Isolation And Identification Of Bovine Enterovirus And Constrution Of Infectious CDNA Of HLJ-3531 Strain

The bovine enteroviruses(BEVs) are members of the family Picornaviridae that fall within the genus Enterovirus. BEV virions consist of a naked capsid that surrounds a core of positive-sense, single-stranded RNA that approximately 7.5kb long. Hydrated native particles are 25-30 nm in diameter in electron micrographs. BEVs are consist of Enterovirus species E(EV-E) and Enterovirus species F(EV-F) according to the Ninth Report of the International Committee on Taxonomy of Viruses states(ICTV). EV-E comprises four serotypes(E1–E4), and EV-F contains six serotypes(F1–F6).The ?rst bovine enteroviruses were collected in the late 1950 s, isolated from feces of healthy cattle by Kunin and Mc Ferran respectively. Since then, many other bovine enteroviruses have been isolated and studied. They do not appear to cause major disease in cattle, generally cause mild diarrhea or recessive through. However, they can result the emergence of a variety of clinical syndromes because sometimes they can invade other organs. Members of the bovine enterovirus species show a wider range of natural hosts, including cattle, water buffalo, African buffalo, sheep, goats, horses, dogs, alpaca and other animals. The pathogenicity and virulence of BEVs is still largely unknown. Failure to experimentally reproduce BEVs infection in calves showing obvious clinical signs led to the conclusion that BEVs are avirulent.In this study, first batch fecal samples were collected from bovine diagnosed with BVD, the second batch fecal samples were collected from bovine with complex clinical symptoms. Several serum samples were obtained from the same farm. BVDV Antigen Test Kit/Serum Plus(IDEXX) was failed to identify BVDV antigens in serum and enzyme-linked immunosorbent assay(I-ELISA) for the detection of BVDV antibodies in serum was also failed. A total of 10 cytolytic strains were isolated from two batches fecal samples, nested RT-PCR for BVDV failed to detect viral genomes. These unknown isolated virus were identified by nucleic acid type and the results indicated they were RNA virus; negative-staining EM revealed the presence of numerous uniformly shaped non-enveloped virus particles approximately 25~30 nm in diameter, which is consistent with the size of picornaviruses; biological and physiochemical properties showed that these isolates wre resistant to treatment of organic solvent and acid, and unstable at 56℃for 30 min; RT-PCR was used to amplify underlying viral sequences and identify the isolated virus. Phylogenetic analysis indicated that the two isolated virus closely matches EV-E and EV-F respectively.The virus isolated from fecal sample 3531 and 4149 were named HLJ-3531 and HLJ-4149 and used in subsequent experiments.Serum neutralization(SN) test showed that two of nine bovine serum samples had neutralizing antibody for HLJ-3531, five of nine bovine serum had neutralizing antibody for HLJ-4149. Compared with electron microscopy of virus, the aggregation of virus were obviously seen in immunoelectron microscopy. Comparision of the two strains of cell tropism showed that the two strains both can infect cattle, pig, rabbit, monkey, chicken, dog, hamster, cat and other animal cells. Interferon showed antiviral activity for the two virus.To analyze sequences and the evolutionary relationships of two strain,degenerate primers were designed for overlap RT-PCR, for amplification of HLJ-3531 and HLJ-4149 sequences respectively, according to sequence published online. To ensure the authenticity and accuracy of gene sequences, 5’RACE was used for amplification of 5’UTR, the other fragments were amplified by high-fidelity DNA polymerase. The complete genome sequence of HLJ-3531 strain was 7448 bp, HLJ-4149 strain was 7437 bp, consisting of 5’UTR, ORF, 3’UTR and ploy(A) tail. The nucleotide homology of two strain was 68.40%; amino acid of ORF homology was 73.40%. Phylogenetic trees were constructed according to nucleotide and amino acid sequence of VP1, the results showed that HLJ-3531 belongs to EV-E2 branch, while HLJ-4149 belongs to EV-F1 branch.ICR suckling mice were used for buliding suckling mouse infection model, for analyzing the pathogenicity of the two strains. Sulking mice were inoculated intraperitoneally with 50μL(108TCID50/0.1m L) of virus, and the mice were observed daily for clinical illness until 21 d after treatment. The heart, liver, intestine, lung tissues of sulking mice were collected on 4, 7, 14, 21 days p.i. Virus infection was veri?ed by RT-PCR, HE staining, SN testing. Compared with the control group, hemorrhagic intestinal mucosa were only observed in mice infected with HLJ-3531 21 d p.i.. Tissue RT-PCR results showed that, two strains could be detected in liver, lung, intestine on 4d, 7d and 14 d p.i., detection for HLJ-4149 was failed while HLJ-3531 still could be dected on 21 d p.i. Both two strains couldn’t infect heart of mice. The results of HE staining showed that, HLJ-3531 could infect mice throughout the observation period, lesions of intestines, liver and lung tissues were apparent and gradually increased over time; HLJ-4149 could infect mice throughout the observation period, and lesions of intestines, liver and lung tissues were apparent, the pathological increased over time firstly(0â†'4â†'7d), then began to recover slowly(7â†'14â†'21d). None of the heart tissue lesions appear in mice. Serum neutralization test showed that mice infected with two strains produced antibodies on 7 days, then gradually increased(14 days), serum from mice infected with HLJ-3531 remained high neutralizing titers on 21 days p.i. while the antibody level in serum of mice infected with HLJ-4149 was decline on 21 days p.i. The results of suckling mice infection modle showed that the two strains could infect mice and cause pathological changes respectively, but there are differences in the extent and duration of lesions. Since the two strains were isolated from the same cattle and neutralizing antibodies for two strains were detected in the same bovine serum, it is suspected that there are multiple infection in this farm. To verify whether the two strains can infect the same individual animals and cause pathological changes, ICR suckling mice were used as animal model for simulating the multiple infections. Sulking mice were inoculated intraperitoneally with 50μL mixture with equal volum of HLJ-3531 and HLJ-4149, infected group showed no significant differences compared with the control group throughout the cycle. The most signi?cant gross ?nding of necropsy was marked intestinal mucosal hyperemia, hyperemia on 14 d and 21 d p.i., and swelling of the hepatic lobe on 21 d p.i. The results of RT-PCR and HE staining revealed that two strains can replicate in same tissues of the same individual mice, lesions caused by co-infection with HLJ-3531 and HLJ-4149 are stronger than separate infection with HLJ-3531 or HLJ-4149. Serum neutralization(SN) test showed that neutralizing antibody for two strains were existed in the same individual and increased over time. In this study, suckling mice infection model of bovine enterovirus was established for the first time and preliminary results of BEVs pathogenicity were achieved. This study lays a solid foundation for the development of the pathogenesis of bovine enteroviruses.Primers were designed for construction of the full-length c DNA according to the full genome sequence of HLJ-3531 strain, with p Bluescript SK(-) as the cloning vector. Full-length c DNA construction was based on the methods combination of fusion PCR and enzyme digestion. Full-length sequence of HLJ-3531 is divided into two gene fragments of 3531-A and 3531-B, T7 promoter were added to 5 ’end of 3531-A.The 3531-A fragment was obtained by fusion PCR from three small fragments. The 3531-B fragment was combined by another two small fragments, and mutated Nae I restriction site was introduced as molecule label in overlap portion of those two fragments(synonymous amino acid mutation). After linearization by Eco R V, recombinant plasmid p BSK-3531 was transfected into BSR cell line which can express T7 RNA polymerase stably. The recombinant virus r HLJ-3531 was identified by RT-PCR, sequencing of molecule label, indirect immunofluorescence and immune electron microscopy. By comparison tests, it was shown that the recombinant virus had similarity with the parent strain in host tropism, TCID50 and growth curves. The results of the study demonstrated that the infectious clone of bovine enterovirus HLJ-3531 strain was constructed successfully. This study provided good foundation on the future research of the structure, function, host tropism and pathogenesis of bovine enterovirus.