Study Of Molecular Epidemiology Of Caliciviruses And Inferaction Mechanism Between Viruses And Host

The family Caliciviridae is composed of small (27 to 40 nm), nonenveloped, icosahedral viruses that possess a linear, positive-sense, single-stranded 7 to 8 kb RNA (ssRNA) genome. They are 27-35 nm in diameter. Caliciviruses were classified into 4 genera in 2002 by the International Committee on the Taxonomy of Viruses (ICTV): Norovirus (NoV), Sapovirus (SaV), Vesivirus, and Lagovirus. The major medical pathogens in the family are NoV and SaV, which cause acute gastroenteritis. Important veterinary pathogens include vesiviruses such as feline calicivirus (FCV), which causes a respiratory disease in cats, and lagoviruses such as rabbit hemorrhagic disease virus (RHDV), which causes an often fatal hemorrhagic disease in rabbits. Human caliciviruses include both human NoV and SaV. Human NoV is believed to be one of the major causes of nonbacterial epidemic gastroenteritis, a disease that usually occurs in family or community-wide outbreaks. The impact of the SaV has not been fully established, but to date they do not share a predominant role with the NoV in epidemic gastroenteritis. Researches showed that both NoV and sapoviruses have been associated with gastroenteritis in infants and young children. Although no inter-species recombinant strain has been found, an inter-genogroup human calicivirus recombinant has been described. Additional, sometimes human strains genetically closely related to the animal strains. Specifically, porcine NoV are genetically closely related to some human NoV in the GII. All of these finding suggests a potential zoonotic risk and pig is considered as an animal reservoir for such viruses.Although some of great progress have been made in the research field of caliciviruses, there are still some unclear points, including (1) The interaction mechanism between caliciviruses and host cells; (2) The replication mechanism of caliciviruses in host cell and animal model; (3) Are caliciviruses zoonotic agents; (4) Pathogenesis of caliciviruses; (5) Recombinants between animal and human caliciviruses; (5) The receptor of SaV. In order to elucidate some points mentioned above, we performed the following researches: 1 Study of molecular epidemiology of caliciviruses(1) The first Chinese porcine sapovirus strain that contributed to an outbreak of gastroenteritis in pigletsWe report the first outbreak of gastroenteritis caused by porcine SaV in piglets in China. This outbreak occurred in February 2008 on a small commercial pig farm that lies in Shanghai suburb. Seven stool specimens were collected from seven piglets which showed symptoms of diarrhea and vomiting in two neighboring farrows on the farm. At the beginning, we first examined the seven specimens from the ill piglets for porcine circovirus, porcine rotavirus, porcine transmissible gastroenteritis virus, and porcine epidemic diarrhea virus using reverse transcription-PCR (RT-PCR), and all specimens showed negative results. All specimens were then examined for SaV by using RT-PCR as described previously. Our results showed that all seven piglets that showed clinical symptoms were positive for SaV RNA, suggesting this outbreak of gastroenteritis may due to porcine SaV infection.In order to elucidate whether the fecal samples contained infectious SaV, the eight SaV-positive fecal specimens were used to infect piglets. Results indicated that all of those experimental infection piglets showed symptoms of diarrhea and vomiting, and shed virus after inoculation.(2) Molecular detection and prevalence of porcine caliciviruses in eastern China Caliciviruses that causes diarrhea were reported in both industrial and developing countries including China recent years. Porcine caliciviruses closely related to human sapovirus and norovirus were also detected in swine group, which caused discussions about the animal reservoir and the potential risk for zoonotic transmission to humans. The objective of this work was to determine the frequency and distribution in the different age of swine for porcine sapovirus and norovirus, and characterize the strains prevalent in eastern China. A total of 904 stool samples from different ages of pigs were collected from eastern China from April 2008 to March 2009, and tested for both SaVs and NoVs using reverse transcription-polymerase chain reaction (RT–PCR). Our results indicated that 8 (0.9%) stool samples were positive for SaV and 2 (0.2%) for NoV. Phylogenetic analyses based on the partial RNA polymerase gene sequences indicated that all of the SaV isolates belonged to GIII, while the 2 NoV isolates belonged to GII. The 8 SaV isolates were further divided into 2 different groups in the GIII cluster, one of which clustered closely with the Netherland isolate (AY615804) and the other one with the Chinese strain (EU599212). Our results suggested that SaV infection was more frequently (p <0.01) in 0-1 month age of swine than that of other ages. In conclusion, the present study provided evidence that infection of PoSaV and PoNoV existed in pig groups of eastern China. (3) Prevalence of porcine caliciviruses and Hepatitis E Virus in pig farms of Guizhou province, ChinaA total of 209 stool samples from pigs of different age groups were collected from 6 pig farms in Guizhou province from May to June 2009, and tested for HEV, SaV and NoV using reverse transcription-polymerase chain reaction (RT–PCR). The overall prevalence of porcine HEV and porcine SaV were 6.7% (15/209) and 1.0% (2/209), respectively. No NoV infection was detected in the current study. The prevalence rates of porcine HEV infection for different ages of pigs were 15.4% (4/26; piglet, age<1 month), 6.8% (3/44; 4 month <age>1 month), 12.5% (6/48, age?4 month), and 1.1% (2/91, sow, 14<age>6 month), respectively. Porcine SaV was only detected in piglets (7.7%, 2/26). All 10 HEV isolates in the current study belong to genotype 4, clustering with a human HEV strain (AF103940) isolated from an adjacent province.This is the first report that porcine SaV exists in swine groups of Guizhou province, China. The porcine HEV isolates clustering with a human strain suggests the potential cross-species transmission between swine and human in this area.(4) Molecular characterization and phylogenetic analysis of the complete genome of a porcine sapovirus from Chinese swine The whole genome of Ch-sw-sav1 was amplified by RT-PCR and was sequenced. Sequence alignment of the complete genome or RNA dependent RNA polymerase (RdRp) gene was done. 3?end of ORF2 with 21-nt nucleotide insertion was further analyzed using softwares. Sequence analysis indicated that the genome of Ch-sw-sav1 was 7541 nucleotide long with two ORFs, excluding the 17 nucleotides ploy (A) at the 3?end. Phylogenetic analysis based on part of RdRp gene of this strain showed that it was classified into subgroup GIII. Sequence alignment indicated that there was an inserted 21-nt long nucleotide sequence at the 3?end of ORF2. The insertion showed high antigenicity index comparing to other regions in ORF2.(5) Prevalence and recombination of NoV in outpatients of Shanghai 452 stool samples from outpatients were collected from Shanghai, China. The fecal samples were tested for human caliciviruses. The full-genome of representative strains were then chosen and determined. Results showed that 20 NoV RNA positive samples were identified and the overall prevalence of NoV was 4.4%. No SaV positive were found in current study. In the present study, three full-length genomes of human NoV from China were determined and the genomic organization and recombination were analyzed. They had similar genome organization and contained three predicted ORFs, though the 5'UTR of those three strains were 2, 4 and 8 nucleotides, respectively. Phylogenetic analysis showed that the HU/GII/SHANGHAI/SH312/2008/CHN strain may be a recombinant of GII-3 capsid and GII-4 polymerase. To confirm the finding and detect the breakpoints where the recombination event occurred, we performed recombination analysis based on the genomic sequences of HU/GII/SHANGHAI/SH312/2008/CHN as the query sequence, and AB220921/NOV/JP/GII-4 and AB365435/NOV/US/GII-3 as the background sequences, using RPD software. Results indicated that the two parental strains were AB220921/NOV/JP/GII-4 and AB365435/NOV/US/GII-3. The breakpoint for this recombination event located at position 5107nt of the genome (in the ORF1 and ORF2 overlap).2 The interaction mechanism between SaV and host(1) Construction of animal models of porcine SaV infection In this study, 2mL of porcine SaV strain (Ch-sw-sav1) was orally inoculated to 11 Bama miniature pigs. Control group was orally inoculated with 2 mL of PBS. Clinical symptoms were supersied for each day. Piglets in both control group and tested groups were euthanized at 1,2,3,5,7,9,11,14,21,30,36 days post infection (PID) to observe possible pathological changes in heart, liver, spleen, lung, kidney, stomach, different part of intestines and inguinal lymph nodes. Pathological sections were inspected by optimal microscopy indirect immunofluorescence, scanning electron microscope and transmission electron microscopy. Results showed that comparing to control group, experimental groups piglets presented clinical symptoms such as diarrhea and vomiting. Pathological sections showed that mild to severe villous atrophy, mild tomoderate and multifocal villous fusion, and crypt hyperplasia were observed in the small intestine (mainly in the duodenum and jejunum). Viruses were detected by indirect immunofluorescence in epithelial cells of the mucosa. Transmission electron microscopy observations indicated that mitochondria of small intestine and stomach had visible. RT-PCR detection of virus in tissues showed that the piglets shed virus from PID 2 to the end of the experiment (PID36).(2) Relationship between porcine SaV and JAM-AThe available data strongly suggest that the recognition of a carbohydrate receptor may be a common feature of caliciviruses, even though they have adapted to different host species after a long course of evolution. Researches showed that histo-blood group antigens (HBGAs) and junctional adhesion molecule A (JAM-A) were the receptor of NoV and feline calicivirus (FCV), respectively. The binding assay confirmed that SaV VLPs can't bind the HBGAs molecular. In order to identify the relationship between SaV and JAM-A, rabbit fJAM-A-specific antiserum was used to block the infection. RT-PCR showed that none of the different diluted antiserum can block or decrease the infection, which mean that the JAM-A was not the receptor of SaV.(3) Screening the receptor of SaV from LLC-PK cells using VOPBA and Co-IP methods; MHC-I interacts with SaV in vitro To date, the receptor of SaV is still unknown. To screening and identify the receptor, both VOPBA and Co-IP assays were performed to screen interactive proteins from susceptible cell LLC-PK. After screening, target protein bands were determined using Mass Spectrum. Several proteins were isolated and MHC-I was chosen for the further analysis. Co-IP results showed that MHC-I interacted with Sav in vitro.3 VLPs expression of human NoV GII-4 and GII-12 strainsMost recombinant EIAs developed for human caliciviruses are based on use of baculovirus-expressed viral capsid antigens. The baculovirus-expressed human calicivirus capsid antigens self-assemble into virus-like particles that are morphologically and antigenically similar to the authentic viruses found in stool specimens, providing excellent reagents for development of immunologic assays. Results showed that both GII-4 and GII-12 capsid gene fragments were successfully expressed, moreover, those proteins can self-assemble into virus-like particles. Western-blot results indicated that those VLPs can be recognized by the antibodies which product from native virion. However, the VP1 fragment fusion expressed with EGFP can not be purified well though they express in Sf9 cells.