Coronaviruses are etiologic agents of respiratory and enteric diseases in humans and in animals. In this study, a one-step real-time reverse transcription-polymerase chain reaction (RT-PCR) assay ...based on SYBR Green chemistry and degenerate primers was developed for the generic detection of coronaviruses. The primers, designed in the open reading frame 1b, enabled the detection of 32 animal coronaviruses including strains of canine coronavirus, feline coronavirus, transmissible gastroenteritis virus (TGEV), bovine coronavirus (BCoV), murine hepatitis virus (MHV) and infectious bronchitis virus (IBV). A specific amplification was also observed with the human coronaviruses (HCoV) HCoV-NL63, HCoV-OC43, HCoV-229E and severe acute respiratory syndrome coronavirus (SARS-CoV). The real-time RT-PCR detected down to 10 cRNA copies from TGEV, BCoV, SARS-CoV and IBV. In addition, the assay exhibited a high sensitivity and specificity on clinical samples from different animal species. The developed assay represents a potential tool for laboratory diagnostics and for detecting still uncharacterized coronaviruses.
Canine herpes virus-1 (CHV1) causes a fatal hemorrhagic disease in neonatal puppies and is associated with reproductive problems in female dogs. This serologic study was conducted to assess the ...seroprevalence of CHV1 infection in Norway. Blood samples were collected from clinically healthy dogs (n = 436) one yr of age and older of both genders, supplied by four small animal clinics (A, B, C and D) in different parts of the country. The immunoperoxidase monolayer assay was used for testing of CHV1 antibodies. Serum titers were recorded as the reciprocal value of the highest dilution producing specific cell staining. Titers equal to or above 80 were considered positive for exposure to CHV1. In total, 80.0% of the dogs had titers ≥80 and were classified as positive. Mean age for seronegative dogs was 4.7 yrs (95% CI 4.1–5.4) and for seropositive dogs 5.0 yrs (95% CI 4.7–5.4). Of the dogs, 32.8% displayed a weakly positive titer of 80, whereas 41.5 and 5.7% fell into the moderately (titer 160 and 320) and strongly (titer ≥640) positive categories, respectively. No association was demonstrated when comparing CHV1 antibody titers to gender or reproductive parameters like previous matings, pregnancies, births or number of puppies born. Age, visit in foreign countries and clinic explained together 78% of the variation in antibody titer categories. The percentage of positive samples differed significantly between the four clinics (A 98%, B 58.5%, C 74.6%, D 89.5%). A reasonable explanation for this finding has not been established. No information about an ongoing outbreak of CHV1 infection was available. In conclusion, this study strongly indicates that CHV1 infection is endemic in the dog population of Norway. There are significant differences in seroprevalence between geographic regions in the country.
Field canine coronaviruses (CCVs) identified during a series of outbreaks of gastroenteritis in Swedish dogs were subjected to genetic analysis involving the open reading frame 1b (ORF1b) and the ...membrane (M) and spike (S) protein genes. Four field viruses originating from the Stockholm region presented identical sequences and segregated separately from other CCVs characterized so far and from GOT/05, the variant recovered in Western Sweden. A recombinant origin of the fifth virus identified in the Stockholm region is suggested. In addition, the five viruses originating from the same geographical area displayed atypical 5' S gene sequences.
The acute phase protein serum amyloid A (SAA) has proven potentially useful as an inflammatory marker in the horse, but the knowledge of SAA responses in viral diseases is limited. The aim of this ...study was to evaluate SAA as a marker for acute equine influenza A2 (H3N8) virus infection. This is a highly contagious, serious condition that inflicts suffering on affected horses and predisposes them to secondary bacterial infections and impaired performance. Seventy horses, suffering from equine influenza, as verified by clinical signs and seroconversion, were sampled in the acute (the first 48 h) and convalescent (days 11-22) stages of the disease, and SAA concentrations were determined. Clinical signs and rectal temperature were recorded. Secondary infections, that could have influenced SAA concentrations, were clinically suspected in 4 horses. SAA concentrations were higher in the acute stage than in the convalescent stage, and there was a statistically positive relationship between acute stage SAA concentrations and clinical signs and between acute stage SAA concentrations and maximal rectal temperature. Horses sampled early in the acute stage had lower SAA concentrations than those sampled later, indicating increasing concentrations during the first 48 h. There was a statistically positive relationship between convalescent SAA concentrations and degree of clinical signs during the disease process. The results of this investigation indicate that equine SAA responds to equine influenza infection by increasing in concentration during the first 48 h of clinical signs and returning to baseline within 11-22 days in uncomplicated cases.
Equid herpes virus 1 (EHV-1) related isolates from a captive blackbuck (strain Ro-1) and Grevy's zebra (strain T965) behaved similarly to EHV-1 and EHV-9 in respect to their host cell range. ...Restriction enzyme analysis and a phylogenetic tree confirmed that Ro-1 and T965 were identical and more closely related to EHV-1 than to EHV-9. Differences from EHV-1 became obvious firstly, by amino acid alignments revealing two unique substitutions in the gB protein of Ro-1 and T965. Secondly, an EHV-1 type-specific monoclonal antibody did not detect its antigen on Ro-1, T965 or EHV-9 infected cells by immunohistochemistry. The results support the view that Ro-1 and T965 isolates represent a distinct, previously unrecognized species of equid herpesviruses.
Equine arteritis viruses (EAV) from Europe and America were compared by phylogenetic analysis of 43 isolates obtained over four decades. An additional 22 virus sequences were retrieved from GenBank. ...Fragments of the glycoprotein G(L) and the replicase genes were amplified by RT-PCR, prior to sequencing and construction of phylogenetic trees. The trees revealed many distinctive lineages, consistent with prolonged diversification within geographically separated host populations. Two large groups and five subgroups were distinguished. Group I consisted mainly of viruses from North America, whilst group II consisted mainly of European isolates. In most instances, where the geographic origin of the viruses appeared to be at variance with the phylogenetically predicted relationships, the horses from which the viruses were recovered had been transported between Europe and America or vice versa. Analysis of the replicase gene revealed similar phylogenetic relationships although not all of the groups were as clearly defined. Virus strains CH1 (Switzerland, 1964) and S1 (Sweden, 1989) represented separate 'outgroups' based on analysis of both genomic regions. The results of this study confirm the value of the G(L) gene of EAV for estimating virus genetic diversity and as a useful tool for tracing routes by which EAV is spread. In addition, computer-assisted predictions of antigenic sites on the G(L) protein revealed considerable variability among the isolates, especially with respect to regions associated with neutralization domains.
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