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Emerging canine coronavirus (CCoV) variants that are associated with systemic infections have been reported in the European Union; however, CCoV-associated disease in the United States is incompletely characterized. The purpose of this study was to correlate the clinicopathological findings and viral antigen distribution with the genotypic characteristics of CCoV in 11 puppies from nine premises in five states that were submitted for diagnostic investigation at Cornell University between 2008 and 2013. CCoV antigen was found in epithelial cells of small intestinal villi in all puppies and the colon in 2 of the 10 puppies where colon specimens were available. No evidence of systemic CCoV infection was found. Comparative sequence analyses of viral RNA extracted from intestinal tissues revealed CCoV-II genotype in 9 out of 11 puppies. Of the nine CCoV-IIs, five were subtyped as group IIa and one as IIb, while three CCoVs could not be subtyped. One of the CCoV-IIa variants was isolated in cell culture. Infection with CCoV alone was found in five puppies, of which two also had small intestinal intussusception. Concurrent infections with either parvovirus ( n = 1), attaching-effacing Escherichia coli ( n = 4), or protozoan parasites ( n = 3) were found in the other six puppies. CCoV is an important differential diagnosis in outbreaks of severe enterocolitis among puppies between 4 days and 21 weeks of age that are housed at high population density. These findings will assist with the rapid laboratory diagnosis of enteritis in puppies and highlight the need for continued surveillance for CCoV variants and intestinal viral diseases of global significance.

Canine pneumovirus (CnPnV) was recently identified during a retrospective survey of kenneled dogs in the United States. In this study, archived samples from pet and kenneled dogs in the United Kingdom were screened for CnPnV to explore the relationship between exposure to CnPnV and the development of canine infectious respiratory disease (CIRD). Within the pet dog population, CnPnV-seropositive dogs were detected throughout the United Kingdom and Republic of Ireland, with an overall estimated seroprevalence of 50% ( n = 314/625 dogs). In the kennel population, there was a significant increase in seroprevalence, from 26% ( n = 56/215 dogs) on the day of entry to 93.5% ( n = 201/215 dogs) after 21 days ( P P < 0.001), and dogs with preexisting antibodies to CnPnV on the day of entry were significantly less likely to develop respiratory disease than immunologically naive dogs ( P < 0.001). CnPnV was detected in the tracheal tissues of 29/205 kenneled dogs. Detection was most frequent in dogs with mild to moderate respiratory signs and histopathological changes and in dogs housed for 8 to 14 days, which coincided with a significant increase in the risk of developing respiratory disease compared to the risk of those housed 1 to 7 days ( P < 0.001). These findings demonstrate that CnPnV is present in the United Kingdom dog population; there is a strong association between exposure to CnPnV and CIRD in the kennel studied and a potential benefit in vaccinating against CnPnV as part of a wider disease prevention strategy.

Although parvoviruses are commonly described in domestic carnivores, little is known about their biodiversity in nondomestic species. A phylogenetic analysis of VP2 gene sequences from puma, coyote, gray wolf, bobcat, raccoon, and striped skunk revealed two major groups related to either feline panleukopenia virus (“FPV-like”) or canine parvovirus (“CPV-like”). Cross-species transmission was commonplace, with multiple introductions into each host species but, with the exception of raccoons, relatively little evidence for onward transmission in nondomestic species.

Understanding the mechanisms of cross-species virus transmission is critical to anticipating emerging infectious diseases. Canine parvovirus type 2 (CPV-2) emerged as a variant of a feline parvovirus when it acquired mutations that allowed binding to the canine transferrin receptor type 1 (TfR). However, CPV-2 was soon replaced by a variant virus (CPV-2a) that differed in antigenicity and receptor binding. Here we show that the emergence of CPV involved an additional host range variant virus that has circulated undetected in raccoons for at least 24 years, with transfers to and from dogs. Raccoon virus capsids showed little binding to the canine TfR, showed little infection of canine cells, and had altered antigenic structures. Remarkably, in capsid protein (VP2) phylogenies, most raccoon viruses fell as evolutionary intermediates between the CPV-2 and CPV-2a strains, suggesting that passage through raccoons assisted in the evolution of CPV-2a. This highlights the potential role of alternative hosts in viral emergence.

Many of our fatal “civilization” infectious diseases have arisen from domesticated animals. Although picornaviruses infect most mammals, infection of a companion animal is not known. Here we describe the identification and genomic characterization of the first canine picornavirus. Canine kobuvirus (CKoV), identified in stool samples from dogs with diarrhea, has a genomic organization typical of a picornavirus and encodes a 2,469-amino-acid polyprotein flanked by 5′ and 3′ untranslated regions. Comparative phylogenetic analysis using various structural and nonstructural proteins of CKoV confirmed it as the animal virus homolog most closely related to human Aichivirus (AiV). Bayesian Markov chain Monte Carlo analysis suggests a mean recent divergence time of CKoV and AiV within the past 20 to 50 years, well after the domestication of canines. The discovery of CKoV provides new insights into the origin and evolution of AiV and the species specificity and pathogenesis of kobuviruses.

Canine influenza virus (CIV) emerged around 2000 when an equine influenza virus (EIV) was transmitted to dogs in Florida. After 2003, the canine virus was carried by infected greyhounds to various parts of the United States and then became established in several large animal shelters, where it has continued to circulate. To better understand the evolution of CIV since its emergence, and particularly its microevolution in spatially restricted populations, we examined multiple gene segments of CIV from dogs resident in two large animal shelters in New York City during the period 2006 to 2009. In particular, we focused on viruses circulating in the two shelters in 2008 and 2009, which we found shared a common ancestor. While viruses in each shelter were generally monophyletic, we observed some gene flow between them. These shelter sequences were compared to earlier CIV isolates. The shelter viruses differed in 1 to 6 amino acids in each gene segment compared to viruses isolated in Florida between 2003 and 2005 and in Colorado in 2006 and 2008. A comparison of the sequences of equine and canine viruses revealed amino acid replacements that distinguished the viruses from the two hosts, but no clear evidence of positive selection indicative of host adaptation was detected, suggesting that any host range adaptation in CIV occurred early in the emergence of this virus or even before it transferred to dogs.

The patterns and dynamics of evolution in acutely infecting viruses within individual hosts are largely unknown. To this end, we investigated the intrahost variation of canine influenza virus (CIV) during the course of experimental infections in naïve and partially immune dogs and in naturally infected dogs. Tracing sequence diversity in the gene encoding domain 1 of the hemagglutinin (HA1) protein over the time course of infection provided information on the patterns and processes of intrahost viral evolution and revealed some of the effects of partial host immunity. Viral populations sampled on any given day were generally characterized by mean pairwise genetic diversities between 0.1 and 0.2% and by mutational spectra that changed considerably on different days. Some observed mutations may have affected antigenicity or host range, including reversions of CIV host-associated mutations. Patterns of sequence diversity differed between naïve and vaccinated dogs, with some presumably antigenic mutations transiently reaching high frequency in the latter. CIV populations are therefore characterized by the rapid generation and clearance of genetic diversity. Potentially advantageous mutations arise readily during the course of single infections and may give rise to antigenic escape or host range variants.

The objective of this study was to develop and evaluate new TaqMan real-time reverse transcription-PCR (rRT-PCR) assays by the use of the minor groove binding probe to detect a wide range of equine influenza virus (EIV) strains comprising both subtypes of the virus (H3N8 and H7N7). A total of eight rRT-PCR assays were developed, targeting the nucleoprotein (NP), matrix (M), and hemagglutinin (HA) genes of the two EIV subtypes. None of the eight assays cross-reacted with any of the other known equine respiratory viruses. Three rRT-PCR assays (EqFlu NP, M, and HA3) which can detect strains of the H3N8 subtype were evaluated using nasal swabs received for routine diagnosis and swabs collected from experimentally inoculated horses. All three rRT-PCR assays have greater specificity and sensitivity than virus isolation by egg inoculation (93%, 89%, and 87% sensitivity for EqFlu NP, EqFlu M, and EqFlu HA3 assays, respectively). These assays had analytical sensitivities of ≥10 EIV RNA molecules. Comparison of the sensitivities of rRT-PCR assays targeting the NP and M genes of both subtypes with egg inoculation and the Directigen Flu A test clearly shows that molecular assays provide the highest sensitivity. The EqFlu HA7 assay targeting the H7 HA gene is highly specific for the H7N7 subtype of EIV. It should enable highly reliable surveillance for the H7N7 subtype, which is thought to be extinct or possibly still circulating at a very low level in nature. The assays that we developed provide a fast and reliable means of EIV diagnosis and subtype identification of EIV subtypes.

In this investigation a population of dogs at a rehoming center was monitored over a period of 2 years. Despite regular vaccination of incoming dogs against distemper, canine adenovirus type 2 (CAV-2), and canine parainfluenza virus (CPIV), respiratory disease was endemic. Tissue samples from the respiratory tract as well as paired serum samples were collected for analysis. The development of PCR assays for the detection of CPIV, canine adenovirus types 1 and 2, and canine herpesvirus (CHV) is described. Surprisingly, canine adenovirus was not detected in samples from this population, whereas 19.4% of tracheal and 10.4% of lung samples were positive for CPIV and 12.8% of tracheal and 9.6% of lung samples were positive for CHV. As reported previously, a novel canine respiratory coronavirus (CRCoV) was detected in this population (K. Erles, C. Toomey, H. W. Brooks, and J. Brownlie, Virology 310: 216-223, 2003). Infections with CRCoV occurred mostly during the first week of a dog's stay at the kennel, whereas CPIV and CHV were detected at later time points. Furthermore, the evaluation of an enzyme-linked immunosorbent assay for detection of antibodies to CPIV and an immunofluorescence assay for detection of antibodies to CHV is described. This study shows that CPIV is present at kennels despite vaccination. In addition, other agents such as CHV and CRCoV may play a role in the pathogenesis of canine respiratory disease, whereas CAV-2 and canine distemper virus were not present in this population, indicating that their prevalence in the United Kingdom is low due to widespread vaccination of dogs.

Cattle immunotolerant to and persistently infected (PI) with bovine viral diarrhea (BVD) virus (BVDV) constitute the mechanism by which BVDV persists in cattle herds. Two procedures for using serum to detect PI cattle were developed and evaluated. BVDV was found to remain viable for 7 days in serum samples stored at room temperature. The tests use cell culture virus isolation (VI) in 96-well microtiter plates, followed by immunostaining of cell monolayers with monoclonal antibodies. One technique, the immunoperoxidase monolayer assay (IPMA), forms a red intracellular precipitate while the other, the monolayer enzyme-linked immunosorbent assay (M-ELISA) produces a yellow color in solution. The optimal incubation period for microtiter VI was determined to be 4 days. Optimal IPMA staining was obtained by fixing cell monolayers with 20 to 30% acetone, whereas a simple dry-rehydrate-dry cycle provided optimal M-ELISA staining. The M-ELISA and IPMA had the same sensitivities and specificities, but the M-ELISA was a more rapid procedure and use of a spectrophotometer for reading samples allowed for greater objectivity. When compared to standard VI with routine samples submitted for the diagnosis of BVD, M-ELISA and IPMA had a relative sensitivity of 85% and a relatively specificity of 100%. When only samples from cattle suspected of being PI were considered, these two parameters were 100% for both IPMA and M-ELISA. The two procedures, especially the M-ELISA, are suitable for whole-herd testing to identify PI cattle. The appeal of these tests is derived from the convenience of using serum as a diagnostic sample and the ability to rapidly screen large numbers of samples at low cost.

Genetic and biological characterization of new hepaciviruses infecting animals contributes to our understanding of the ultimate origins of hepatitis C virus (HCV) infection in humans and dramatically enhances our ability to study its pathogenesis using tractable animal models. Animal homologs of HCV include a recently discovered canine hepacivirus (CHV) and GB virus B (GBV-B), both viruses with largely undetermined natural host ranges. Here we used a versatile serology-based approach to determine the natural host of the only known nonprimate hepacivirus (NPHV), CHV, which is also the closest phylogenetic relative of HCV. Recombinant protein expressed from the helicase domain of CHV NS3 was used as antigen in the luciferase immunoprecipitation system (LIPS) assay to screen several nonprimate animal species. Thirty-six samples from 103 horses were immunoreactive, and viral genomic RNA was present in 8 of the 36 seropositive animals and none of the seronegative animals. Complete genome sequences of these 8 genetically diverse NPHVs showed 14% (range, 6.4% to 17.2%) nucleotide sequence divergence, with most changes occurring at synonymous sites. RNA secondary structure prediction of the 383-base 5′ untranslated region of NPHV was refined and extended through mapping of polymorphic sites to unpaired regions or (semi)covariant pairings. Similar approaches were adopted to delineate extensive RNA secondary structures in the coding region of the genome, predicted to form 27 regularly spaced, thermodynamically stable stem-loops. Together, these findings suggest a promising new nonprimate animal model and provide a database that will aid creation of functional NPHV cDNA clones and other novel tools for hepacivirus studies.

Equine herpesvirus 1 (EHV-1) induces a variety of disease manifestations, including respiratory disease, abortions, and myeloencephalopathy. Several vaccines are commercially available but could not previously be distinguished by serologic testing from infection with EHV-1 (or the closely related EHV-4). Currently available vaccines are not reliably protective against the severe manifestations of the disease, including fatal myeloencephalopathy. We determined immunological parameters that can differentiate vaccinated from previously infected animals by comparing humoral and cellular EHV-1-specific responses in clinically healthy horses 10 months after vaccination. Forty-seven horses with known histories of vaccination and infection were studied, including a group of horses that survived a severe neurological outbreak 5 years prior to vaccination. Results of serum virus neutralization (SN), serum IgG isotyping, and cytokine profiling of lymphocyte subsets were compared. IgG4/7 levels strongly correlated with virus neutralization ( P < 0.0001). IgG1/3 and SN values distinguished vaccinated/outbreak-exposed (vacc/outbreak) horses from vaccinated horses ( P < 0.05). EHV-1-specific gamma interferon (IFN-γ)-producing CD4 + (but not CD8 + ) T-cell numbers were also increased in vacc/outbreak horses, which distinguished them from vaccinated horses ( P < 0.01). IFN-α secretion was similar between all groups and independent of previous exposure or vaccination. Our data suggest that IgG isotype responses to EHV-1 are more diverse under field conditions than is revealed by experimental studies and that the current modified-live virus (MLV) vaccine induces a more restricted IgG isotype response than does natural exposure to EHV-1. Since these parameters can be assessed in a high-throughput manner, they may prove useful in screening future vaccine candidates and assessing levels of protection.

A monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (S-ELISA) was developed for specific detection of peste des petits ruminants virus. Compared with virus isolation in Vero cell cultures using 89 paired tissue and secretion samples from six experimentally infected goats, S-ELISA was significantly more sensitive (71.9% versus 65.2%; P < 0.05). The S-ELISA is a suitable alternative to virus isolation.

A blocking enzyme-linked immunosorbent assay (B-ELISA), using two neutralizing monoclonal antibodies (MAbs), was established and compared with the virus neutralization test (VNT) for detecting specific peste-des-petits-ruminants virus (PPRV) antibody in caprine and ovine sera. This technique was developed because VNT, the only available specific serological test for PPRV and the cross-reactive rinderpest virus (RPV), is time-consuming and unaffordable for most laboratories in regions where both peste des petits ruminants and rinderpest occur. The test depends on the blocking of the binding of the MAb to a specific epitope in the presence of positive serum. Test conditions were optimized by using peste-des-petits-ruminants and rinderpest sera that were known to be VNT positive and negative. A blocking format, in which serum is preincubated with a solid-phase PPRV antigen and then incubated with the MAb, yielded levels of sensitivity and specificity superior to those of a competitive format, in which the two reagents are added simultaneously. A threshold value of 45% inhibition, representing the mean for a negative population (n = 277) plus 2.7 standard deviations, was adopted for routine screening. A total of 605 serum samples were screened by B-ELISA and the VNT. The sensitivity and specificity of B-ELISA relative to the VNT were 90.4 and 98.9%, respectively. Of 264 field serum samples tested, 11 (4.2%) could not be assayed by the VNT because of contamination or cytotoxicity; the overall agreement quotient between results of the two tests (n = 253) was 0.91. A high correlation (r>/=0.98) was observed between B-ELISA and the VNT for endpoint titration of sera (n=57). Because B-ELISA proved to be nearlyas sensitive and specific as the VNT while being simpler and more rapid, it would be an adequate substitute for the VNT for assessing herd immune status and for epidemiologic surveillance.

Purified defective interfering (DI) particles of vesicular stomatitis virus (VSV) inhibit the replication of a heterologous virus, pseudorabies virus (PSR), in hamster (BHK-21) and rabbit (RC-60) cell lines. In contrast to infectious B particles of VSV, UV irradiation of DI particles does not reduce their ability to inhibit PSR replication. However, UV irradiation progressively reduces the ability of DI particles to cause homologous interference with B particle replication. Pretreatment with interferon does not affect the ability of DI particles to inhibit PSR replication in a rabbit cell line (RC-60) in which RNA, but not DNA, viruses are sensitive to the action of interferon. Under similar conditions of interferon pretreatment, the inhibition of PSR by B particles is blocked. These data suggest that de novo VSV RNA or protein synthesis is not required for the inhibition of PSR replication by DI particles. DI particles that inhibit PSR replication also inhibit host RNA and protein synthesis in BHK-21 and RC-60 cells. Based on the results described and data in the literature, it is proposed that the same component of VSV B and DI particles is responsible for most, if not all, of the inhibitory activities of VSV, except homologous interference.

A panel of monoclonal antibodies that recognize the two major glycoproteins of bovine viral diarrhea virus (BDV) was used to evaluate the antigenic relationship between cytopathic (CP) and noncytopathic (NCP) viruses isolated from cattle dead or dying from fatal BDV infections. Various unrelated BDV isolates were initially screened by indirect immunofluorescence with monoclonal antibodies directed against the 56- to 58- and 48-kilodalton glycoproteins of the virus. A wide spectrum of reactivity that was independent of biotype was found. Biological clones of the same isolate showed only minor variations from the parental isolate, as did isolates taken from different animals located on the same farm. A similar analysis was repeated with pairs of CP and NCP viruses isolated from 16 unrelated clinical cases of BDV infection resulting in fatal disease. The reactivity patterns within individual pairs of isolates taken from the same animals were in most instances very similar and in some cases indistinguishable from one another. The results demonstrate that antigenic similarity between biotypes is a consistent finding in animals dying from fatal BDV infections. In view of the wide degree between biotypes is a consistent finding in animals dying from fatal BDV infections. In view of the wide degree of variation in reactivity patterns between unrelated BDV isolates, the close antigenic similarity of CP BDV to the homologous NCP BDV of a given pair strongly suggests that CP BDV arises by mutation from NCP BDV.

To delineate the proximity and spatial arrangement of the major structural proteins of intact vesicular stomatitis (VS) virions, protein complexes formed by oxidation or by bivalent cross-linkers were analyzed by two-dimensional electrophoresis on polyacrylamide slab gels. H2O2 oxidation of VS virions produced an N-polypeptide dimer (molecular weight, approximately equal to 110,000) on a first dimension gel that could be reduced to N monomers (molecular weight, approximately equal to 50,000). Proteins extracted from unreduced and unoxidized VS virions contained dimeric and trimeric forms of M-protein complexes as well as a heterodimer of M and N protein. Qualitatively similar VS viral protein complexes were generated by exposing VS virions to the reversible protein cross-linkers methyl-4-mercaptobutyrimidate (MMB), tartryl diazide (TDA), and dithiobis(succinimidyl proprionate) (DTBSP); cross-linked complexes on first-dimension gels were cleaved by reduction with 2-mercaptoethanol (MMB or DTBSP cross-linked) or by periodate oxidation (TDA cross-linked). In addition to covalently linked homodiamers of M and N proteins and a protein M-N heterodimer, the protein cross-linkers also generated homo-oligomers of G protein and a G-M heterodimer. These data suggest that the glycoprotein spike of VS virus is composed of more than one G protein. The existence of N-M and G-M heterodimers is consistent with the hypothesis that the matrix (M) protein may serve as a bridge between the G and N proteins in assembly of the VS virion.

Noncytocidal persistent infections at 37 C of mouse L cells (Lvsv) with infective B particles of vesicular stomatitis virus (VSV) could be established only in the presence of large numbers of defective interfering (DI) particles. Under these conditions, there was a rapid spontaneous selection of temperature-sensitive (ts) virus. At 10 days there was an increase to 17.8% in the frequency of ts clones in the virus population; by 17 days this frequency had reached 85.2%, and by 63 days 100% of the clones isolated were ts at 39.5 C, the nonpermissive temperature used. All 34 of the clones isolated from the 84-day fluid had an RNA-phenotype, and 8 clones that were tested all belonged to VSV complementation group I. When tested by an interference assay, Lvsv fluids did not contain significant numbers of DI particles (less than 1 DI/PFU). Furthermore, persistent infection of L cells at 37 C could be initiated under conditions in which few, if any, DI particles were present by using low input multiplicities (10(-4) and 10(-5) of a clonal isolate of an RNA-group I mutant obtained from Lvsv cells. On the basis of these and other results, a mechanism is proposed to explain the role of ts mutants in both the establishment and maintenance of the persistently infected state.

Studies were undertaken to determine if a prewetting device (humidifier bulb) used in combination with an all glass impinger (AGI-30) would increase the recovery of airborne mengovirus-37A, vesicular stomatitis virus (VSV), and the S-13 coliphage. Suspensions of T3 coliphage with mengovirus-37A, VSV, or S-13 were aerosolized and collected by using the AGI-30-humidifier bulb combination to sample the aerosols before and after shifts in relative humidities (RH). These studies revealed the following. (i) At low RH values there was a 3 to 4 log increase in recovery of airborne T3 phage; (ii) concomitantly, the recovery of mengovirus-37A and VSV decreased; and (iii) only at the mid-range RH values was the recovery of S-13 enhanced. The prehumidification technique significantly increased the recovery of airborne T3 phage but decreased the recovery of the two animal viruses tested.

The influence of relative humidity on the airborne survival of simian virus 40 (SV40) was studied by allowing virus aerosols to age in rotating drums at 21 or 32 C and at a relative humidity (RH) value ranging from 22 to 88%. Airborne SV40 virus was stable at every RH tested at 21 C, but aerosols maintained at 32 C were inactivated within 60 min at mid-range RH values. The unusual stability at 21 C over a broad RH range indicates that potentially biohazardous situations may occur under laboratory conditions if this virus becomes accidentally airborne.

The infectivity of the nucleic acids of πX-174 and MS-2 was examined after aerosolization. It was found that the nucleic acids were undamaged by aerosolization.