Isolation,Identification And Pathogenicity Of Subgroup B Avian Metapneumovirus

The natural host of avian Metapneumovirus(aMPV)is turkey,followed by chicken.Avian Metapneumovirus caused Turkey rhinotracheitis(TRT)in turkeys,the disease was the second most serious respiratory disease affecting turkeys.aMPV was also associated with swollen head syndrome(SHS)of chickens.When single aMPV infection occurred,the mortality rate was low(5%-10%)and egg production decreased(10%-30%).The greatest harm was that it was easy to cause other bacterial infections after aMPV infection,causing serious respiratory symptoms,and the mortality rate was as high as 25%.Up to now,aMPV has been reported in all over the world except Oceania.In 1999,aMPV was firstly isolated in China,and subsequently many researchers carried out a series epidemiological investigation.These epidemiological data showed that the seropositive rate of chicken farms was very high,the harm of this disease to poultry industry in China was increasing day by day.Due to it was difficult to isolate aMPV,there were few studies on the biological characteristics and pathogenicity of aMPV in China,which was not conducive to the effective prevention and control of the disease.In this study,to isolate virus from the turbinate bone and lungs of suspected SHS chickens,we blind-passaged all samples five times in Vero cells.Only one turbinate sample showed CPE in Vero cells.The Vero cells were observed to become rounding and pile up to form syncyties,which have typical cytopathic(CPE)characteristics caused by aMPV.The isolate was further confirmed using negative staining and EM.The virus showed an irregular and spherical.Enveloped partic les ranged from 50 nm to 200 nm in diameter,which has typical morphological characteristics of paramyxovirus,we named the isolated strain LN16.The Subtype-specific RT-PCR results indicated that the virus corresponded to subtype B aMPV.We further amplified and sequenced the G gene of LN16 strain,sequence analysis showed that LN16 shared above 93.8 %identity with subtype B aMPV,but less than 63.7% identity with subtype A,C and D aMPV isolates.Indirect immunofluorescence assay was performed with specific positive serum of subtype B aMPV.The results of indirect immunofluorescence observed by fluorescence inverted microscope showed that the infected Vero cells had green fluorescence signal.Taken together,the data confirmed that the LN16 strain belongs to subtype B aMPV.In order to understand the sequence characteristics of LN16 strain,seven pairs of primers were designed to detect the genome of LN16.Genome of the subtype B aMPV strain LN16 was 13,513 bp with eight genes,which encode nine protein.The genomic sequence of strain LN16 was deposited in GenBank under the accession number MH745147,which was the first complete genome of subtype B aMPV isolated from chicken.Sequence analysis showed that strain LN16 was 97.6% identical to the strain isolated from turkey.The aa sequences of strain LN16 were also highly homologous to those of the strain isolated from turkey(92.8–100%).However,gene insertion and deletion exist in the intergenic region,which was obvious difference.In order to further evaluate the pathogenicity of LN16 to chickens,chickens infected with LN16 strain at 3,6,9 and 15 weeks of age all had clinical symptoms such as nasal discharge,shaking,tearing and dispirited.The incidence rate was basically the same,all of which were above 70%.Shedding virus was observed from 2 to 5 days after infection,the shedding rate was highest at 4 dpi,all of which were above 83.6%.The results of virus tissue distribution showed that aMPV could be detected in turbinate bone,trachea,throat,occasionally in thymus,which was immune organ,from 2 to 5 days after infection.Histopathological analysis showed hemorrhage in the cortex and medulla,inflammation in the turbinate and lungs.These results indicated that LN16 was pathogenic.In this study,a strain of aMPV/B was successfully isolated from infected chickens.The first complete genomic sequence of aMPV/B isolated from chickens was obtained.Strain LN16 was therefore pathogenic to chicken.Which enriched the epidemiological data of aMPV and laid a foundation for the development of vaccine and other prevention and control products.