10(6) mol. wt.) to accommodate these genes, whereas the RNA from the late passage DI particles (20S, 1.0 x 10(6) mol. wt.) is not. Late (15th) passage DI particles do not induce interferon formation.

Four lambs and 3 calves, seronegative to bluetongue virus (BTV), were inoculated intravenously with a highly plaque-purified strain of BTV Serotype 10. A single calf and lamb served as controls and were inoculated with uninfected cell culture lysate. All BTV-inoculated lambs exhibited mild clinical manifestations of bluetongue, whereas infected calves were asymptomatic. Viremia persisted in BTV-infected lambs for 35-42 days, and for 42-56 days in BTV-infected calves. Neutralizing antibodies were first detected in sera collected at Day 14 post-inoculation (PI) from 2 BTV-infected calves and all 4 infected lambs, and at Day 28 PI in the remaining calf. The appearance of neutralizing antibody in serum did not coincide with clearance of virus from blood; BTV and specific neutralizing antibody coexisted in peripheral blood of infected lambs and calves for as long as 28 days. The sequential development, specificity and intensity of virus protein-specific humoral immune responses of lambs and calves were evaluated by immunoprecipitation of [35S]-labelled proteins in BTV-infected cell lysates by sera collected from inoculated animals at bi-weekly intervals PI. Sera from infected lambs and calves reacted most consistently with BTV structural proteins VP2 and VP7, and nonstructural protein NS2, and less consistently with structural protein VP5, and nonstructural protein NS1. Lambs developed humoral immune responses to individual BTV proteins more rapidly than calves, and one calf had especially weak virus protein-specific humoral immune responses; viremia persisted longer in this calf than any other animal in the study. The clearance of virus from the peripheral blood of BTV-infected lambs and calves is not caused simply by the production of virus-specific neutralizing antibody, however the intensity of humoral immune responses to individual BTV proteins might influence the duration of viremia in different animals.

We have measured the amounts of interferon formed by chick cells 'aged' in vitro in response to different amounts of infectious wild-type Sindbis virus. Our results suggest that one plaque-forming unit is enough to induce maximum interferon formation. With higher m.o.i. the yield of interferon is less. To inactivate the interferon-inducing activity of Sindbis virus, four times more u.v.-radiation was needed than to inactivate the infectivity of the virus. This suggests that only 25% of the virus genome need be intact in order to induce interferon. Temperature-sensitive Sindbis virus mutants from the three RNA+ complementation groups, C. D and E, gave rise to interferon in chick cells incubated at a non-permissive temperature, Similarly, mutants from two of the RNA- groups, B and F, gave rise to interferon, but not mutants from groups G and A. We conclude that no pre-formed inducer of interferon is present in Sindbis virus. It appears, however, that genes G and A represent a special one-quarter of the genome which must be functional in order to synthesize an interferon-inducing moiety. We suggest that this moiety is a double-stranded RNA molecule formed after synthesis of a segment of RNA complementary to the genome.

The envelope glycoproteins of Newcastle disease virus (NDV), hemagglutinin-neuraminidase (HN) and fusion (F) proteins, play important roles in determining the host immune response and the virulence of that particular virus strain. The complete nucleotide sequence of the HN and F genes of a highly neurovirulent strain of NDV (Texas G. B., 1948) was determined in an effort to study the molecular basis of this strain's neurotropic properties. Comparison of the predicted amino acid sequences for the HN and F among the American NDV strains revealed that the Texas G. B. and Beaudette C envelope genes are closely related to each other and are less closely related to the avirulent B1 Hitchner strain. We have found 11 amino acid changes in the predicted HN protein between the Beaudette C and Texas G. B. strain but only 2 conservative amino acid changes (amino acids 11 and 197) in the F protein between these two strains. Although the virulence of NDV strains has been related to sequences at the cleavage site of F0, the property of neurovirulence cannot depend solely upon these sequences because there are no sequence differences between the Beaudette C and Texas G. B. strains. We suggest that the neurovirulence phenotype could be due to the molecular properties of the HN protein; however, we cannot exclude the possibility that the two conservative amino acid differences between the two F proteins could also play a role in determining the phenotypic differences between these two virus strains.

Newborn calves were inoculated IV with highly plaque-purified bluetongue virus (BTV), serotype 10. The electrophoretic migration patterns of RNA segments and proteins of viruses isolated from calves at intervals after inoculation were compared. In addition, sera collected from calves at intervals after inoculation were compared for their abilities to neutralize several virus isolates from the same calf. Viremia persisted in calves for up to 56 days. Differences were not detected in the electrophoretic migration pattern of RNA segments or proteins of any of the BTV isolates. All calves produced high titers of neutralizing antibody to the original BTV inoculum by 28 days after inoculation, and significant (greater than or equal to 4-fold) differences were not detected in the neutralizing titers of sera to viruses collected at intervals after inoculation. The plaque-purified strain of BTV appeared to be stable genetically in infected calves, and failure to demonstrate antigenic variation among isolates indicated that antigenic shift was not the mechanism that allowed viremia to persist in BTV-infected calves.

The complete nucleotide sequence of the fifth largest segment of single-stranded RNA of the tick-borne, orthomyxo-like Dhori/Ind/1313/61 virus was determined by using cloned cDNA derived from infected cell mRNA and dideoxynucleotide sequencing of viral RNA. The fifth RNA contains 1479 nucleotides and can code for a protein of 477 amino acids with a molecular weight of 53,679 Da. The RNA 5 protein of the Dhori/Ind/1313/61 virus possesses five short regions (16-26 amino acids) which share a high degree (50-59%) of amino acid sequence homology with a computer-aligned consensus sequence of the influenza nucleoprotein gene family. These and other structural features of the RNA 5 protein suggest that RNA 5 of Dhori viruses codes for the nucleoprotein. The data also suggest that Dhori viruses are orthomyxoviruses, but that they are more distantly related to the influenza viruses than type A, B, and C viruses are to each other.

Plasmids were removed from pathogenic Bordetella avium using a variety of treatments. The plasmid-cure rates depended on the treatment and isolate. Pathogenicity of B. avium in turkey poults was not altered by removal of plasmids.

Humoral immune responses of 7 calves to bluetongue virus (BTV) infection were evaluated by plaque-reduction assay and immunoblotting. Most readily interpretable results were obtained with the immunoblot assay when colostrum-deprived calves were used, and sera were reacted with proteins in partially purified extracts of BTV. Viremia persisted in calves for 35 to 56 days, and BTV coexisted in blood for several weeks with virus-specific neutralizing antibody. Calves developed antibody to virus protein 2, the major determinant of virus neutralization, at 14 to 28 days after inoculation; this time interval also coincided with the appearance of neutralizing antibody in serum. Virus clearance in BTV-infected calves did not coincide with humoral immune responses to protein 2 or other virion proteins.

A defective-interfering (DI) particle of Sindbis virus was generated from a ts mutant of RNA-complementation group A by serially undiluted passages at 30 degrees C. This mutant induced interferon at a permissive temperature (30 degrees C), but not at a non-permissive temperature (40.5 degrees C); it also expressed homotypic interference throughout the range 30 to 40.5 degrees C. This demonstrates for the first time in a DI particle a ts function, namely, the ability to induce interferon. In addition, our data provide further evidence that the RNA genome of a Sindbis DI particle can be translated within the cell. We postulate that the products of translation function to produce the putative inducer of interferon, namely a molecule of dsRNA.

The nucleotide sequence of the glycoprotein genes of fully virulent Sindbis virus and derived mutants that have reduced neurovirulence for neonatal mice (attenuated mutants) has been determined. A single amino acid difference, arginine instead of serine at position 114 of the mature E2 glycoprotein, distinguished the prototype attenuated mutant from its virulent wild-type parent. Virulent revertants of the attenuated mutant showed same-site reversion to the wild-type sequence. An identical single amino acid substitution, an arginine for the serine at E2 position 114, was found in a second independently selected attenuated mutant. The strains are characterized by genetic linkage between attenuation, accelerated penetration of baby hamster kidney cells, and efficient neutralization by the E2-specific monoclonal antibodies R6 and R13; selection for change in one property simultaneously selected for change in the other two (Olmsted, R. A., Baric, R. S., Sawyer, B. A. & Johnston, R. E. (1984) Science 225, 424-427 and Olmsted, R. A., Meyer, W. J. & Johnston, R. E. (1986) Virology 148, 1-10). The nucleotide sequence data suggest that a single mutation in the E2 gene is sufficient to cause these coordinate phenotypic changes. These findings identify a single locus in a Sindbis virus surface glycoprotein gene that determines both efficiency of interaction with cultured baby hamster kidney cells and degree of virulence in neonatal mice.