Canine H3N8 influenza virus infection in dogs and mice.

An H3N8 influenza virus closely related to equine influenza virus was identified in racing greyhound dogs with respiratory disease in 2004 and subsequently identified in shelter and pet dogs. Pathologic findings in dogs spontaneously infected with canine influenza virus were compared with lesions induced in beagle and mongrel dogs following experimental inoculation with influenza A/canine/Florida/43/2004. BALB/c mice were inoculated with canine influenza virus to assess their suitability as an experimental model for viral pathogenesis studies. All dogs inoculated with virus developed necrotizing and hyperplastic tracheitis and bronchitis with involvement of submucosal glands as well as mild bronchiolitis and pneumonia. Viral antigen was identified in bronchial and tracheal epithelial cells of all dogs and in alveolar macrophages of several dogs. Many dogs that were spontaneously infected with virus also developed bacterial pneumonia, and greyhound dogs with fatal spontaneous infection developed severe pulmonary hemorrhage with hemothorax. Virus-inoculated BALB/c mice developed tracheitis, bronchitis, bronchiolitis, and mild pneumonia in association with viral antigen in airway epithelial cells and in type 2 alveolar epithelial cells. Virus was not detected in extrarespiratory sites in any animals. The results indicate that canine influenza virus infection consistently induces acute tracheitis and bronchitis in dogs. Mice may be a useful model for some pathogenesis studies on canine influenza virus infection.

Delivery of epitopes by the Salmonella type III secretion system for vaccine development.

Avirulent strains of Salmonella typhimurium are being considered as antigen delivery vectors. During its intracellular stage in the host, S. typhimurium resides within a membrane-bound compartment and is not an efficient inducer of class I-restricted immune responses. Viral epitopes were successfully delivered to the host-cell cytosol by using the type III protein secretion system of S. typhimurium. This resulted in class I-restricted immune responses that protected vaccinated animals against lethal infection. This approach may allow the efficient use of S. typhimurium as an antigen delivery system to control infections by pathogens that require this type of immune response for protection.

Unusually High Mortality in Waterfowl Caused by Highly Pathogenic Avian Influenza A(H5N1) in Bangladesh.

Mortality in ducks and geese caused by highly pathogenic avian influenza A(H5N1) infection had not been previously identified in Bangladesh. In June-July 2011, we investigated mortality in ducks, geese and chickens with suspected H5N1 infection in a north-eastern district of the country to identify the aetiologic agent and extent of the outbreak and identify possible associated human infections. We surveyed households and farms with affected poultry flocks in six villages in Netrokona district and collected cloacal and oropharyngeal swabs from sick birds and tissue samples from dead poultry. We conducted a survey in three of these villages to identify suspected human influenza-like illness cases and collected nasopharyngeal and throat swabs. We tested all swabs by real-time RT-PCR, sequenced cultured viruses, and examined tissue samples by histopathology and immunohistochemistry to detect and characterize influenza virus infection. In the six villages, among the 240 surveyed households and 11 small-scale farms, 61% (1789/2930) of chickens, 47% (4816/10 184) of ducks and 73% (358/493) of geese died within 14 days preceding the investigation. Of 70 sick poultry swabbed, 80% (56/70) had detectable RNA for influenza A/H5, including 89% (49/55) of ducks, 40% (2/5) of geese and 50% (5/10) of chickens. We isolated virus from six of 25 samples; sequence analysis of the hemagglutinin and neuraminidase gene of these six isolates indicated clade 2.3.2.1a of H5N1 virus. Histopathological changes and immunohistochemistry staining of avian influenza viral antigens were recognized in the brain, pancreas and intestines of ducks and chickens. We identified ten human cases showing signs compatible with influenza-like illness; four were positive for influenza A/H3; however, none were positive for influenza A/H5. The recently introduced H5N1 clade 2.3.2.1a virus caused unusually high mortality in ducks and geese. Heightened surveillance in poultry is warranted to guide appropriate diagnostic testing and detect novel influenza strains.

No virological evidence for an influenza A - like virus in European bats.

New members of the influenza A virus genus have been detected recently in bats from South America. By molecular investigations, using a generic real-time RT-PCR (RT-qPCR) that detects all previously known influenza A virus subtypes (H1-H16) and a newly developed RT-qPCR specific for the South American bat influenza-like virus of subtype H17, a total of 1571 samples obtained from 1369 individual bats of 26 species from Central Europe were examined. No evidence for the occurrence of such influenza viruses was found. Further attempts towards a more comprehensive evaluation of the role of bats in the ecology and epidemiology of influenza viruses should be based on more intense monitoring efforts. However, given the protected status of bats, not only in Europe, such activities need to be embedded into existing pathogen-monitoring programs.

Induction of apoptosis and cleavage of poly(ADP-ribose) polymerase by cytopathic bovine viral diarrhea virus infection.

The Pestivirus bovine viral diarrhea virus (BVDV) causes the fatal diarrheal syndrome, mucosal disease, because of mutations in the viral genome which convert the common noncytopathic (ncp) BVDV into a cytopathic (cp) biotype. We examined the nature of the cytopathic effect of cp-BVDV in cultured bovine cells in order to accurately describe the process and to gain insight into the mechanism of cp-BVDV-induced cell death. The findings demonstrate that cells infected with cp-BVDV in vitro die by apoptosis, but cells infected with ncp-BVDV do not. Analysis of nuclear morphology by staining with fluorescent DNA dye and cpi-fluorescence microscopy showed chromatin condensation and margination in cells infected with cp-BVDV. Transmission electron microscopy (TEM) confirmed the condensation of chromatin, as well as cell shrinkage and generation of apoptotic bodies. The chromosomal DNA of cells infected with cp-BVDV undergoes fragmentation, generating the typical oligonucleosomal fragments commonly noted during apoptosis. The fragmented DNA was released from the nucleus to the cytoplasm, and eventually to the culture supernatant. Infection with cp-BVDV activates cellular proteases of the ICE family leading to cleavage of poly(ADP-ribose) polymerase (PARP), a nuclear enzyme implicated in genome maintenance. This demonstration that cp-BVDV kills cells by triggering apoptosis suggests the possibility that cp-BVDV is associated with a fatal disease by the acquisition of a new apoptosis-inducing activity. We consider BVDV to be an excellent model system for studies of the biological and medical relevance of apoptosis in viral infections.

Bovine viral diarrhea virus induced apoptosis correlates with increased intracellular viral RNA accumulation.

Non-cytopathic (NCP) and cytopathic (CP) parent-daughter pairs are often isolated from cattle with bovine viral diarrhea virus (BVDV) induced mucosal disease. Alignment of these pair genomes revealed that genetic changes in CP BVDV involve the NS2-3 coding region and correlate with expression of NS3. However, additional mutations are present elsewhere in the genomes of these natural pairs, precluding unambiguous mapping of this function to the NS2-3 region. To evaluate this phenomenon in identical genetic backgrounds, we have constructed an NCP isogenic pair of the NADL by deletion of the cIns from NS2 region. The levels of viral protein synthesis in infected cells revealed no marked difference between the CP and the isogenic NCP BVDV mutant. In contrast, RNA accumulation in cells infected with CP virus was up to 25 times higher than that in cells infected with NCP BVDV. No significant difference in growth kinetics and viral yields were observed between the CP BVDV and the isogenic NCP pair. Analyses of additional NCP/CP parent-daughter field BVDV isolates revealed a similar pattern of macromolecular synthesis, suggesting the generality of this phenomenon. These results implicate increased levels of RNA accumulation in CP BVDV infected cells, along with the production of NS3 as potential contributors to viral cytopathogenicity.

Identification of bovine viral diarrhea virus nonstructural polypeptide NS4B/P38.

The bovine pestivirus polyprotein is processed into at least 11 mature polypeptides. Previous studies with polyprotein region-specific antiserum raised against beta-galactosidase fusion proteins or synthetic peptides allowed the assignment of viral non-structural proteins to specific segments of the BVDV genome. However, the gene product from the NS4B/P38 region of the viral genome remained to be demonstrated directly. BVDV cDNA fragments predicted to encode part of NS4B/P38 (from codon 2521 to 2838 of the BVDV open reading frame) and a portion of NS5A/P58 (from codon 3008 to 3340) were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. Polyclonal rabbit antibodies prepared against each of the purified fusion proteins, GST-2521-2838 and GST-3008-3340, were used to immunoprecipitate viral polypeptides present in BVDV-infected cell lysates. Rabbit antiserum to GST-2521-2838 bound a polypeptide of 38 kDa identified as the mature NS4B/P38 polypeptide; while anti GST-3008-3340 lacked this specificity and bound NS5A/P58. Moreover, both antisera recognized a 96 kDa polypeptide, previously identified as a NS4B-NS5A/PP96 precursor. The function of the newly identified and highly conserved NS4B/P38 protein in viral replication remains to be determined.

Influence of host species on the evolution of the nonstructural (NS) gene of influenza A viruses.

The matrix (M) and nonstructural (NS) genes of influenza A viruses each encode two overlapping proteins. In the M gene, evolution of one protein affects that of the other. To determine whether or not this evolutionary influence operating between the two M proteins also occurs in the NS gene, we sequenced the NS genes of 36 influenza A viruses isolated from a broad spectrum of animal species (wild and domestic birds, horses, pigs, humans, and sea mammals) and analyzed them phylogenetically, together with other previously published sequences. These analyses enabled us to conclude the following host species-related points that are not found in the other influenza A virus genes and their gene products. (1) The evolution of the two overlapping proteins encoded by the NS gene are lineage-dependent, unlike the M gene where evolutionary constraints on the Ml protein affect the evolution of the M2 protein (Ito et al.. J. Virol. 65 (1991) 5491 5498). (2) The gull-specific lineage contained nonH13 gull viruses and the non-gull avian lineage contained H13 gull viruses, indicating that the gull-specific lineage does not link to the H13 HA subtype in the NS gene unlike findings with other genes. (3) The branching topology of the recent equine lineage (H7N7 viruses isolated after 1973 and H3N8) indicates recent introduction of the NS, M, and PB2 genes into horses from avian sources by genetic reassortment.

Presence of bovine viral diarrhea virus (BVDV) E2 glycoprotein in VSV recombinant particles and induction of neutralizing BVDV antibodies in mice.

We generated a recombinant vesicular stomatitis virus (VSV-E2) encoding the bovine viral diarrhea virus (BVDV) E2 glycoprotein with the VSV-G protein signal peptide. Infection of BHK21 cells with VSV-E2 induced the synthesis of a recombinant E2 (rE2) that comigrated with authentic BVDV-E2 in PAGE-SDS gels. Non-reducing immunoblots showed that rE2 is a disulfide bond-linked homodimer with at least 10-fold higher avidity for conformation-dependent anti-BVDV-E2 antibodies than its reduced monomeric counterpart. Immunofluorescence microscopy also showed that rE2 was transported to the plasma membrane of infected cells and analysis of purified particles demonstrated that dimeric rE2 was incorporated into VSV-E2 virions in approximately 1:10 ratio with respect to the G glycoprotein. BALB/c mice inoculated intranasally with VSV-E2 doses of up to 10(7) plaque forming units (pfu) showed no symptoms of viral-induced disease and developed a specific BVDV neutralizing response that lasted for at least 180 days post inoculation.

Bovine viral diarrhea virus-specific neutralizing antibodies induced by anti-idiotypic antibodies.

Two murine neutralizing monoclonal antibodies (MAbs), 4D8 and 6D11, recognizing epitopes on gp53, a surface glycoprotein of bovine viral diarrhea virus (BVDV), were used to generate anti-idiotypic antibodies (anti-ids) in a calf. The polyclonal anti-ids were isolated from serum by affinity chromatography on their respective Ab-1-Sepharose columns, followed by repeated adsorption on isotype-matched antibody-Sepharose columns. The anti-ids reacted specifically with their respective Ab-1, but not with isotype-matched controls. They also inhibited the binding of their Ab-1 to BVDV in a concentration-dependent manner. Mice immunized with the two anti-id preparations developed antibodies to BVDV, which neutralized the virus in vitro.

Bovine viral diarrhea virus proteins and their antigenic analyses.

Bovine Viral Diarrhea Virus (BVDV) polypeptides present in infected cells are the result of the processing of the polyprotein translated from the large single open reading frame of the BVDV genomic RNA. The presence of these proteins in infected cells was studied by radiolabeling under hypertonic conditions and with the aid of radioimmunoprecipitation. The genomic mapping of these polypeptides suggests a complex pattern of processing which involves cellular and viral proteases. The consistent absence of 80k in noncytopathic isolates of BVDV suggests that the processing of the viral polyprotein is different in cytopathic and noncytopathic biotypes of BVDV. The antigenic structure of BVDV was studied with a panel of monoclonal antibodies (MABs) prepared against the Singer isolate of BVDV. Neutralizing MABs were found to bind the 56-58k polypeptide, providing evidence that this glycoprotein is present on the surface of the virion and carries neutralization epitopes. Antigenic analyses with the panel of MABs reveals extensive antigenic heterogeneity among BVDV field isolates. MABs were used to determine the frequency of neutralization escape mutants in stocks of BVDV. Plaque-purified BVDV stocks contain neutralization escape mutants with a frequency of 10(-2.47).

Recombinant polypeptide from the gp48 region of the bovine viral diarrhea virus (BVDV) detects serum antibodies in vaccinated and infected cattle.

To characterize the immune response of cattle to bovine viral diarrhea virus (BVDV) glycoprotein gp48, we have produced a large amount of recombinant glutathione-s-transferase-gp48 (GST-gp48) fusion protein in Escherichia coli. Antibodies to gp48 were present in cattle vaccinated with killed or modified-live virus vaccination, or following natural infection. These results were in agreement with results of serum neutralization (SN) test which detected gp53 of BVDV.

Production and characterization of monoclonal antibodies to Brazilian isolates of bovine viral diarrhea virus.

Three Brazilian isolates of bovine viral diarrhea virus (BVDV), antigenically distinct from the standard North American isolates, were selected to immunize BALB/c mice in order to obtain hybridoma cells secreting anti-BVDV monoclonal antibodies (mAbs). Two hybridoma clones secreting mAbs, reacting specifically with BVDV-infected cells (mAbs 3.1C4 and 6.F11), were selected after five fusions and screening of 1001 hypoxanthine-aminopterin-thymidine-resistant clones. These mAbs reacted in an indirect fluorescent antibody (IFA) assay with all 39 South and North American BVDV field isolates and reference strains available in our laboratory, yet failed to recognize other pestiviruses, namely the hog cholera virus. The mAbs reacted at dilutions up to 1:25,600 (ascitic fluid) and 1:100 (hybridoma culture supernatant) in IFA and immunoperoxidase (IPX) staining of BVDV-infected cells but only mAb 3.1C4 neutralized virus infectivity. Furthermore, both mAbs failed to recognize BVDV proteins by IPX in formalin-fixed paraffin-embedded tissues and following SDS-PAGE and immunoblot analysis of virus-infected cells, suggesting they are probably directed to conformational-type epitopes. The protein specificity of these mAbs was then determined by IFA staining of CV-1 cells transiently expressing each of the BVDV proteins: mAb 3. 1C4 reacted with the structural protein E2/gp53 and mAb 6.F11 reacted with the structural protein E1/gp25. Both mAbs were shown to be of the IgG2a isotype. To our knowledge, these are the first mAbs produced against South American BVDV isolates and will certainly be useful for research and diagnostic purposes.

Experimental infection of calves with bovine viral diarrhea virus genotype II (NY-93).

To ascertain the virulence of bovine viral diarrhea virus (BVDV) genotype II, isolate NY-93 was inoculated intranasally into 3 calves, 2 of which were treated with a synthetic glucocorticoid prior to and after virus inoculation. Anorexia, fever (up to 42 C), dyspnea, and hemorrhagic diarrhea developed 6 days after intranasal inoculation with BVDV NY-93. The condition of all calves deteriorated further until the end of the study on day 14 postinoculation. The most significant postmortem macroscopic changes in all calves were limited to the gastrointestinal tract and consisted of moderate to severe congestion of the mucosa with multifocal hemorrhages. Microscopic lesions found in the gastrointestinal tract were similar to those observed in mucosal disease, including degeneration and necrosis of crypt epithelium and necrosis of lymphoid tissue throughout the ileum, colon, and rectum. The basal stratum of the epithelium of tongue, esophagus, and rumen had scattered individual necrotic cells. Spleen and lymph nodes had lymphocytolysis and severe lymphoid depletion. Severe acute fibrinous bronchopneumonia was present in dexamethasone-treated calves. Abundant viral antigen was detected by immunohistochemistry in the squamous epithelium of tongue, esophagus, and forestomachs. BVDV antigen was prominent in cells of the media of small arteries and endothelial cells. The presence of infectious virus in tissues correlated with an absence of circulating neutralizing antibodies. These findings highlight the potential of BVDV genotype II to cause severe disease in normal and stressed cattle.

Molecular specificity of the antibody responses of cattle naturally and experimentally infected with cytopathic and noncytopathic bovine viral diarrhea virus biotypes.

The specificity of the humoral IgG response of cattle naturally or experimentally infected with bovine viral diarrhea virus (BVDV) was studied by radioimmunoprecipitation. Serum samples were tested against radiolabeled lysates of cells infected with cytopathic and noncytopathic biotypes of BVDV. A biotype-specific serologic marker was not detected. The specificity of the IgG induced in cows naturally or experimentally infected with either BVDV biotype was essentially the same. A strong IgG response to the 2 glycoproteins (56 to 58 kilodaltons, [kD] and 48 kD) of both biotypes and to the major polypeptides was induced in infected cells: 118 kD and 80 kD by cytopathic BVDV and only 118 kD by noncytopathic BVDV. The most consistent difference among cattle was the presence of IgG specific for the 37-kD polypeptide. Sequential serum sample collection after spontaneous and induced infections with either BVDV biotype did not indicate specific IgG markers for determining infection history. Sera from cattle with a confirmed diagnosis of mucosal disease and lacking neutralizing antibodies to BVDV usually lacked (greater than 80%) nonneutralizing BVDV-specific IgG. One animal had substantial amounts of IgG to the 80-kD polypeptide. Other cattle had less readily detectable amounts of IgG specific for 80-kD or 37-kD viral polypeptides.

Characterization of a panel of monoclonal antibodies and their use in the study of the antigenic diversity of bovine viral diarrhea virus.

A panel of 40 monoclonal antibodies (MAb) specific for bovine viral diarrhea virus (BVDV) was produced, and each MAb was characterized and grouped according to its viral protein specificity, immunoglobulin subclass, virus-neutralizing activity, and immunoreactivity with a large collection of BVDV isolates. The MAb were found to be specific for 1 of 3 sets of related viral-induced proteins found in cells infected with the Singer strain of BVDV. Group-1 MAb were specific for the 80- and 118-kilodalton (kD) proteins of BVDV. Group-2 MAb recognized 3 proteins with molecular sizes of 54, 56, and 58 kD. Group-3 MAb recognized a 43- and a 65-kD protein. The MAb belonged to either the IgG1, IgG2a, IgG2b, IgG3 subclasses or the IgE class of mouse immunoglobulin. All MAb in group 2 were able to neutralize BVDV and had neutralization titers that ranged from 24 to 1,600,000. The reactivity of the MAb with numerous field isolates of BVDV was highly variable. Both cytopathic and noncytopathic biotypes of BVDV were examined and had the same degree of antigenic variation. The greatest degree of variation was detected with group-2 MAb. The data demonstrate that BVDV isolates have a high degree of antigenic variation that is largely confined to the envelope glycoproteins associated with virus neutralization. The results also suggest that antigenic variability of this virus is important in the development and severity of the disease it causes.

Herd problem of abortions and malformed calves attributed to bovine viral diarrhea.

A herd problem of abortions and the birth of malformed calves developed subsequent to a transient febrile illness in adult cows. The herd was not vaccinated against bovine viral diarrhea (BVD). Bovine viral diarrhea virus was isolated from blood of one calf. The problem continued for 8 months. The entire herd was vaccinated with a killed BVD virus product. No further problems developed. This herd problem illustrates one of the many recognized clinical manifestations of BVD.

Molecular biology of bovine viral diarrhea virus and its interactions with the host.

The contributions of pestivirus molecular biology research to our understanding of Bovine Viral Diarrhea Virus (BVDV) biology and disease have been remarkable. Completion of nucleotide sequence information for genomes of NCP and CP-BVDV isolates was an important milestone. Subsequent work on the protein map of BVDV and polyprotein processing pathways paved the way for the interpretation of many other virologic and immunologic studies. Discovery of a correlation between genotype II and virulence (hemorrhagic syndrome) will help to clarify previously controversial data and to improve disease control. Description of multiple pathways of p80 expression in CP-BVDV offered insight into the pathogenesis of mucosal disease. Identification of gp53/ E2 as the target of neutralizing antibodies and source of antigenic hypervariability helped us to understand immunity to BVDV. Collectively, the advances described contribute to the implementation of improved diagnostic and control strategies to reduce losses inflicted by the bovine pestivirus.

Review of influenza A virus in swine worldwide: a call for increased surveillance and research.

Pigs and humans have shared influenza A viruses (IAV) since at least 1918, and many interspecies transmission events have been documented since that time. However, despite this interplay, relatively little is known regarding IAV circulating in swine around the world compared with the avian and human knowledge base. This gap in knowledge impedes our understanding of how viruses adapted to swine or man impacts the ecology and evolution of IAV as a whole and the true impact of swine IAV on human health. The pandemic H1N1 that emerged in 2009 underscored the need for greater surveillance and sharing of data on IAV in swine. In this paper, we review the current state of IAV in swine around the world, highlight the collaboration between international organizations and a network of laboratories engaged in human and animal IAV surveillance and research, and emphasize the need to increase information in high-priority regions. The need for global integration and rapid sharing of data and resources to fight IAV in swine and other animal species is apparent, but this effort requires grassroots support from governments, practicing veterinarians and the swine industry and, ultimately, requires significant increases in funding and infrastructure.

Real-time RT-PCR assay to differentiate clades of H5N1 avian influenza viruses circulating in Vietnam.

Continued circulation and geographical expansion of highly pathogenic avian influenza H5N1 virus have led to the emergence of numerous clades in Vietnam. Although viral RNA sequencing and phylogenetic analysis are the gold standard for H5N1 HA clade designation, limited sequencing capacity in many laboratories precludes rapid H5N1 clade identification and detection of novel viruses. Therefore, a Taqman real-time RT-PCR assay for rapid differentiation of the four major H5N1 clades detected in Vietnam was developed. Using HA sequence alignments of clades 1.1, 2.3.2.1, 2.3.4, and 7 viruses, primers and FAM-labeled probes were designed to target conserved regions characteristic of each clade. The assay was optimized and evaluated using circulating clades of H5N1 collected in Vietnam from 2007 to 2012 and shown to be both sensitive and specific for the differentiation of the four H5N1 clades. The assay provides a useful tool for screening of large specimen collections for HA gene sequencing and phylogenetic analysis and for the rapid identification of molecular clade signatures to support outbreak investigations and surveillance activities. Finally, this assay may be useful to monitor for the emergence of novel or variant clades of H5N1 in Vietnam in the future or in other countries where these particular clades may circulate.