病毒宏基因組學(xué)分析兒童和豬腸道病毒群落及23株病毒的初步研究
發(fā)布時(shí)間：2018-09-09 12:01
【摘要】：新發(fā)病毒性傳染病危害人和動(dòng)物的健康。挖掘和鑒定新病毒是預(yù)防、診斷和治療新發(fā)病毒性傳染病的首要任務(wù)。病毒宏基因組學(xué)是在宏基因組學(xué)的基礎(chǔ)上發(fā)展起來(lái)的一門研究特定環(huán)境中病毒群落的新興技術(shù)。該技術(shù)已經(jīng)被廣泛地應(yīng)用到挖掘新病毒,并且取得可喜的成就。腹瀉是各個(gè)年齡段的人群中常見的疾病,也是影響?zhàn)B豬業(yè)經(jīng)濟(jì)效益的主要疾病之一。在世界范圍內(nèi),每年有將近15億人腹瀉,其中兒童和年長(zhǎng)者發(fā)病率高;有200多萬(wàn)5歲以下腹瀉兒童死亡,并且發(fā)展中國(guó)家占有較大的比例。目前,現(xiàn)有的檢測(cè)技術(shù)只能診斷出60%的腹瀉病例的病因,而近40%的病例查不出病因。引起未知病因的腹瀉的致病因素,可能是目前暫未被發(fā)現(xiàn)的病毒。豬作為經(jīng)濟(jì)動(dòng)物,為人類提供了大量的肉類,同時(shí)也是攜帶人獸共患病的宿主。腹瀉是仔豬的常發(fā)疾病,給養(yǎng)豬業(yè)造成嚴(yán)重的經(jīng)濟(jì)損失。我國(guó)是發(fā)展中國(guó)家。腹瀉是影響我國(guó)人民的健康主要疾病之一,尤其是兒童的健康。分析腹瀉兒童糞便中的病毒群落和發(fā)掘潛在的新病毒將為診斷不明腹瀉的病因提供新的途徑。克隆新病毒的基因組和流行病學(xué)調(diào)查將為預(yù)防和治療新病毒引起的腹瀉奠定基礎(chǔ)。養(yǎng)豬業(yè)在世界養(yǎng)殖業(yè)占有重要的地位。預(yù)防和控制豬病是提高養(yǎng)豬效益的最好的解決辦法,同時(shí)也是控制豬源性人獸共患病的有效途徑。研究腹瀉豬和健康豬糞便中的病毒群落及挖掘潛在的新病原,將為診斷豬病毒性疾病提供可靠的資料;克隆豬源性新病毒的基因組和流行病學(xué)調(diào)查將為預(yù)防和治療豬病毒性疾病奠定基礎(chǔ)。 本論文主要研究?jī)?nèi)容和結(jié)果如下: (一)病毒宏基因組學(xué)分析人和豬糞便中的病毒群落 (1)病毒宏基因組學(xué)分析腹瀉兒童糞便中的病毒群落 本實(shí)驗(yàn)應(yīng)用病毒宏基因組學(xué)方法,構(gòu)建了12份上海腹瀉兒童糞便中病毒群落的文庫(kù),通過sanger測(cè)序法分析了總文庫(kù)中的840個(gè)克隆。結(jié)果顯示:在上海腹瀉兒童糞便文庫(kù)中,31.4%的序列匹配到病毒,5.8%的序列匹配到噬菌體,2%的序列匹配到細(xì)菌和60.7%的序列是未知序列;病毒序列主要匹配到腸道病毒、博卡病毒、星狀病毒、Aichi virus、雙埃克病毒、Human coavirus和Klassevirus/Salivirus; (2)病毒宏基因組學(xué)分析我國(guó)豬群糞便中的病毒群落 本實(shí)驗(yàn)應(yīng)用病毒宏基因組學(xué)方法,構(gòu)建了17份我國(guó)豬糞便中病毒群落的文庫(kù),通過sanger測(cè)序技術(shù)分析了總文庫(kù)中的1190個(gè)克隆。結(jié)果顯示:32.9%的序列匹配到真核病毒,7.0%的序列匹配到噬菌體,2.2%的序列匹配到細(xì)菌和57.9%的序列沒有匹配到任何序列。病毒序列匹配到得豬腸道病毒、博卡病毒和星狀病毒。 (3)病毒宏基因組學(xué)分析美國(guó)豬群糞便中的病毒群落 本實(shí)驗(yàn)應(yīng)用病毒宏基因組學(xué)方法,通過454高通量測(cè)序分析美國(guó)18個(gè)健康豬和18個(gè)腹瀉豬糞便中病毒群落文庫(kù)。結(jié)果顯示:該文庫(kù)包含652,614個(gè)序列,最小測(cè)序長(zhǎng)度為40bp,最大測(cè)序長(zhǎng)度為699bp,測(cè)序平均長(zhǎng)度為236bp。去除引物標(biāo)簽后,文庫(kù)中的有效序列大于57萬(wàn)條,平均長(zhǎng)度為192bp。序列比對(duì)結(jié)果顯示: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%的序列匹配到細(xì)菌、10%的序列匹配到噬菌體、13%的序列是未知序列及68%的序列匹配到病毒。病毒序列匹配到腸道病毒、Sapelovirus、星狀病毒、Kobuvirus、Sapovirus、Teschovirus、環(huán)狀類病毒、博卡病毒和Rosavirus。 (二)23株病毒的初步研究 (1)6株星狀病毒的初步研究 本實(shí)驗(yàn)克隆了上海第一株HAstV1的基因組,其大小為6808bp。分析ORF2的核苷酸系統(tǒng)進(jìn)化樹,結(jié)果顯示本分離株與日本分離株AB000285比較相近。通過RT-PCR檢測(cè)了上海腹瀉兒童的279份樣品,結(jié)果顯示:HAstV感染率為8.2%,主要為HAstV1。 本實(shí)驗(yàn)克隆了我國(guó)第一株P(guān)AstV的基因組,其大小為6610bp。分析ORF2編碼的氨基酸的系統(tǒng)進(jìn)化樹,結(jié)果顯示:本株P(guān)AstV屬于PAstV1。通過RT-nPCR檢測(cè)了來(lái)自我國(guó)5省市的340份豬糞便樣品,結(jié)果顯示有PAstV1感染率為22.9%。 本實(shí)驗(yàn)克隆了美國(guó)豬群中4株新的PAstVs(PAstV2-43、PAstV2-51、PAstV4-35和PAstV5-33)。分析ORF2編碼的氨基酸的系統(tǒng)進(jìn)化樹,結(jié)果顯示:PAstV2-43和PAstV2-51屬于PAstV2,與加拿大的聚為一支;PAstV4-35屬于PAstV4,與匈牙利的聚為一支;PAstV5-33單獨(dú)聚為一支,并且其與所有已知的AstV的氨基酸同源性都小于26%,其可能為PAstV的一個(gè)新種。 (2)2株HPeVs的初步研究 通過RT-nPCR檢測(cè)了220份上海腹瀉兒童樣品和116份腹瀉猴糞便樣品中的HPeVs。結(jié)果顯示:上海腹瀉兒童糞便中的HPeVs的陽(yáng)性率為20.5%;腹瀉猴糞便中的陽(yáng)性率為5.2%。本研究首次在猴糞便中發(fā)現(xiàn)HPeV。 本實(shí)驗(yàn)克隆了我國(guó)第一株HPeV1的基因組,其大小為7259bp。系統(tǒng)進(jìn)化樹分析P1區(qū)核苷酸序列結(jié)果顯示該株與德國(guó)株(EF051629)和荷蘭株(FM178558)的HPeV1聚為一支�？寺×藖�(lái)自猴糞便中的第一株HPeV6的基因組,基因組大小為7311bp。系統(tǒng)進(jìn)化樹分析HPeV6的全基因組結(jié)果顯示該株和日本株(AB252582)和荷蘭株(EU077518)聚為一支。 (3) Salivirus/Klassevirus的初步研究 本實(shí)驗(yàn)克隆了我國(guó)第一株Salivirus/Klassevirus的基因組,其大小為7798bp。系統(tǒng)進(jìn)化樹分析P1區(qū)氨基酸結(jié)果顯示該株與已報(bào)道的Salivirus/Klassevirus聚為一支。該可能病毒為picornaviridae中一個(gè)新的屬。 通過RT-nPCR檢測(cè)了216份上海腹瀉兒童樣品和96份健康兒童樣品。結(jié)果顯示上海腹瀉兒童糞便中的Salivirus/Klassevirus的陽(yáng)性率為4.2%。通過數(shù)理統(tǒng)計(jì)結(jié)果顯示:Salivirus/Klassevirus和腹瀉的關(guān)聯(lián)分析差異顯著(p=0.03)。結(jié)果表明該病毒可能引起腹瀉。 (4)8株博卡病毒的初步研究 本實(shí)驗(yàn)克隆了我國(guó)2株HBoV1的基因組和1株HBoV2,它們的基因組大小分別為:5288bp、5277bp和5244bp。系統(tǒng)進(jìn)化樹分析HBoV1和HBoV2的基因組,結(jié)果顯示HBoV1與我國(guó)已報(bào)道的分離株聚為一起,HBoV2與巴基斯坦(FJ170279)分離株聚為一起。通過PCR檢測(cè)了上海腹瀉兒童的320份樣品,結(jié)果顯示HBoV1和HBoV2的陽(yáng)性率分別為5.3%和5.0%。 通過PCR檢測(cè)來(lái)自上海3家寵物醫(yī)院的158份腹瀉犬的糞便樣品,結(jié)果顯示一個(gè)樣品為CnMV陽(yáng)性。根據(jù)美國(guó)株(FJ214110)的基因組設(shè)計(jì)引物,克隆了我國(guó)第一株CnMV的基因組,其大小為5132bp。 通過基因組步移,本實(shí)驗(yàn)克隆了我國(guó)2株P(guān)BoV(PBoV1-H18和PBoV2-A6),它們的基因組大小為5267 bp和5117 bp。系統(tǒng)進(jìn)化樹分析了全基因組和3個(gè)基因的核苷酸和氨基酸序列結(jié)果顯示PBoV1-H18與豬boca-like virus聚為一支,而PBoV2-A6與PBoV1(HM053693)和PBoV2(HM053694)聚為一起。流行病學(xué)調(diào)查結(jié)果顯示PBoV1-H18和PBoV2-A6陽(yáng)性率高達(dá)75%和70.1%。 本實(shí)驗(yàn)克隆了美國(guó)2株P(guān)BoV3的基因組,其大小為4710bp和4689bp。系統(tǒng)進(jìn)化樹分析VP1蛋白結(jié)果顯示:PBoV3與6V和7V聚為一支。初步推測(cè)PBoV可能存在3個(gè)種(PBoV1、PBoV2和PBoV3)。 (5)4株P(guān)o-Circo-like Virus的初步研究 本實(shí)驗(yàn)克隆了4株新的Po-Circo-like Viruses,其基因組大小分別為3912bp、3923 bp、2904bp和2833bp。系統(tǒng)進(jìn)化樹分析了Rep蛋白,結(jié)果顯示:4株P(guān)o-Circo-like Viruses聚為一支,其可能是環(huán)狀病毒家族中一個(gè)新的屬。這4株環(huán)狀類病毒基因組中含有的特征結(jié)構(gòu)為:莖環(huán)結(jié)構(gòu)和Rep蛋白的保守區(qū)域(viral Rep和RNA helicase)。 (6)Posavirus 1和Posavirus2的初步研究 本實(shí)驗(yàn)克隆了2株微小RNA病毒超級(jí)家族中新的成員,暫且命名為Posavirus 1和Posavirus 2。其基因組大小為9816bp和10191bp。根據(jù)不同宿主的單鏈RNA病毒的堿基排列規(guī)律劃分,posavirus 1和posavirus 2被劃分到昆蟲區(qū)域范圍內(nèi)。系統(tǒng)進(jìn)化樹分析RNA病毒的RdRp區(qū)域,結(jié)果顯示posavirus 1和posavirus 2分別單獨(dú)聚為兩支。初步推測(cè)posavirus 1和posavirus 2可能為微小RNA病毒超級(jí)家族中的新的不同科或?qū)�。通過RT-nPCR檢測(cè)36份豬的樣品。結(jié)果顯示posavirus 1和posavirus 2陽(yáng)性率分別為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.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R346
本文編號(hào)：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.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R346
【引證文獻(xiàn)】
相關(guān)會(huì)議論文 前1條
1 何彪;涂長(zhǎng)春;;病毒宏基因組學(xué)的研究現(xiàn)狀及應(yīng)用[A];中國(guó)畜牧獸醫(yī)學(xué)會(huì)獸醫(yī)公共衛(wèi)生學(xué)分會(huì)第三次學(xué)術(shù)研討會(huì)論文集[C];2012年
相關(guān)碩士學(xué)位論文 前1條
1 韓文;基于宏基因組學(xué)的動(dòng)物病毒偵測(cè)方法的建立[D];中國(guó)農(nóng)業(yè)科學(xué)院;2012年
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