Studies On The Pathogenesis Of Shandong Strain Of Bovine Parainlfuenza Virus Type3and Development Of A Real-time Lfuorescent Quanittative RT-PCR

Bovine parainfluenza virus type3(BPIV3) is recognized as one of the most important of the knownviral respiratory pathogens of both young and adult cattle and has also been associated with bovinerespiratory disease complex (BRDC) development in feedlot cattle. To date, genotype A(BPIV3a),genotype B(BPIV3b)and genotype C(BPIV3c)have been isolated from cattle. During2008there weremany cattle identified with clinical signs considered to be consistent with BPIV3infection. Four BPIV3strains were isolated from four of63bovine nasal swabs collected from Shandong Province. In order tofurther genotyping of the four Shandong isolates, the complete genome for one strain which namedSD0835was determined. The sequence alignment and phylogenetic analysis implicated that the fourShandong BPIV3isolates were distinct from the previously reported genotype A and genotype B ofBPIV3and may represent a new genotype, which was tentatively classified as genotype C (BPIV3c). Todate, the pathogenesis of the genotypes B and C of BPIV3infection in calves and laboratory animalshave not been reported. And only limited studies on the pathogenesis of the genotype A of BPIV3wereconducted and reported.In order to ascertain the pathogenesis of the genotype C of BPIV3SD0835isolate from Shandong Province, animal experimental infections were conducted and the virusreplication, virus excretion period, and pathological changes in animals were evaluated. The studydemonstrated that the BPIV3strain SD0835were pathogenic to Balb/c mice of5to6weeks old andalbino guinea pigs. The pathogenesis of SD0835was characterized by virus isolation and titration,real-time fluorescence quantitative RT-PCR, immunofluorescence staining, and immunohistochemicalstaining. Virus replications in Balb/c mice had occurred in the respiratory tissues as early as24h afterintranasal inoculation and the viral RNA copies of lung were higher than trachea tissues. The results ofimmunofluorescent staining and IHC implicated that the virus replication in lungs and tracheas andlocalization of viral antigens was seen in the cytoplasms of peribronchovascular cells in lungs. Thehistopathologic examinations revealed that alveoli septa thickening and focal cellulose pneumonia wereseen in the lungs of experimentally infected mice. Serologic tests showed the antibodies against BPIV3were detected from6to14days PI and the HI titers reached1:80.With the same methods, the pathogenesis of SD0835to albino guinea pigs was studied. The notedclinical signs are sneezing, nasal discharges in experimentally infected guinea pigs. The virus isolationresults showed that BPIV3replication occurred mainly in lungs and tracheas and the virus excretionlasted until6days post-infection. The results of real-time fluorescence quantitative RT-PCR suggestedthat the highest copies of lungs and tracheas appeared in3days post-infection and then continued todecline, but the viral RNA could be detected from lungs and trachea of guinea pigs during the14daysof observation. The virus replication in lungs and tracheas and localization of viral antigens in thecytoplasms of alveolar septum cells were observed by using immunofluorescent staining and IHC. Thehistopathologic examinations revealed that interstitial pneumonia was the notable histopathological change along with the disease development, and seriflux and cellulose exudation were also seen.Trachea appeared mucosa atrophy and mucosal epithelium exfoliation. The liver appeared vacuolardegeneration. Lymphocytes were obviously reduced in white pulp of spleen. The submandibular lymphnodes showed suppurative lymphadenitis. HI test revealed that the antibodies against BPIV3weredetected at3days post-infection, and the mean titer of HI antibodies reached1:60.At the same time, a fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR)was established for BPIV3. It is much more sensitive than conventional RT-PCR and could detect theleast template of17.6copies/μl. Using the plasmid was detected for5times and standard curve wasdrawn. The related coefficient was0.9948and amplification efficiency was1.942. These results showedexcellent correlations. At the same time, the intra-and inter-batch repeatability tests were conductedusing the standard plasmid, respectively and the coefficients of variation were less than2.5%. There wasno cross reaction with the other respiratory pathogens in the fluorescence quantitative RT-PCRdetections. The method had a very good specificity. The results of fluorescence quantitative RT-PCRdetections for clinical samples correlated well with those of median tissue culture infective dose(TCID50) measurement. This indicated that this method was suitable for clinical sample detection forBPIV3.In sum, the aforementioned results indicated that the SD0835of the genotype C was pathogenic toBalb/c mice of5~6weeks old and albino guinea pigs and could infect them experimentally. Virusreplication occurred mainly in lungs and tracheas and induced the histopathologic changes in theexperimentally infected Balb/c mice and albino guinea pigs with SD0835. At the same time, SD0835infections in Balb/c mice and albino guinea pigs could stimulate the host to generate HI antibodies. Thisindicated that Balb/c mice of5~6weeks old and albino guinea pigs were susceptible to the BPIV3strainSD0835. The Balb/c mice and albino guinea pigs experimental infection models would shed light on thegenotype C of BPIV3infection process and pathogenesis. This research indicated that obvious clinicalsigns were observed in experimentally infected guinea pigs with SD0835for the first time and laid afoundation for further studies of the genotype C of BPIV3in guinea pigs infection model.