Genome characterization of Salem virus reveals its evolutionary intermediate status in the subfamily Paramyxovirinae.

Salem virus (SalPV) was originally isolated from a horse during a disease outbreak in 1992 in the USA. In this study, we complete the genome characterization of SalPV and confirm the classification of this virus as a member of the subfamily Paramyxovirinae. The SalPV genome is 16,698 nucleotides in length, with six transcriptional units in the order 3'-N/P/V/C-M-F-G-L-5'. SalPV has a fixed 3-nt intergenic region, which is the same as in all Paramyxovirinae members except rubulaviruses and avulaviruses. The genome size of SalPV is greater than those of the "old" members of the subfamily, such as morbilliviruses and respiroviruses, but smaller than those of the "new" members, including henipaviruses and several unclassified novel viruses. Interestingly, this evolutionary "middle point" was also reflected in the phylogeny, suggesting that SalPV represents an important intermediate between the previously well-characterized paramyxoviruses and the recently emerged members of this subfamily.

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.

Detection of equine herpesvirus-1 in nasal swabs of horses by quantitative real-time PCR.

BACKGROUND: Early identification of inhalation-transmitted equine herpesvirus type 1 (EHV-1) infections has been facilitated by the availability of a number of real-time quantitative PCR (qPCR) tests. A direct comparison between nasal swab qPCR and traditional virus isolation (VI) requires a method for normalizing the qPCR samples and controlling for PCR inhibitors present in some clinical samples. OBJECTIVES: To quantify EHV-1 shedding in viral swabs using an internal control and to compare fast qPCR to VI for the detection of EHV-1 in nasal swabs from horses. ANIMALS: Fifteen horses experimentally infected with EHV-1. METHODS: Experimental study: Nasal swab samples were collected daily after experimental infection for up to 21 days. VI was performed by conventional methods. The DNA was prepared for qPCR with the addition of a known quantity DNA of Marek's disease virus as an internal control. qPCR was performed. RESULTS: The qPCR method detected virus up to day 21 after challenge, whereas VI detected virus only to day 5. The median Kaplan-Meier estimates for EHV-1 detection were 12 days for qPCR and 2 days for VI (P< .0001). When compared with VI, the sensitivity and specificity of qPCR were 97 (95% CI: 86-100) and 27% (95% CI: 20-35). CONCLUSIONS AND CLINICAL IMPORTANCE: We conclude that fast qPCR of nasal swab samples should be chosen for diagnosis and monitoring of herpesvirus-induced disease in horses. Recommended reference ranges of C(T) values are provided as well as justification of a minimum 10-day quarantine period.

Infectious diseases of dogs and cats on Isabela Island, Galapagos.

BACKGROUND: Vaccination and importation of dogs and cats are prohibited in the Galapagos, resulting in a uniquely isolated population. The purpose of this study was to determine the prevalence of infectious diseases of dogs and cats that impact their health, could spill over to native wildlife, or sentinel diseases of concern to humans. HYPOTHESIS: The isolation of dogs and cats in the Galapagos protects them from diseases common in mainland populations. ANIMALS: Ninety-five dogs and 52 cats presented during a neutering campaign. METHODS: A prospective cross-sectional study was performed. Blood was collected for serological and DNA evaluation of a panel of infectious diseases. RESULTS: Antibodies against parvovirus (100%), parainfluenza virus (100%), adenovirus 1/2 (66-67%), and distemper virus (22%) were present in dogs. Dirofilaria immitis was also common in dogs (34%), with lower prevalences of Wolbachia pipiens (22%), Bartonella sp. (13%), Ehrlichia/Anaplasma spp. (1%), and Mycoplasma haemocanis (1%) observed. Antibodies against panleukopenia virus (67%), Toxoplasma gondii (63%), calicivirus (44%), and herpesvirus 1 (10%) were detected in cats. Feline leukemia virus antigen, feline immunodeficiency virus antibody, or coronavirus antibodies were not detected. Bartonella sp. (44%) infections were common in cats, but only one was infected with M. haemofelis. CONCLUSIONS AND CLINICAL IMPORTANCE: Despite their relative seclusion from the rest of the world, cats and dogs of Isabela were exposed to many pathogens found in mainland South America. Parasite prophylaxis, neutering, and strict enforcement of animal movement restrictions would control a majority of the diseases. In the absence of vaccination, a reservoir of susceptible animals remains vulnerable to new disease introductions.

Bovine herpesvirus-1: Genetic diversity of field strains from cattle with respiratory disease, genital, fetal disease and systemic neonatal disease and their relationship to vaccine strains.

Bovine herpesvirus-1 (BoHV-1) causes disease in cattle with varied clinical forms. In the U.S. there are two BoHV1 subtypes, BoHV-1.1 and BoHV-1.2b. Control programs in North America incorporate modified live (MLV) or killed (KV) viral vaccines. However, BoHV-1 strains continue to be isolated from diseased animals or fetuses after vaccination. It is possible to differentiate BoHV-1 wild-type from MLV vaccine strains by determining their single nucleotide polymorphism (SNP) patterns through either whole-genome sequencing or PCR sequencing of genomic regions containing vaccine-defining SNPs. To determine the BoHV-1 subtype in clinical isolates and their relationship to MLV strains, 8 isolates from varied clinical disease at three different laboratories in the U.S. were sequenced and phylogenetically analyzed. Five samples were isolated within the past 5 years from New York and 3 were archived samples recovered 35 years prior from Oklahoma and Louisiana. Based on phylogenetic analysis, four of the cases appeared to be due to an MLV vaccine: 3 cases of aborted fetuses and one neonate with systemic BoHV-1 disease. One aborted fetus was from a herd with no reported history of MLV vaccination in two years. The remaining four isolates did not group with any MLV vaccines: two were associated with bovine respiratory disease, one with vulvovaginitis, and a fourth was determined to be a BoHV-1.2b respiratory isolate. Recovery of BoHV-1.1 that is very closely related to an MLV vaccine virus from a herd not receiving vaccines in an extended period prior to its isolation suggests that MLV viruses may remain latent or circulate within herds for long periods.

Aromatic amidines: comparison of their ability to block respiratory syncytial virus induced cell fusion and to inhibit plasmin, urokinase, thrombin, and trypsin.

Two series of amidine derivatives consisting of a total of 24 compounds were examined for a correlation between their blocking effect on respiratory syncytial virus induced cell fusion and their inhibitory activity against selected trypsin-like protease. Although no correlation was evident between the two activities, several potentially important discoveries were made. A highly selective inhibitor of plasmin over thrombin (compound 10) was obtained, and a potent new blocker of virus-induced cell fusion (compound 22) was identified.

Evaluation of a new sandwich ELISA kit that uses serum for detection of cattle persistently infected with BVD virus.

Cattle immunotolerant to and persistently infected (PI) with bovine viral diarrhea (BVD) virus (BVDV) constitute the mechanism by which BVDV persists in and spreads among cattle herds. Detection and elimination of PI cattle are necessary for control of BVD. Serum is an excellent specimen for BVD PI testing because of high survivability of BVDV in serum and ease of collection, storage, and handling. Currently, microtiter virus isolation (VI) employing serum and sandwich ELISAs (S-ELISA) on tissues or leukocytes are used for BVDV PI screening. This paper evaluates a new S-ELISA kit that uses serum as the diagnostic sample. Cattle sera (n = 408) were tested using VI and the S-ELISA. The VI detected 172 BVDV-positive sera. Of these, 18 were confirmed PI cattle. The S-ELISA was positive on all PI samples. Considering only the PI animals, and using VI as the gold standard, the relative sensitivity of S-ELISA was 100%. The overall relative sensitivity was 93.6% and the agreement quotient (kappa) was 0.94. The relative specificity of the kit, based on 236 VI-negative sera, was 100%. These data indicate that the new kit is very adequate for detection of BVDV PI cattle.

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).

Insertion of cellular sequences in the genome of bovine viral diarrhea virus.

The genomic sequences of four pestiviruses, two BVDV strains (Osloss and NADL, both of which are cytopathogenic) and two HCV strains, were analyzed. Comparative studies revealed the presence of small insertions of cellular sequences in the genomes of both BVDV strains; the insertions are located in a region coding for a nonstructural protein. Such insertions are not present in the HCV sequences. The insertion identified in BVDV Osloss encodes a complete ubiquitin-like element. The sequence inserted in the BVDV NADL genome shows no homology to a ubiquitin gene but is almost identical with another bovine mRNA sequence. Molecular characterization of a BVDV "pair", isolated from an animal with mucosal disease, led to the detection of a ubiquitin-like sequence in the genome of the cytopathogenic strain, but not of the noncytopathogenic strain. It is proposed that recombination between viral and cellular RNA leads to formation of cpBVDV genomes. This hypothesis has direct implications for the pathogenesis of mucosal disease.

Detection of bovine herpesvirus-1 in bovine semen by a nested PCR assay.

A nested PCR assay targeting a portion of the glycoprotein IV gene has been developed for the detection of Bovine Herpesvirus-1 (BHV-1). Rapid and sensitive detection of the PCR products was achieved using a nonisotopic reverse dot-blot format with a visible color readout. Cross-reactivity of this PCR assay was not observed with the closely related BHV-3. The sensitivity of this assay when tested on a supernatant from a BHV-1 cell culture was approximately 4.5 TCID50 (50% tissue culture infectious dose). A procedure using the chelating resin Chelex 100 was used to prepare viral DNA from artificially inoculated samples of extended and raw semen for use in the PCR assay. In combination with nested PCR and reverse dot blot, this method allowed the detection of 5 x 10(3) TCID per 0.5 ml of semen, which is comparable to the detection in the Cornell Semen Test. The whole procedure can be completed in approximately 8 h. This assay has therefore the potential of replacing the currently available yet time consuming and costly detection methods for BHV-1 in bovine semen.

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.

Isolation of Salem virus, a novel equine paramyxovirus, and assessment of its etiologic role in a disease outbreak.

Salem virus (SalV) is a recently identified equine virus belonging to the family Paramyxoviridae. The only known isolate was obtained from a horse that was involved in a disease outbreak of undetermined nature and the circumstances of its isolation suggested an etiologic role. However, the experimental infection of a colostrum-deprived foal failed to reproduce the disease; only mild neutropenia and temperature elevation were recorded. An additional attempt to establish an etiological relationship with the disease was made by conducting a retrospective evaluation of the serological profiles of animals involved in the outbreak. Animals reported as being affected by the disease according to a comprehensive United States Department of Agriculture (USDA) database were found to be 48% (n=27) positive for antibodies to SalV, but the percent positive for all horses, affected and unaffected, was actually higher at 56% (n=62). For 15 affected horses for which paired acute and convalescent serum specimens were available, no unequivocal seroconversions to SalV were identified. Furthermore, the horse from which SalV was isolated was not listed as one of the animals affected by the disease. In total, the evidence suggests that SalV was not the etiological agent of the disease and that its isolation was fortuitous.

Detection of bovine immunodeficiency virus DNA in the blood and semen of experimentally infected bulls.

Five 18- to 24-month-old bulls were inoculated with either a cell suspension containing bovine immunodeficiency virus (BIV-FL112; 3 bulls) or a BIV-free cell suspension (2 bulls). Blood and semen specimens were collected once a week for 14 weeks, and seroconversion was confirmed by indirect immunofluorescent antibody (IFA) testing. The presence of BIV in blood and semen was determined by virus isolation and/or polymerase chain reaction (PCR) assays. Antibodies to BIV were detected in the 3 experimentally infected bulls as early as day post inoculation (DPI) 17, and levels peaked at DPI 37-58. BIV was isolated from the peripheral blood mononuclear cells (MNCs) of the infected bulls at DPI 9 (2 bulls) and DPI 23 (1 bull), and could be isolated from one animal up to DPI 65. PCR analysis of MNC DNA, using BIV pol gene primers, detected virus in all three of the experimentally infected bulls from DPI 9 until the termination of the experiment at DPI 98. Efforts to isolate a significant number of non-spermatozoal cells (NSC) by gradient separation from the semen of the experimentally infected bulls were unsuccessful. Two methods for the extraction of total NSC DNA from up to 2 ml of non-extended semen were employed; however, no BIV pol fragment was amplified from these DNA preparations. Additionally, 30 bulls from artificial insemination (AI) centers were evaluated for BIV infection by PCR. No amplification products were obtained from MNC DNA from the AI submissions using primer sets for both the BIV pol and env genes.

Effects of sodium taurodihydrofusidate on nasal absorption of insulin in sheep.

To investigate the utility of a novel adjuvant, sodium taurodihydrofusidate (STDHF), as an enhancer of mucosal permeation of drugs, experiments involving intranasal insulin:STDHF administration in sheep were performed. Rabbit erythrocyte lysis assays were employed to assess the relative membrane lytic activity of STDHF, as well as that of its glycine-conjugated analogue, compared with a nonionic detergent and a common bile salt. Equivalent weight concentrations of the fusidates were found to be 5- to 10-fold less lytic than the bile salt and at least 100-fold less lytic than the nonionic detergent laureth-9. Provided the concentration of STDHF was greater than its critical micellar concentration, formulations of insulin with STDHF greatly enhanced intranasal insulin absorption. Optimal nasal insulin absorption was attained at a molar ratio of STDHF to insulin of 5:1. In addition, intranasal absorption was linearly related to insulin dose. Compared with intravenous administration, the mean bioavailability of intranasal insulin was 16.4%. Interovine variability was low, with a coefficient of variation of 14% for 12 animals. It was found that intranasal absorption of sodium insulin was not significantly different from that of zinc insulin. However, formulations of both crystalline insulin preparations were absorbed more efficiently than a formulation prepared using commercially available solutions of U-500 insulin. The results taken together indicate that STDHF is an excellent enhancer of insulin absorption from the nasal mucosa.

Genetic diversity and BVD virus.

The various measures of genetic variation of BVD virus was reviewed with emphasis on the implications for future control of virus-induced disease and diagnosis. While experimental data does not support unique serotypes for BVDV, there is substantial antigenic variation among the isolates examined. This variation may permit fetal infections even in animals assumed to be well vaccinated. The genetic differences between cytopathic and noncytopathic strains of BVDV are expressed in infected cells by the production of a p80 protein by cytopathic strains. In addition, cellular gene inserts have been detected in cytopathic strains. Monoclonal antibodies have demonstrated a high degree of diversity with the pestivirus population. Grouping of BVDV isolates by monoclonal antibody analysis is suggestive at best. The use of nucleic acid probes as diagnostic reagents has been compromised by the nucleic acid sequence variation found in the BVDV isolates tested.

Comparison of virus isolation and reverse transcription polymerase chain reaction assay for detection of bovine viral diarrhea virus in bulk milk tank samples.

The use of a reverse transcriptase polymerase chain reaction (RT-PCR) assay to screen bulk milk tank samples for bovine viral diarrhea virus (BVDV) has proven to be a sensitive and economical means to evaluate the lactating animals in a herd. The assay is capable of detecting the presence of a single persistently infected animal within a group of several hundred cows. Over a 3-year period, 144 samples from 97 farms were tested for BVDV using an RT-PCR assay in conjunction with a classical virus isolation (VI) procedure to measure the relative effectiveness of the techniques. Virus could be detected with both methods when the milk from a single persistently infected animal was diluted 1:600 with the milk from a herd of BVDV-negative animals. Based on individual farms, there was an overall prevalence of 12.4% BVDV infection, and the correlation between the 2 assays was 95.9%. In terms of sensitivity, specificity, and turnaround time, RT-PCR was superior to VI. However, of the 17 samples that were VI positive, 4 were RT-PCR negative. RT-PCR may not detect all naturally occurring BVDV isolates because they may contain minor sequence variations in the primer regions. VI and RT-PCR are both suitable for detection of BVDV in bulk milk samples when used independently, but to increase the probability of successful detection and to provide cross-checks against assay contamination, it is desirable to utilize both methods in parallel.

Experimental equine herpesvirus-1 infection in llamas (Lama glama).

Three llamas (Lama glama) were experimentally infected intranasally with an isolate of equine herpesvirus-1 (EHV-1) from the brain of an alpaca that had experienced severe neurologic signs. Two of the 3 llamas developed severe neurologic disorders following inoculation; 1 died, and 1 was euthanized in a moribund state. The third llama showed only mild neurologic signs. The euthanized llama had preexisting antibodies to EHV-1, and the remaining 2 llamas were seronegative (virus neutralization titer less than 6) at the time of inoculation. One of the seronegative llamas died acutely without production of detectable antibodies, and the other developed antibodies typical of a primary immune response. The EHV-1 virus was recovered only from a sample of the thalamus of the llama that died acutely. Histopathologic lesions were observed in the brain and retina of the dead and euthanized animals. This study verifies the pathogenic potential of EHV-1 for llamas.

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.

Equine herpesvirus type 2: prevalence and seroepidemiology in foals.

Whole blood and serum were collected from foals to determine the prevalence of Equine herpesvirus type 2 (EHV 2) infection in foals, age at which infection can first be identified and serological responses to infection. Equine herpesvirus type 2 was isolated from peripheral blood mononuclear cells (PBMC) from 68 of 69 foals, 1-8-months-old, sampled once. Virus isolation was performed twice at intervals of 2-7 months on PBMCs from 33 foals and EHV2 was isolated on both occasions in all but one foal (negative, then positive). Regression analysis of log2-transformed reciprocal serum EHV2 virus neutralising (VN) titres revealed that in foals age 1-7 months, EHV2 VN antibody titre was positively correlated with age (r = 0.94). Paired serum samples were obtained from 58 foals, with the first samples collected age 1-6 months and the second samples collected 2-4 months later. There were significant (P < 0.05) increases in mean VN titres to EHV2 in foals sampled initially at age 1-4. Eight foals had blood sampled prior to sucking and at age 7, 20, 30 and 45 days. Each foal was negative for EHV2 in PBMC and each foal had a negative serum EHV2 VN titre immediately after birth. Each foal was positive for EHV2 in PBMC by age 45 days, with the earliest isolation at 25 days. Tracheal aspirate fluid and peripheral blood were collected from 20 foals without clinical signs of respiratory disease and from 30 foals with clinical signs of lower respiratory disease. In 20 foals without clinical signs of respiratory disease, EHV2 was isolated from tracheal aspirates (1/20 foals) and PBMC (20/20 foals) and in 30 foals with such clinical signs, from trachea aspirates (20/30 foals: P < 0.01) and from PBMC (30/30 foals). In one 6-month-old foal, EHV1, but not EHV2, was isolated from the tracheal aspirate, 3 months after EHV2 had been isolated from a tracheal aspirate. These results demonstrate a greater prevalence of EHV2 in lower respiratory secretions in foals with clinically apparent lower respiratory disease, but a cause and effect relationship between the virus and lower respiratory disease remains to be elucidated. It is noteworthy, however, that of virus isolations performed on 50 tracheal aspirates, a virus (EHV1) other than EHV2 was isolated only once.

Equine viral arteritis in newborn foals: clinical, pathological, serological, microbiological and immunohistochemical observations.

Clinical, pathological, immunohistochemical, serological and microbiological findings are described for 2 geographically and temporally distinct equine arteritis virus (EAV) epidemics in newborn foals. Outbreak A occurred at a commercial Standardbred breeding facility; Outbreak B began in a group of research animals. Clinical signs were severe and primarily referable to the respiratory tract. Fever and leucopenia and/or thrombocytopenia were observed in foals surviving for more than 24 h. The most common gross pathological findings were limited to the respiratory tract. Common histopathological findings included interstitial pneumonia, lymphocytic arteritis and periarteritis with fibrinoid necrosis of the tunica media. Renal tubular necrosis was noted in 2 foals. Immunoperoxidase histochemistry combined with virus isolation was diagnostic in all cases.