Molecular Characterization Of Chinese H16/h165 Of Porcine Transmissible Gastroenteritis Virus And Preliminary Exploration On Development Of The Reverse Genetics System

Porcine transmissible gastroenteritis virus (TGEV) was initially identified as the etiological agent of transmissible gastroenteritis (TGE) in swine in 1946 in the United States. In neonates, TGEV infects the epithelial cells of the small intestines, leading to potentially fatal gastroenteritis. The virus can also lead to infection in the upper respiratory tract and less often, in the lungs. In adults, TGEV causes mild disease. In swine, it is the major cause of viral enteritis and fetal diarrhea in neonates, resulting in significant economic losses. TGEV was reported in many swine-producing countries between the late 1980s and the 1990s. TGEV strains of varying virulence have been isolated and characterized worldwide. Some strains have been used to develop modified live vaccines with limited success. In China, a TGE outbreak was first reported in the 1970s. Since then it has been prevalent in many provinces and has become one of the most important viral diarrhea diseases in China. The Chinese TGEV vaccine strain H165 was derived from a virulent field strain H16 by 165 passages in PK15 cells. Vaccines based on the H165 strain are currently commercially available to prevent and control TGEV infections in China. H165 virus was proven to be safe in piglets and pregnant sows and efficacious against TGEV infection. Whole genome sequences of strains H165 and H16 will help us to understand the genetic basis of TGEV attenuation and enhance the geographic differentiation information among TGEV strains.Up to now, there were only seven TGEV strains and one PRCV strain PRCV-ISU-1 had been fully sequenced, though partial sequences of TGEV strains were available in the GenBank. Moreover, only two virulent and attenuated TGEV pairs were reported with difference in the gene 3 region. Previously studies have shown that there is a genetic diversity in the genomes of TGEV, especially in the gene 3. However, this report is a first one dealing with the genetic diversity in the gene 3 of Chinese field TGEV strains and reference TGEV strains. Our findings showed that 8 Chinese TGEV strains were genetically diverse in the gene 3 among themselves as well as in comparison with the reference strains. The nucleotide sequence data revealed genetic diversity in the gene 3 region of the Chinese field TGEV strains, even though the viruses were isolated from the same place. These results also indicated that different strains were present in the same farm.There were a total of 27 nt mutations identified in strain H165, resulting in a total of 16 aa mutations mainly located within proteins 1a, 1ab, S, 3a, 3b, and E. Furthermore, six nt mutations (G6014TORF1a, T12388CORF1b, T21937CS, T21969AS, A26025CE, and C27507TN) could be the makers used to differentiate the Chinese vaccine strain from other strains of TGEV in the GenBank. Vaccines based on passages 155 to 165 in cell cultures are available commercially as vaccines for the prevention and control of infections with TGEV in China. Nucleoprotein (N) sequences of the virus at passages 155 and 165 were aligned and compared using a computer software program. The suitability of restriction fragment length polymorphism (RFLP) analysis for differentiation of the vaccine strain from the other TGEVs was investigated. The RFLP analysis identified a change in the cleavage sites of Acl I at passages 155 and 165. This RFLP pattern of the N gene differentiated the Chinese vaccine strain from its parental strain, the TGEVs studied and the other reported TGEVs in the GenBank. Phylogenetic and sequence analysis results showed that the Chinese TGEVs were divided into three groups with several specific nucleotides and amino acids among them. These findings suggest that Chinese strains of TGEV are evolving continuously.In order to determine the genetic basis of TGEV attenuation for virulent H, we would like to construction the reverse genetics system based on the pBAC-5`- 3` vector, which was kindly provided by the Prof. Luis Enjuanes in Campus Universidad Autonoma. Five Intermediate vector plasmid with the corrected sequences were prepared to insert into the pBAC-5`- 3` vector. Up to now, three fragments had been inserted into the vector, In order to construct the reverse genetics system of attenuated H, it is necessary to insert the left two fragments into the vector in the following study.