病毒宏基因组学分析儿童和猪肠道病毒群落及23株病毒的初步研究
发布时间：2018-09-09 12:01
【摘要】：新发病毒性传染病危害人和动物的健康。挖掘和鉴定新病毒是预防、诊断和治疗新发病毒性传染病的首要任务。病毒宏基因组学是在宏基因组学的基础上发展起来的一门研究特定环境中病毒群落的新兴技术。该技术已经被广泛地应用到挖掘新病毒,并且取得可喜的成就。腹泻是各个年龄段的人群中常见的疾病,也是影响养猪业经济效益的主要疾病之一。在世界范围内,每年有将近15亿人腹泻,其中儿童和年长者发病率高;有200多万5岁以下腹泻儿童死亡,并且发展中国家占有较大的比例。目前,现有的检测技术只能诊断出60%的腹泻病例的病因,而近40%的病例查不出病因。引起未知病因的腹泻的致病因素,可能是目前暂未被发现的病毒。猪作为经济动物,为人类提供了大量的肉类,同时也是携带人兽共患病的宿主。腹泻是仔猪的常发疾病,给养猪业造成严重的经济损失。我国是发展中国家。腹泻是影响我国人民的健康主要疾病之一,尤其是儿童的健康。分析腹泻儿童粪便中的病毒群落和发掘潜在的新病毒将为诊断不明腹泻的病因提供新的途径。克隆新病毒的基因组和流行病学调查将为预防和治疗新病毒引起的腹泻奠定基础。养猪业在世界养殖业占有重要的地位。预防和控制猪病是提高养猪效益的最好的解决办法,同时也是控制猪源性人兽共患病的有效途径。研究腹泻猪和健康猪粪便中的病毒群落及挖掘潜在的新病原,将为诊断猪病毒性疾病提供可靠的资料;克隆猪源性新病毒的基因组和流行病学调查将为预防和治疗猪病毒性疾病奠定基础。 本论文主要研究内容和结果如下: (一)病毒宏基因组学分析人和猪粪便中的病毒群落 (1)病毒宏基因组学分析腹泻儿童粪便中的病毒群落 本实验应用病毒宏基因组学方法,构建了12份上海腹泻儿童粪便中病毒群落的文库,通过sanger测序法分析了总文库中的840个克隆。结果显示:在上海腹泻儿童粪便文库中,31.4%的序列匹配到病毒,5.8%的序列匹配到噬菌体,2%的序列匹配到细菌和60.7%的序列是未知序列;病毒序列主要匹配到肠道病毒、博卡病毒、星状病毒、Aichi virus、双埃克病毒、Human coavirus和Klassevirus/Salivirus; (2)病毒宏基因组学分析我国猪群粪便中的病毒群落 本实验应用病毒宏基因组学方法,构建了17份我国猪粪便中病毒群落的文库,通过sanger测序技术分析了总文库中的1190个克隆。结果显示:32.9%的序列匹配到真核病毒,7.0%的序列匹配到噬菌体,2.2%的序列匹配到细菌和57.9%的序列没有匹配到任何序列。病毒序列匹配到得猪肠道病毒、博卡病毒和星状病毒。 (3)病毒宏基因组学分析美国猪群粪便中的病毒群落 本实验应用病毒宏基因组学方法,通过454高通量测序分析美国18个健康猪和18个腹泻猪粪便中病毒群落文库。结果显示:该文库包含652,614个序列,最小测序长度为40bp,最大测序长度为699bp,测序平均长度为236bp。去除引物标签后,文库中的有效序列大于57万条,平均长度为192bp。序列比对结果显示:0.2%的序列匹配到真核基因、0.2%的序列匹配到其他序列、9%的序列匹配到细菌、10%的序列匹配到噬菌体、13%的序列是未知序列及68%的序列匹配到病毒。病毒序列匹配到肠道病毒、Sapelovirus、星状病毒、Kobuvirus、Sapovirus、Teschovirus、环状类病毒、博卡病毒和Rosavirus。 (二)23株病毒的初步研究 (1)6株星状病毒的初步研究 本实验克隆了上海第一株HAstV1的基因组,其大小为6808bp。分析ORF2的核苷酸系统进化树,结果显示本分离株与日本分离株AB000285比较相近。通过RT-PCR检测了上海腹泻儿童的279份样品,结果显示:HAstV感染率为8.2%,主要为HAstV1。 本实验克隆了我国第一株PAstV的基因组,其大小为6610bp。分析ORF2编码的氨基酸的系统进化树,结果显示:本株PAstV属于PAstV1。通过RT-nPCR检测了来自我国5省市的340份猪粪便样品,结果显示有PAstV1感染率为22.9%。 本实验克隆了美国猪群中4株新的PAstVs(PAstV2-43、PAstV2-51、PAstV4-35和PAstV5-33)。分析ORF2编码的氨基酸的系统进化树,结果显示:PAstV2-43和PAstV2-51属于PAstV2,与加拿大的聚为一支;PAstV4-35属于PAstV4,与匈牙利的聚为一支;PAstV5-33单独聚为一支,并且其与所有已知的AstV的氨基酸同源性都小于26%,其可能为PAstV的一个新种。 (2)2株HPeVs的初步研究 通过RT-nPCR检测了220份上海腹泻儿童样品和116份腹泻猴粪便样品中的HPeVs。结果显示:上海腹泻儿童粪便中的HPeVs的阳性率为20.5%;腹泻猴粪便中的阳性率为5.2%。本研究首次在猴粪便中发现HPeV。 本实验克隆了我国第一株HPeV1的基因组,其大小为7259bp。系统进化树分析P1区核苷酸序列结果显示该株与德国株(EF051629)和荷兰株(FM178558)的HPeV1聚为一支。克隆了来自猴粪便中的第一株HPeV6的基因组,基因组大小为7311bp。系统进化树分析HPeV6的全基因组结果显示该株和日本株(AB252582)和荷兰株(EU077518)聚为一支。 (3) Salivirus/Klassevirus的初步研究 本实验克隆了我国第一株Salivirus/Klassevirus的基因组,其大小为7798bp。系统进化树分析P1区氨基酸结果显示该株与已报道的Salivirus/Klassevirus聚为一支。该可能病毒为picornaviridae中一个新的属。 通过RT-nPCR检测了216份上海腹泻儿童样品和96份健康儿童样品。结果显示上海腹泻儿童粪便中的Salivirus/Klassevirus的阳性率为4.2%。通过数理统计结果显示:Salivirus/Klassevirus和腹泻的关联分析差异显著(p=0.03)。结果表明该病毒可能引起腹泻。 (4)8株博卡病毒的初步研究 本实验克隆了我国2株HBoV1的基因组和1株HBoV2,它们的基因组大小分别为:5288bp、5277bp和5244bp。系统进化树分析HBoV1和HBoV2的基因组,结果显示HBoV1与我国已报道的分离株聚为一起,HBoV2与巴基斯坦(FJ170279)分离株聚为一起。通过PCR检测了上海腹泻儿童的320份样品,结果显示HBoV1和HBoV2的阳性率分别为5.3%和5.0%。 通过PCR检测来自上海3家宠物医院的158份腹泻犬的粪便样品,结果显示一个样品为CnMV阳性。根据美国株(FJ214110)的基因组设计引物,克隆了我国第一株CnMV的基因组,其大小为5132bp。 通过基因组步移,本实验克隆了我国2株PBoV(PBoV1-H18和PBoV2-A6),它们的基因组大小为5267 bp和5117 bp。系统进化树分析了全基因组和3个基因的核苷酸和氨基酸序列结果显示PBoV1-H18与猪boca-like virus聚为一支,而PBoV2-A6与PBoV1(HM053693)和PBoV2(HM053694)聚为一起。流行病学调查结果显示PBoV1-H18和PBoV2-A6阳性率高达75%和70.1%。 本实验克隆了美国2株PBoV3的基因组,其大小为4710bp和4689bp。系统进化树分析VP1蛋白结果显示:PBoV3与6V和7V聚为一支。初步推测PBoV可能存在3个种(PBoV1、PBoV2和PBoV3)。 (5)4株Po-Circo-like Virus的初步研究 本实验克隆了4株新的Po-Circo-like Viruses,其基因组大小分别为3912bp、3923 bp、2904bp和2833bp。系统进化树分析了Rep蛋白,结果显示:4株Po-Circo-like Viruses聚为一支,其可能是环状病毒家族中一个新的属。这4株环状类病毒基因组中含有的特征结构为:茎环结构和Rep蛋白的保守区域(viral Rep和RNA helicase)。 (6)Posavirus 1和Posavirus2的初步研究 本实验克隆了2株微小RNA病毒超级家族中新的成员,暂且命名为Posavirus 1和Posavirus 2。其基因组大小为9816bp和10191bp。根据不同宿主的单链RNA病毒的碱基排列规律划分,posavirus 1和posavirus 2被划分到昆虫区域范围内。系统进化树分析RNA病毒的RdRp区域,结果显示posavirus 1和posavirus 2分别单独聚为两支。初步推测posavirus 1和posavirus 2可能为微小RNA病毒超级家族中的新的不同科或属。通过RT-nPCR检测36份猪的样品。结果显示posavirus 1和posavirus 2阳性率分别为47.2%和30.6%。
[Abstract]:New viral infectious diseases endanger human and animal health. Mining and identifying new viruses is the primary task of preventing, diagnosing and treating new viral infectious diseases. Viral macrogenomics is a new technology developed on the basis of macrogenomics to study viral communities in specific environments. This technology has been widely used. Diarrhea is a common disease among people of all ages and one of the major diseases affecting the economic benefits of pig farming. Around 1.5 billion people suffer from diarrhea every year worldwide, with a high incidence of children and the elderly; more than 2 million children under the age of 5 die of diarrhea; and China is developing. At present, the current detection technology can only diagnose 60% of the causes of diarrhea, and nearly 40% of the cases can not find out the cause of the disease. The pathogenic factors causing diarrhea of unknown etiology may be the virus that has not been discovered yet. * pigs, as economic animals, provide a large quantity of meat for human beings, and at the same time they also carry animals and animals together. Diarrhea is a common disease of piglets, causing serious economic losses to pig industry. China is a developing country. Diarrhea is one of the major diseases affecting the health of our people, especially children's health. The genome and epidemiological investigation of new cloned viruses will lay the foundation for prevention and treatment of diarrhea caused by new viruses. * the pig industry plays an important role in the world aquaculture industry. Prevention and control of pig diseases is the best way to improve the efficiency of * * pigs, and is also an effective way to control swine zoonosis. The viral community and fecal * * * s of diarrhoea pigs and healthy pigs will provide reliable information for the diagnosis of swine viral diseases. The genome and epidemiological investigation of cloned new swine viruses will lay the foundation for the prevention and treatment of porcine viral diseases.
The main contents and results of this paper are as follows:
(1) virus genomics analysis of virus communities in human and pig feces *
(1) viral metagenomics analysis of viral communities in feces of diarrhoea children.
In this study, 12 viral community libraries from Shanghai diarrhea children's feces were constructed by viral macrogenomics method, and 840 clones were analyzed by Sanger sequencing. The results showed that 31.4% of the viral sequences were matched to the virus, 5.8% to the phage, and 2% to the fine. Bacteria and 60.7% of the sequences were unknown; viral sequences were mainly matched to enterovirus, Boca virus, stellate virus, Aichi virus, double Eckovirus, Human coavirus and Klassevirus/Salivirus.
(2) viral metagenomics analysis of virus communities in pig feces in China
In this study, a total of 17 libraries of pig fecal virus communities * were constructed by using the virus metagenomic method. 1190 clones in the total library were analyzed by Sanger sequencing technology. The results showed that 32.9% of the sequences matched to eukaryotic viruses, 7% of the sequences matched to phages, 2.2% of the sequences matched to bacteria and 57.9% of the sequences did not match. * to any sequence. The virus sequence is matched to the porcine enterovirus, Boka virus and stellate virus.
(3) viral metagenomics analysis of virus communities in feces of American swine
The viral metagenomic method was used to analyze the viral community library of feces in 18 healthy pigs and 18 * * diarrhea pigs in the United States by using 454 high throughput sequencing. The results showed that the library contained 652614 sequences, the smallest sequencing length was 40bp, the maximum sequencing length was 699bp, and the average length of sequencing was 236bp.. Sequence alignment showed that 0.2% of the sequences were matched to eukaryotic genes, 0.2% to other sequences, 9% to bacteria, 10% to bacteriophages, 13% to unknown sequences and 68% to viruses. Virus, stellate virus, Kobuvirus, Sapovirus, Teschovirus, cyclic viroid, Boka virus and Rosavirus.
(two) preliminary study of 23 strains of virus
(1) preliminary study of 6 strains of stellate virus
The first strain of HAstV1 in Shanghai was cloned and its size was 6808bp. The nucleotide phylogenetic tree of ORF 2 was analyzed. The results showed that the isolate was similar to the Japanese strain AB000285. 279 samples of children with diarrhea in Shanghai were detected by RT-PCR. The results showed that HAstV infection rate was 8.2%, mainly HAstV1.
The genome of the first strain of PAstV in China was cloned, and its size was 6610bp. phylogenetic tree of amino acid encoded by ORF2. The results showed that the strain PAstV belonged to PAstV1., and 340 pig feces samples from 5 provinces and cities in China were detected by RT-nPCR. The results showed that PAstV1 infection rate was 22.9%..
Four new strains of PAstVs (PAstV2-43, PAstV2-51, PAstV4-35 and PAstV5-33) were cloned from American pigs. The phylogenetic tree of amino acids encoded by ORF2 was analyzed. The results showed that PAstV2-43 and PAstV2-51 belonged to PAstV2 and clustered with Canada, PAstV4-35 belonged to PAstV4 and clustered with Hungary, and PAstV5-33 belonged to one branch. Its homology with all known AstV amino acids is less than 26%, which may be a new species of PAstV.
(2) preliminary study of 2 strains of HPeVs
HPeVs were detected by RT-nPCR in 220 children with diarrhea in Shanghai and 116 fecal samples of diarrhea monkeys. The results showed that the positive rate of HPeVs was 20.5% in children with diarrhea in Shanghai and 5.2% in fecal samples of diarrhea monkeys.
The genome of the first HPeV1 strain in China was cloned. The size of the genome was 7259 bp. The nucleotide sequence of P1 region was analyzed by phylogenetic tree. The results showed that the HPeV1 strain was clustered with the German strain (EF051629) and the Dutch strain (FM178558). The genome of the first HPeV6 strain from monkey feces was cloned. The genome size was 7311 bp. The whole genome of eV6 showed that the strain was clustered together with Japanese strain (AB252582) and Holland strain (EU077518).
(3) preliminary study of Salivirus/Klassevirus
The genome of the first Salivirus/Klassevirus strain in China was cloned and its size was 7798 bp. The phylogenetic tree analysis showed that the strain was clustered with the reported Salivirus/Klassevirus in the P1 region.
The results showed that the positive rate of Salivirus/Klassevirus in the feces of Shanghai diarrhea children was 4.2%. The correlation analysis between Salivirus/Klassevirus and diarrhea was significant (p=0.03).
(4) preliminary study of 8 strains of Boka virus
The genomes of two HBoV1 strains and one HBoV2 strain in China were cloned. The genomes of HBoV1 and HBoV2 were 5 288 bp, 5 277 BP and 5 244 bp, respectively. The phylogenetic tree analysis showed that HBoV1 and HBoV2 were clustered together with the reported isolates in China, and HBoV2 and FJ170279 were clustered together. The Shanghai isolates were detected by PCR. 320 samples of diarrhea children showed that the positive rates of HBoV1 and HBoV2 were 5.3% and 5.0%. respectively.
A total of 158 stool samples of diarrhea dogs from three pet hospitals in Shanghai were detected by PCR. One of the samples was positive for CnMV. According to the primers of FJ214110, the genome of the first Chinese strain of CnMV was cloned. The size of CnMV was 5132 bp.
Through genome walking, 2 Chinese strains of PBoV (PBoV1-H18 and PBoV2-A6) were cloned, and their genome size was 5267 BP and 5117 bp.. Phylogenetic tree analysis of nucleotide and amino acid sequences of the genome and 3 genes showed that PBoV1-H18 and pig * boca-like virus were clustered together, and PBoV2-A6 and PBoV1 (HM053693) and PBoV2-A6 ( 694) get together. Epidemiological findings show that the positive rates of PBoV1-H18 and PBoV2-A6 are as high as 75% and 70.1%. respectively.
The genomes of two PBoV3 strains from the United States were cloned and their sizes were 4710 BP and 4689 bp. Phylogenetic tree analysis of VP1 protein showed that PBoV3 was clustered with 6V and 7V. Three species of PBoV3 (PBoV1, PBoV 2 and PBoV3) were presumed to exist.
(5) preliminary study on 4 strains of Po-Circo-like Virus
Four new strains of Po-Circo-like Viruses were cloned and their genome sizes were 3912 bp, 3923 bp, 2904 BP and 2833 bp, respectively. Rep proteins were analyzed by phylogenetic tree analysis. The results showed that four strains of Po-Circo-like Viruses were clustered into one branch and might be a new genus in the family of cyclic viruses. They are: stem ring structure and conserved region of Rep protein (viral Rep and RNA helicase).
(6) preliminary study of Posavirus 1 and Posavirus2
Two new members of the superfamily of microRNA viruses were cloned and temporarily named Posavirus 1 and Posavirus 2. Their genome sizes were 9816 BP and 10191 bp. According to the sequence of single-stranded RNA viruses in different hosts, posavirus 1 and posavirus 2 were classified into insect regions. The RdRp region showed that posavirus 1 and posavirus 2 were separately clustered into two branches. It was preliminarily speculated that posavirus 1 and posavirus 2 might be new families or genera in the super RNA virus superfamily * 36 samples of pigs were detected by RT-nPCR. The results showed that the positive rates of posavirus 1 and posavirus 2 were 47.2% and 30.6%., respectively.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R346
本文编号：2232307
[Abstract]:New viral infectious diseases endanger human and animal health. Mining and identifying new viruses is the primary task of preventing, diagnosing and treating new viral infectious diseases. Viral macrogenomics is a new technology developed on the basis of macrogenomics to study viral communities in specific environments. This technology has been widely used. Diarrhea is a common disease among people of all ages and one of the major diseases affecting the economic benefits of pig farming. Around 1.5 billion people suffer from diarrhea every year worldwide, with a high incidence of children and the elderly; more than 2 million children under the age of 5 die of diarrhea; and China is developing. At present, the current detection technology can only diagnose 60% of the causes of diarrhea, and nearly 40% of the cases can not find out the cause of the disease. The pathogenic factors causing diarrhea of unknown etiology may be the virus that has not been discovered yet. * pigs, as economic animals, provide a large quantity of meat for human beings, and at the same time they also carry animals and animals together. Diarrhea is a common disease of piglets, causing serious economic losses to pig industry. China is a developing country. Diarrhea is one of the major diseases affecting the health of our people, especially children's health. The genome and epidemiological investigation of new cloned viruses will lay the foundation for prevention and treatment of diarrhea caused by new viruses. * the pig industry plays an important role in the world aquaculture industry. Prevention and control of pig diseases is the best way to improve the efficiency of * * pigs, and is also an effective way to control swine zoonosis. The viral community and fecal * * * s of diarrhoea pigs and healthy pigs will provide reliable information for the diagnosis of swine viral diseases. The genome and epidemiological investigation of cloned new swine viruses will lay the foundation for the prevention and treatment of porcine viral diseases.
The main contents and results of this paper are as follows:
(1) virus genomics analysis of virus communities in human and pig feces *
(1) viral metagenomics analysis of viral communities in feces of diarrhoea children.
In this study, 12 viral community libraries from Shanghai diarrhea children's feces were constructed by viral macrogenomics method, and 840 clones were analyzed by Sanger sequencing. The results showed that 31.4% of the viral sequences were matched to the virus, 5.8% to the phage, and 2% to the fine. Bacteria and 60.7% of the sequences were unknown; viral sequences were mainly matched to enterovirus, Boca virus, stellate virus, Aichi virus, double Eckovirus, Human coavirus and Klassevirus/Salivirus.
(2) viral metagenomics analysis of virus communities in pig feces in China
In this study, a total of 17 libraries of pig fecal virus communities * were constructed by using the virus metagenomic method. 1190 clones in the total library were analyzed by Sanger sequencing technology. The results showed that 32.9% of the sequences matched to eukaryotic viruses, 7% of the sequences matched to phages, 2.2% of the sequences matched to bacteria and 57.9% of the sequences did not match. * to any sequence. The virus sequence is matched to the porcine enterovirus, Boka virus and stellate virus.
(3) viral metagenomics analysis of virus communities in feces of American swine
The viral metagenomic method was used to analyze the viral community library of feces in 18 healthy pigs and 18 * * diarrhea pigs in the United States by using 454 high throughput sequencing. The results showed that the library contained 652614 sequences, the smallest sequencing length was 40bp, the maximum sequencing length was 699bp, and the average length of sequencing was 236bp.. Sequence alignment showed that 0.2% of the sequences were matched to eukaryotic genes, 0.2% to other sequences, 9% to bacteria, 10% to bacteriophages, 13% to unknown sequences and 68% to viruses. Virus, stellate virus, Kobuvirus, Sapovirus, Teschovirus, cyclic viroid, Boka virus and Rosavirus.
(two) preliminary study of 23 strains of virus
(1) preliminary study of 6 strains of stellate virus
The first strain of HAstV1 in Shanghai was cloned and its size was 6808bp. The nucleotide phylogenetic tree of ORF 2 was analyzed. The results showed that the isolate was similar to the Japanese strain AB000285. 279 samples of children with diarrhea in Shanghai were detected by RT-PCR. The results showed that HAstV infection rate was 8.2%, mainly HAstV1.
The genome of the first strain of PAstV in China was cloned, and its size was 6610bp. phylogenetic tree of amino acid encoded by ORF2. The results showed that the strain PAstV belonged to PAstV1., and 340 pig feces samples from 5 provinces and cities in China were detected by RT-nPCR. The results showed that PAstV1 infection rate was 22.9%..
Four new strains of PAstVs (PAstV2-43, PAstV2-51, PAstV4-35 and PAstV5-33) were cloned from American pigs. The phylogenetic tree of amino acids encoded by ORF2 was analyzed. The results showed that PAstV2-43 and PAstV2-51 belonged to PAstV2 and clustered with Canada, PAstV4-35 belonged to PAstV4 and clustered with Hungary, and PAstV5-33 belonged to one branch. Its homology with all known AstV amino acids is less than 26%, which may be a new species of PAstV.
(2) preliminary study of 2 strains of HPeVs
HPeVs were detected by RT-nPCR in 220 children with diarrhea in Shanghai and 116 fecal samples of diarrhea monkeys. The results showed that the positive rate of HPeVs was 20.5% in children with diarrhea in Shanghai and 5.2% in fecal samples of diarrhea monkeys.
The genome of the first HPeV1 strain in China was cloned. The size of the genome was 7259 bp. The nucleotide sequence of P1 region was analyzed by phylogenetic tree. The results showed that the HPeV1 strain was clustered with the German strain (EF051629) and the Dutch strain (FM178558). The genome of the first HPeV6 strain from monkey feces was cloned. The genome size was 7311 bp. The whole genome of eV6 showed that the strain was clustered together with Japanese strain (AB252582) and Holland strain (EU077518).
(3) preliminary study of Salivirus/Klassevirus
The genome of the first Salivirus/Klassevirus strain in China was cloned and its size was 7798 bp. The phylogenetic tree analysis showed that the strain was clustered with the reported Salivirus/Klassevirus in the P1 region.
The results showed that the positive rate of Salivirus/Klassevirus in the feces of Shanghai diarrhea children was 4.2%. The correlation analysis between Salivirus/Klassevirus and diarrhea was significant (p=0.03).
(4) preliminary study of 8 strains of Boka virus
The genomes of two HBoV1 strains and one HBoV2 strain in China were cloned. The genomes of HBoV1 and HBoV2 were 5 288 bp, 5 277 BP and 5 244 bp, respectively. The phylogenetic tree analysis showed that HBoV1 and HBoV2 were clustered together with the reported isolates in China, and HBoV2 and FJ170279 were clustered together. The Shanghai isolates were detected by PCR. 320 samples of diarrhea children showed that the positive rates of HBoV1 and HBoV2 were 5.3% and 5.0%. respectively.
A total of 158 stool samples of diarrhea dogs from three pet hospitals in Shanghai were detected by PCR. One of the samples was positive for CnMV. According to the primers of FJ214110, the genome of the first Chinese strain of CnMV was cloned. The size of CnMV was 5132 bp.
Through genome walking, 2 Chinese strains of PBoV (PBoV1-H18 and PBoV2-A6) were cloned, and their genome size was 5267 BP and 5117 bp.. Phylogenetic tree analysis of nucleotide and amino acid sequences of the genome and 3 genes showed that PBoV1-H18 and pig * boca-like virus were clustered together, and PBoV2-A6 and PBoV1 (HM053693) and PBoV2-A6 ( 694) get together. Epidemiological findings show that the positive rates of PBoV1-H18 and PBoV2-A6 are as high as 75% and 70.1%. respectively.
The genomes of two PBoV3 strains from the United States were cloned and their sizes were 4710 BP and 4689 bp. Phylogenetic tree analysis of VP1 protein showed that PBoV3 was clustered with 6V and 7V. Three species of PBoV3 (PBoV1, PBoV 2 and PBoV3) were presumed to exist.
(5) preliminary study on 4 strains of Po-Circo-like Virus
Four new strains of Po-Circo-like Viruses were cloned and their genome sizes were 3912 bp, 3923 bp, 2904 BP and 2833 bp, respectively. Rep proteins were analyzed by phylogenetic tree analysis. The results showed that four strains of Po-Circo-like Viruses were clustered into one branch and might be a new genus in the family of cyclic viruses. They are: stem ring structure and conserved region of Rep protein (viral Rep and RNA helicase).
(6) preliminary study of Posavirus 1 and Posavirus2
Two new members of the superfamily of microRNA viruses were cloned and temporarily named Posavirus 1 and Posavirus 2. Their genome sizes were 9816 BP and 10191 bp. According to the sequence of single-stranded RNA viruses in different hosts, posavirus 1 and posavirus 2 were classified into insect regions. The RdRp region showed that posavirus 1 and posavirus 2 were separately clustered into two branches. It was preliminarily speculated that posavirus 1 and posavirus 2 might be new families or genera in the super RNA virus superfamily * 36 samples of pigs were detected by RT-nPCR. The results showed that the positive rates of posavirus 1 and posavirus 2 were 47.2% and 30.6%., respectively.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R346
【引证文献】
相关会议论文 前1条
1 何彪;涂长春;;病毒宏基因组学的研究现状及应用[A];中国畜牧兽医学会兽医公共卫生学分会第三次学术研讨会论文集[C];2012年
相关硕士学位论文 前1条
1 韩文;基于宏基因组学的动物病毒侦测方法的建立[D];中国农业科学院;2012年
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