Molecular Evolution Analysis Of Group C Rotavirus And Genogroup1Picobirnavirus In Shenzhen

BackgroundWorld Health Organization data show that80million children (less than5years old) worldwide died from diarrheal diseases each year. In China, the2010national healthy report cases of infectious diarrhea in740,000cases, and viral infections accounted for92.7%of laboratory-confirmed cases. Group A rotavirus, Human astrovirus, Calicivirus and Human adenoviruses are common diarrhea pathogens, Torovirus, Coronavirus, Group C rotavirus, Picobirnavirus, which were gradually identified as diarrhea emerging pathogens. Group C rotavirus and Picobirnavirus were detected in a wide range of global and all were segmental virus, both are prone to recombine and reassortment was great potential dangerous to human health. Shenzhen is a big migrant city, a high likelihood of viral genetic exchange occurs, and two viruses has not been detected in Shenzhen before, so the research of great significance.Group C rotavirus (GCRV) belonging to Reovirus families Rotavirus genera, can cause acute gastroenteritis in humans and animals, including11genome segments of double-stranded RNA, encoding six viral structural proteins (VP1-VP4, VP6, VP7) and six non-structural proteins (NSP1-NSP6). GCRV worldwide are detected, the host including people, pigs, cattle, dogs, chickens, also was detected from sewage. GCRV mainly caused sporadic diarrhea epidemic, currently outbreaks only were reported in Japan, China and the United Kingdom before. Global Serological monitoring showed, national population infection rate of GCRV33%～39%, the elderly population was highest. Contrast urban areas, GCRV infection infection rates of rural areas, younger and higher. In addition to VP3genes, all human GCRV are highly conserved RNA segments, of which the highest homology between VP6gene, rather than the nonstructural protein gene. Mix infection of GCRV from different hosts has been reported, suggesting that GCRV may exceed the species restriction, to achieve cross-species transmission between humans, pigs, cattle. Rotavirus of different groups’co-infection may result in gene transfer and will increase the intestinal damage. Compared with GARV causing severe diarrhea, symptoms caused by GCRV was relatively mild, but GCRV may cause infants biliary atresia and may result in reduction in production of Milk from cow, human health and the economy has adversely impact.Picobirnaviruses (PBV) is a small, nonenveloped, double-stranded RNA vir uses, a large segment of the genome encoding the capsid protein and a small segment of genome encoding an RNA-dependent RNA polymerase. PBV host, various vertebrates including humans. Since PBV was detected in diarrhea sam ples from an acute gastroenteritis children of an outbreak in1988. Role of PB V caused diarrhea has been the focus of research. PBV also be frequently dete cted in healthy nondiarrhea hosts, some speculated PBV may be a chance path ogen in immunocompromised individuals. Early detection of PBV mainly by P AGE-SS method, PBV was divided into large-and small-genome type. Base on the RdRp conserve region of two prototype strains (GGI/PVV/human/China/ 1-CHN-97/1997and GGII/PBV/human/USA/4-GA-91/1991), the genotyping prim er was design and was verified in multiple studies in the world. PBV were de tected in human fecal samples in a number of countries, from as low as0.09%to13%. Currently PBV was detected from the feces and the environment is most of GGI PBV, also, at least95%sequences of PBV on GenBank wer e about GGI PBV. GGI PBV has been the predominant genogroup of PBV, bu t it might be the primer of GGII PBV lack of detection capabilities. Currently phylogenetic studies about PBV did not show any clustering based on geogra phical distribution, suggesting that the prevalence of PBV and simultaneous tra nsmission. All Pig PBV belong to GGI PBV, suggesting that PBV may exist a s quasispecies. Phylogenetic analysis showed that porcine PBV associated with human PBV that also has a relationship with the pig PBV, which may be sign ificant cross-species transmission. Early studies showed that the nucleotide sequ ence not found the same in PBV strains isolates from the same location, even compared to a number of isolates from the same acute gastroenteritis outbrea ks, the relationship between them was closer. Later, someone detected PBV iso late in diarrhea foal in Calcutta, its sequence was fully consistent with the seq uence of human PBV isolated from the same area, and was closely related to the swine PBV reported in Hungary. Latest research about environmental virus show the pathway of PBV get through the water seems to be a reasonable e xplanation to these seemingly contradictory on the foregoing findings.MethodsViral diarrhea suspected samples from Shenzhen infectious diarrhea pathogen spectrum monitoring network in2011and2012was collected and was extracted RNA to detect GCRV and GGI PBV. Fluorescent real-time PCR and RT-PCR was used to screen Group C rotavirus and Picobirnaviruses, respectively. Group C rotavirus-positive isolates obtained was amplified the complete genome sequence by17pairs of specific primers of Group C rotavirus. Bidirectional Sequence the amplification of specific fragments and splicing them. Download homologous sequences of the GCRV and GGI PBV isolated from this study from the GenBank database, and acquire information about the sequence. Finding the best nucleotide substitution model and using the maximum likelihood method to reconstruct maximum likelihood tree. By BLAST search, finding the most relevant isolates to the GGI PBV isolated. Construction of nucleotide and amino acid similarity similarity matrix to understand the similarities between positive strains of GGI PBV, phylogenetic tree also been used to understand clustering of GGI PBV isolated in Shenzhen.Results1Two GCRV was isolated from1158diarrhea suspected samples in2011, named as94-SZ11strain and272-SZ11strain. Two GCRV positive diarrhea patients were adults, but t there are not statistical difference of detection rate of GCRV in each group of age, gender, season, hospital, the symptoms of diarrhea are slight, no mixed infection.2Successfully amplified and sequenced the complete genome sequence of2GCRV isolates. Nucleotide and amino acid homology of11segments gene was≥96.1%and≥97.0%. Reconstruction of maximum likelihood tree of11segments gene based on the optimal nucleotide substitution model, GCRV iolates in Shenzhen all are G4-P [2]-I2-R2-C2-M3-A2-N2-T2-E2-H2-type.38GGI PBV was screened from1030diarrhea suspected samples, named78-SZ12,184-SZ12,187-SZ12,275-SZ12,406-SZ12,740-SZ12,827-SZ12,1077-SZ12. All GGI PBV positive patients are adult, ratio of male to female are5:3, both from Peking University Shenzhen Hospital and Longgang People’s of Hospital. detection rates of GGI PBV between age, gender, hospitals are significant differences, there is no statistically significant difference in the seasonal distribution. Mix infections of5GGI PBV positive patients with norovirus GII type.4Low genetic similarity of8GGI PBV isolates in Shenzhen, averages similarity of nucleotide and amino acid were70%and74%, respectively. The closest reference strains of8GGI PBVs, four are detected in wastewater from the United States, four are among the United States, India, Hungary detection, display nucleotide homology of79%-100%. Phylogenetic tree shows8GGI PBV in Shenzhen cluster two clades, one clade close to the reference strains from Netherlands and Hungary, where another clade close to the reference strains from India and the United.Conclusion1. GCRV detection rate was0.16percent in Shenzhen, no significant differences in age, gender, seasons and hospital distribution. Two GCRV isolates94-SZ11and272-SZ11belong M3group, the group most closely with the East Asian strains, strains of group relations in South Asia and far.2. GCRV at the time and place of origin of the world is likely to be in Melbourne, Australia in1973. Korea CAU10-312Bristol strain and strain may be recombinant strain V508strain spread to South Korea.3. GGI PBV only detected from adults in Shenzhen, there is no obvious seasonal characteristics, most with mixed infection.8GI type PBV divided into two clades, isolates in the same clade of high similarity, the similarity between different clades was low.