| Emerging viral infecting disease endangers the health of human and animals. Discovery and identity of novel viruses are the primary task for prevention, diagnosis and therapy of novel emerging viral infecting disease. Viral metagenomics coming from metagenomics is a new technology which is mainly used to study the virome of particular environment. This technology has been extensive used to discovery new virus,and the results is wonderful. Diarrhea is common disease in people of different age groups, also is the main cause which reduces the economic performance in pig husbandry. Worldwide, approximately 1.5 billion persons suffer from diarrhea and more than 2 million children less than 5 years die from it annually, mainly in children and elders, especially in developing countries. At present, using existing diagnostic technique, only 60% of cases with diarrhea can be diagnosed, but no etiologic agent can be identified in up to 40% of cases. The etiological factor of diarrhea of unknown origin may be novel viruses that are not discovered. Swine as economic animal produce lots of meat for mankind, but which also is the host of etiologic agent of zoonosis. Diarrhea is common disease in piglets and causes severe economic loss in pig husbandry.Our country is developing country. Diarrhea is one of main diseases which affect the heath of our people, especially the heath of Children. Analysis of the virome of stools from Children with diarrhea and discovery of the potential novel viruses will be the best way to diagnosis the diarrhea of unknown origin. Cloning the genomes and the epidemiological study of novel viruses will lay the foundation of the prevention and therapy of diarrhea causing by novel virus. Pig husbandry plays an important role in animal husbandry. Prevention and control of pig disease is the best way to improve the economic performance, and also to control the zoonosis in swine. Study of viromes of stools from diarrhea pigs and healthy pigs and discovery of the potential novel viruses will provide reliable data for diagnostic of porcine viral diseases; cloning the genomes and the epidemiological study of novel viruses from swine will lay the foundation of the prevention and therapy of porcine viral diseases. The main topics and results of this thesis are as following:(?) Viral metagenomic analysis of the viromes of stools from human and swine(1) Viral metagenomic analysis of the virome of stools from childrenIn this study, using the method of viral metagenomics, the libraries of virome of 12 stool samples from children in shanghai were constructed, and 840 clones of the libraries were sequenced by Sanger sequencing. The results showed that 31.4% of all the reads hit to viruses, 31.4% of all the reads hit to phages, 2%% of all the reads hit to bacterium and 60.7% of all the reads are unknown sequences. Viral sequences mainly hit to enteroviruses, bocaviruses, astroviruses, Aichi viruses, human parechoviruses, Klassevirus/Salivirus and Human coavirus.(2) Viral metagenomic analysis of the virome of stools from swine in our countryIn this study, using the method of viral metagenomics, the libraries of virome of 17 stool samples from swine in our country were constructed, and 1190 clones of the libraries were sequenced by Sanger sequencing. The results showed that 32.9% of all the reads hit to viruses, 7% of all the reads hit to phages, 2.2%% of all the reads hit to bacterium and 57.9% of all the reads are unknown sequences. Viral sequences hit to enteroviruses, bocaviruses and astroviruses.(3) Viral metagenomic analysis of the virome of stools from swine in USAIn this study, using the method of metagenomics,the library of virome of stool samples from 18 piglets with diarrhea and 18 healthy piglets in USA were constructed. A total of 652,614 sequence reads in this library, the length of the smallest read and biggest read and is 40bp and 699bp, and the average length is 236bp.After tags of primers trimmed from the ends, the useful sequences are more than 57 millions and the average length is 192bp in the library. The results showed that 0.2% of all the reads hit to eukaryotic genes, 0.2% of all the reads hit to other sequences, 9%% of all the reads hit to bacterium, 10% of all the reads hit to phages, 13% of all the reads are unknown sequences and 68% of all the reads hit to viruses. Viral sequences hit to enteroviruses, Sapeloviruses, astroviruses, Kobuviruses, Sapoviruses, Teschoviruses, Circo-like viruses, bocaviruses and Rosaviruses.(?) Preliminary study of 23 strains of viruses(1) Preliminary study of 6 strains of astrovirusesIn this study, the genome of the first strain of HAstV1 in shanghai was cloned, and which is 6808bp. Phylogenetic analysis of the nucleotide of ORF2 showed that this strain is more close to Japanese strain (AB000285). Using RT-PCR, 279 samples from children with diarrhea in shanghai were screened and the results showed that infection rate of HAstV is 8.2% and main infection is HAstV1.The genome of the first strain of PAstV in our country was cloned, and which is 6610bp. Phylogenetic analysis of amino acids of ORF2 showed that this strain belonged to PAstV1. Using RT-PCR, 340 samples from 4 provinces and 1 city in our country were screened and the result showed that infection rate of PAstV1 is 22.9%.The genomes of four strains of PAstVs (PAstV2-43?PAstV2-51?PAstV4-35 and PAstV5-33)in USA were cloned. Phylogenetic analysis of amino acids of ORF2 showed that PAstV2-43 and PAstV2-51 are PAstV2,which and the stains of Canada belong to one clade; PAstV4-35 is PAstV4, and which and the stain of Hungary belong to one clade; PAstV5-33 belong to a new clade,and which has less than 26% identity with all known astroviruses; PAstV5-33 may be a new species.(2) Preliminary study of 2 strains of HPeVsUsing RT-PCR, 220 samples from children with diarrhea in shanghai and 116 samples from monkeys with diarrhea were screened. The results showed that infection rate of HPeVs in children and monkeys are 20.5% and 5.2%. In this study, HPeVs were firstly discovered in stools of monkeys.In this study, the genome of the first strain of HPeV1 was cloned, which is 7259bp. Phylogenetic analysis of nucleotide of P1 region showed that this strain and the strains of Germany (EF051629) and Netherland(FM178558)belong to one clade. The genome of the first strain of HPeV6 from monkey was cloned, which is 7311bp. Phylogenetic analysis of the genomes showed that this strain and the strains of Japan (AB252582) and Netherland(EU077518)belong to one clade.(3) Preliminary study of Salivirus/KlassevirusIn this study, the genome of the first strain of Salivirus/Klassevirus was cloned, which is 7798bp. Phylogenetic analysis of nucleotide of P1 region showed that this strain and the strains of Salivirus/Klassevirus reported belong to a new clade. The result showed that salivirus/klassevirus may be a new genus of picornaviridae. Using RT-PCR, 216 samples from children with diarrhea in shanghai were screened. The result showed that infection rate of HPeVs in children is 4.2%. The Fisher exact test showed a significant (p=0.03) association between salivirus/klassevirus detection and diarrhea.(4) Preliminary study of 8 strains of bocavirusesIn this study, the genomes of 2 strains of HBoV1 and one strain of HBoV2 were cloned, which are 5288bp, 5277bp and 5244bp. Phylogenetic analysis showed that HBoV1 and the strains of our country reported belong to a clade and HBoV2 and the strain of Pakistan (FJ170279) belong to another clade. Using PCR, 320 samples from children with diarrhea in shanghai were screened. The result showed that infection rate of HBoV1 and HBoV2 are 5.3% and5.0%, respectively.Using PCR, 320 samples from dogs with diarrhea from 3 pet clinices in shanghai were screened. One sample is positive with CnMV. Based on the genome of American strain (FJ214110), primers designed for cloning the first genome of CnMV in our country, which is 5132bp.In this study, using genomic walking, the genomes of two strains (PBoV1-H18 and PBoV2-A6) of PBoVs in our country were cloned, which are 5267bp and 5117bp. Phylogenetic analysis of the full genomes and the nucleotide and amino acid of 3 genes showed that PBoV1-H18 and porcine boca-like virus belong to a clade and PBoV2-A6, PBoV1 (HM053693) and PBoV2 (HM053694) belong to another clade. Epidemiological study showed that that the high infection rate of HBoV1 and HBoV2 are 75% and 70.1%.In this study, the genomes of two strains of PBoV3 in USA were cloned, which are 4710bp and 4689bp. Phylogenetic analysis of protein of VP1 showed that the two strains of PBoV3 and 6V and 7V belong to a clade. We tentatively presume that PBoV may contain 3 species (PBoV1, PBoV2 and PBoV3).(5) Preliminary study of 4 strains of Po-Circo-like VirusIn this study, the genomes of 4 strains of Po-Circo-like Viruses in USA were cloned, which are 3912bp, 3923 bp, 2904bp and 2833bp, respectively. Phylogenetic analysis of protein of Rep showed that 4 strains of Po-Circo-like Viruses belong to a new clade. they may belong to a new genus. The motif and conserved region found in the 4 strains of Po-Circo-like Viruses are loop-stem structure, viral Rep and RNA helicase.(6) Preliminary study of Posavirus 1 and Posavirus2In this study, the genomes of 2 strains of a new member of the order Picornavirale in USA were cloned, which are 9816bp and 10191bp, tentatively named them as posavirus 1 and posavirus2. Discriminant analysis of mononucleotide and dinucleotide frequencies showed that posavirus 1 and posavirus 2 belong to the region of insects. Phylogenetic analysis of protein of RdRp region showed that t posavirus 1 and posavirus 2 belong to a new clade. They may belong to a new genus or family in the order Picornavirale. Using RT-nPCR, 36 samples from piglet were screened. The infection rate of posavirus 1 and posavirus 2 are 47.2% and 30.6%, respectively. |
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