The Immunogenicity Of The Attenuated Strain Of Bovine Parainfluenza Virus Type 3 In Guinea Pigs And Genetic Basis Of Its Attenuation Phenotype

Bovine parainfluenza caused by bovine parainfluenza virus type 3(BPIV3)is an acute contagious disease,once referring to shipping fever.The clinical signs of cattle were characterized by anorexia,sluggishness and respiratory symptoms including cough and nasal discharge,and sometimes elevated temperature and dyspnea.Under the circumstance of stress factors such as transport and chill,secondary infection with bacteria and mycoplasma resulted in worsening of bronchopneumonia,contributing to the development of bovine respiratory disease complex(BRDC)and increase of mortality.BRDC resulted in huge financial loss of cattle-feeding industry all over the world.Recent both etiology detections and serological surveys demonstrated that BPIV3 infection was widespread in China.Thus there is an urgent need to develop effective vaccines to prevention and control of BPIV3 infection.In the present study,BPIV3 SD0835 isolated in our lab was serially passaged on MDBK cells and the pathogenicity of the high-passage viruses was evaluated in guinea pigs,hoping to select an attenuated virus as a live vaccine candidate.BPIV3 SD0835 was passaged for 207 times in vitro and the pathogenicity of high-passage BPIV3 isolates(P126,P151 and P208)and the parental virus was evaluated in guinea pigs.The results showed that high-passage viruses were attenuated in guinea pigs with fewer animals showing elevated temperatures than the parental virus,as well as lower virus titers in the lungs and fewer gross lung lesions.However the histopathology results revealed that the lung sections from the guinea pigs in P126 and P151 groups were characterized by thicking of alveolar septum and so on.It was notable that the BPIV3 high passage isolate P208was not lethal to guinea pigs and showed a significant reduction in viral load in the lower respiratory tract,and histopathologic lesions of the lungs in this group was reduced compared with the other virus-infected groups.Moreover,all three high-passage viruses could induce neutralizing antibodies against BPIV3 in guinea pigs.Taken together,the results of our study suggested that the attenuated BPIV3 P208 isolate could be a live vaccine candidate and had high immunogenicity and safety.Future tests in cattle might be merited based on these data.Serological survey also demonstrates that BPIV3 infection is widespread in China.However,there is still no available vaccination for BPIV3 prevention in China nowadays.In the above study,the high-passage BPIV3 was attenuated in guinea pigs.Thus,the passage 208 of BPIV3c strain SD0835 was used as a live vaccine candidate to immunize the guinea pigs.The vaccination results revealed that two vaccinations could induce excellent serum neutralizing antibody responses as well as proliferation of T lymphocytes.The vaccinated guinea pigs were well protected against challenge with a low passage of BPIV3c strain SD0835.Additionally,T cell subsets in the T cell population after vaccination and challenge were also detected,and the results showed that the percentages of CD4~+and CD8~+T cell subsets of animals in vaccinated group increased after first immunization as well as boost immunization;T cell subsets on day 2 after challenge in both groups decreased,and the decline of CD4~+and CD8~+T cell subsets levels of four guinea pigs in vaccinated group was relatively moderate,comparing with that of control group.These data supports further testing of the attenuated virus as an effective candidate vaccine.The genetic basis of the attenuation of BPIV3 after serial passage on MDBK cells is still unclear.We determined the sequences of the F and HN genes of BPIV3 P2 and its high-passage derivatives and identified fourteen amino acid mutations that were potentially associated with the attenuation of the BPIV3.However,which of these mutations resulted in the attenuation of BPIV3needs to be studied.An infectious cDNA clone is of benefit to investigate the mechanism of the attenuation.Thus we used the method of splicing of overlapping RT-PCR products to obtain the whole BPIV3 genomic cDNA.Meanwhile,the support plasmids for recovery of virus from cDNA were constructed and immunofluorescence assay demonstrated all these plasmids could be expressed in BHK21 cells.The BPIV3 genomic cDNA together with the support plasmids was transfected the MDBK cells and no infectious virus was recovered up to date.In summary,BPIV3 still replicate effeciently in MDBK cells comparable to the parental virus after passaging 207 times in vitro.The study of pathogenicity demonstrated that the high-passage viruses,espcially BPIV3 P208,were attenuated in guinea pigs,characterized by not being lethal to annimals,decrease of the viral titer in lungs,no gross and histopathology lesions,meanwhile inducing neutralizing antibody,indicating BPIV3 P208 could be used as a live vaccine candidate.Thus the guinea pigs were immunized with the BPIV3 P208,and the animals exhibited no clinical sighs and no viremia was detected,suggesting this attenuated virus was safe as a live vaccine.This vaccine could provoke humoral immunity and simulate the proliferation of lymphocyte in immunized guinea pigs.Challenge test showed that the vaccinted guinea pigs could defense against the virulent virus infection,manifested by no animals dying after infection and significantly low viral titers in respiratory organs,indicating this live vaccine was effective and safe.Many mutations ocurred during the passage in vitro and involved with the virulence attenuation of BPIV3.In order to explain the relationship between the virulence and genetic mutations,the reverse genetics system for BPIV3 should be established.Then the BPIV3 genomic cDNA and three helping plasmids were successfully constructed.This study lays the root for establishment of reverse genetic system of genotype C of BPIV3.