Isolation And Characterization Of A Herpesvirus From Feral Pigeons And Bioinformatics Analysis Of UL27 Gene

Avain herpesvirus exists in many kinds of birds and widely infects chickens and ducks. Now, avain herpesvirus infection presents diversification. The infection is no longer limited to domestic poultry, but also found in pigeons. The naturally infected pigeons exhibited anorexia, lassitude, severed serous conjunctivitis. Anatomic changes included upper respiratory tract inflammation, ulcers, necrotic liver, spleen, kidney, and pancreatic. Small necrotic lessions could be seen on the surface of the liver. Histopathological examination showed intranuclear inclusion bodies in hepatocytes. Necrotic foci and inflammatory cells infiltration could be seen in the liver, spleen, kidney, and pancreas. This brings serious economic loss to pigeons industry. Pigeon herpesvirus (PiHV), also named Columbid herpesvirus-1 (CoHV-1), belongs to the Mardivirus genus within the subfamily Alphaherpesvirinae and the family Herpesviridae.CoHV-1 was first reported in 1945 and became one of major diseases in pigeons industry among the worldwide afterwards. The KangDa Livestock and Poultry Disease Consulting Service Centre received a severe outbreak report occurring in feral pigeons in areas surrounding Heilongjiang Province in 2013. Epidemiology, clinical symptoms, isolation, purification, physicochemical property, molecular biology method, domestic pigeon regression experiment of our isolated strains from infected feral pigeons proved the isolated virus was CoHV-1. gB protein is encoded by UL27 gene and is one of the main capsule membrane glycoproteins of herpesvirus. It plays an important role in the process of virus infection. Thus, the research on UL27 gene of CoHV-1-HLJ plays a guiding role in the molecular biology function, clinical diagnosis, prevention, and genetic engineering vaccine. In this study, we cloned UL25, UL26, and UL27 gene sequences of CoHV-1-HLJ by degenerate primers PCR and gene walking PCR, which is about 1755bp,2328bp, and 2788bp encoding 584,775, and 995 amino acids. The sequences of UL27 gene were submitted to Genbank KU051711. We analysed the antigencity of gB protein. One fragment of gB protein with better antigenicity in the extracellular area was cloned into the expression vector pET28a (+) to construct the recombinant expression plasmid pET28a-CoHV-gB, which was transformed into E.coli BL21 (DE3) to express the recombinant protein. The results of SDS-PAGE showed that the protein was mainly expressed in inclusion bodies, approximately 37.5 kDa. Recombinant protein could react with anti-CoHV-1-HLJ rabbit serum and has relatively strong reactivity.In a word, this is the first study on the isolation and characterization of a herpesvirus from feral pigeons in China. The main clinical characteristic of pigeons infected with CoHV-1-HLJ is difficulty breathing and conjunctivitis. Necrosis was the major pathological change in the liver, kidney, spleen, and pancreas. The main antigen region of UL27 gene of CoHV-1-HLJ was pET28a-CoHV-gB (231-510aa). Our study lay a foundation for us to understand the molecular prevalence of CoHV-1, diagnosis prevention and control, and vaccine strain screening.