旨在从牛、羊监控动物群中,对流行于我国的帕利亚姆血清群病毒(PALV)进行分离、鉴定与序列特征分析,明确我国流行毒株与国外毒株之间的关系。自监控动物采集的PALV阳性血液接种BHK-21细胞进行病毒分离;对所分离PALV毒株的Seg-7与Seg-2...旨在从牛、羊监控动物群中,对流行于我国的帕利亚姆血清群病毒(PALV)进行分离、鉴定与序列特征分析,明确我国流行毒株与国外毒株之间的关系。自监控动物采集的PALV阳性血液接种BHK-21细胞进行病毒分离;对所分离PALV毒株的Seg-7与Seg-2基因节段进行扩增、测序与序列分析;通过高分辨率琼脂糖凝胶电泳分析不同血清型PALV代表毒株基因组的"电泳带型"特征;通过免疫荧光与中和试验分析不同血清型PALV的阳性血清之间的交叉反应特性与中和活性。试验结果如下:2012—2016年,在云南、广西、广东共计分离出19株PALV;Seg-2与Seg-7测序与系统发育分析显示,分离毒株分属Chuzan virus(CHUV)、D’Aguilar virus(DAV)与Bunyip Creek virus(BCV)三种血清型,不同血清型毒株均与日本毒株具有最近的亲缘关系,而与澳洲、非洲毒株亲缘关系较远。病毒基因组电泳结果显示PALV基因组呈现"3-3-4"的电泳带型特征。免疫荧光结果显示针对CHUV的多克隆抗体可使感染CHUV、DAV与BCV的BHK-21细胞呈现特异性荧光。血清学中和试验显示,CHUV、DAV与BCV三种病毒的阳性血清之间无交叉中和保护作用。本研究报道了2012—2016年中国PALV的分离与Seg-2、Seg-7序列特征,并首次报道了PALV的DAV与BCV两种血清型病毒在我国的分离,研究结果将有助于掌握我国PALV的分布与遗传特征,为诊断试剂的开发、流行病学调查与致病性研究提供科学依据。展开更多
Dengue has been well recognized as a global public health threat,but only sporadic epidemics and imported cases were reported in recent decades in China.Since July 2014,an unexpected large dengue outbreak has occurred...Dengue has been well recognized as a global public health threat,but only sporadic epidemics and imported cases were reported in recent decades in China.Since July 2014,an unexpected large dengue outbreak has occurred in Guangdong province,China,resulting in more than 40000 patients including six deaths.To clarify and characterize the causative agent of this outbreak,the acute phase serum from a patient diagnosed with severe dengue was subjected to virus isolation and high-throughput sequencing(HTS).Traditional real-time RT-PCR and HTS with Ion Torrent PGM detected the presence of dengue virus serotype 2(DENV-2).A clinical DENV-2 isolate GZ05/2014 was obtained by culturing the patient serum in mosquito C6/36 cells.The complete genome of GZ05/2014 was determined and deposited in Gen Bank under the access number KP012546.Phylogenetic analysis based on the complete envelope gene showed that the newly DENV-2 isolate belonged to Cosmopolitan genotype and clustered closely with other Guangdong strains isolated in the past decade.No amino acid mutations that are obviously known to increase virulence or replication were identified throughout the genome of GZ05/2014.The high homology of Guangdong DENV-2 strains indicated the possibility of establishment of local DENV-2 circulation in Guangdong,China.These results help clarify the origin of this epidemic and predict the future status of dengue in China.展开更多
Using two pairs of primer based on conserved sequences of the NP gene of influenza A virus, a RT-nested PCR assay was developed to detect Swine influenza virus in clinical samples. No cross reactions with three other ...Using two pairs of primer based on conserved sequences of the NP gene of influenza A virus, a RT-nested PCR assay was developed to detect Swine influenza virus in clinical samples. No cross reactions with three other swine pathogens of respiratory disease such as Porcine respiratory and reproductive syndrome virus, Porcine circovirus type 2 and Mycoplasma suipneumoniae. With agarose electrophoresis detection, the RT-nested PCR was 100 times more sensitive compared to that of non-nested RT-PCR. 35 clinical samples including 4 blood samples and 31 lung tissues from Southern China were tested by RT-nested PCR, and 28 (80%) out of 35 sample were positive. By propagated in 9-to 10-day-old SPF embryonated chicken eggs, 23 strains of Swine influenza virus were isolated from 28 positive samples. The result of RT-nested PCR was 82% in agreement with that of virus isolation, and the RT-nested PCR were more sensitive than the virus isolation. No Swine influenza virus was isolated from the all RT-nested PCR negative samples. On the basis of characterization of HA gene sequences, 5 out of 23 strains had H3 and H9 subtypes. It suggested that the RT-nested PCR assay was rapid,specific and sensitive, and it was is advantageous to detect influenza virus in latent infections.展开更多
文摘旨在从牛、羊监控动物群中,对流行于我国的帕利亚姆血清群病毒(PALV)进行分离、鉴定与序列特征分析,明确我国流行毒株与国外毒株之间的关系。自监控动物采集的PALV阳性血液接种BHK-21细胞进行病毒分离;对所分离PALV毒株的Seg-7与Seg-2基因节段进行扩增、测序与序列分析;通过高分辨率琼脂糖凝胶电泳分析不同血清型PALV代表毒株基因组的"电泳带型"特征;通过免疫荧光与中和试验分析不同血清型PALV的阳性血清之间的交叉反应特性与中和活性。试验结果如下:2012—2016年,在云南、广西、广东共计分离出19株PALV;Seg-2与Seg-7测序与系统发育分析显示,分离毒株分属Chuzan virus(CHUV)、D’Aguilar virus(DAV)与Bunyip Creek virus(BCV)三种血清型,不同血清型毒株均与日本毒株具有最近的亲缘关系,而与澳洲、非洲毒株亲缘关系较远。病毒基因组电泳结果显示PALV基因组呈现"3-3-4"的电泳带型特征。免疫荧光结果显示针对CHUV的多克隆抗体可使感染CHUV、DAV与BCV的BHK-21细胞呈现特异性荧光。血清学中和试验显示,CHUV、DAV与BCV三种病毒的阳性血清之间无交叉中和保护作用。本研究报道了2012—2016年中国PALV的分离与Seg-2、Seg-7序列特征,并首次报道了PALV的DAV与BCV两种血清型病毒在我国的分离,研究结果将有助于掌握我国PALV的分布与遗传特征,为诊断试剂的开发、流行病学调查与致病性研究提供科学依据。
基金supported by Guangzhou Science and Technology Program for Public Wellbeing(2014Y2-00185)the Special Program of National Science and Technology of China(2013ZX10004-805)the National Natural Science Foundation of China(81301491 and 31270974)
文摘Dengue has been well recognized as a global public health threat,but only sporadic epidemics and imported cases were reported in recent decades in China.Since July 2014,an unexpected large dengue outbreak has occurred in Guangdong province,China,resulting in more than 40000 patients including six deaths.To clarify and characterize the causative agent of this outbreak,the acute phase serum from a patient diagnosed with severe dengue was subjected to virus isolation and high-throughput sequencing(HTS).Traditional real-time RT-PCR and HTS with Ion Torrent PGM detected the presence of dengue virus serotype 2(DENV-2).A clinical DENV-2 isolate GZ05/2014 was obtained by culturing the patient serum in mosquito C6/36 cells.The complete genome of GZ05/2014 was determined and deposited in Gen Bank under the access number KP012546.Phylogenetic analysis based on the complete envelope gene showed that the newly DENV-2 isolate belonged to Cosmopolitan genotype and clustered closely with other Guangdong strains isolated in the past decade.No amino acid mutations that are obviously known to increase virulence or replication were identified throughout the genome of GZ05/2014.The high homology of Guangdong DENV-2 strains indicated the possibility of establishment of local DENV-2 circulation in Guangdong,China.These results help clarify the origin of this epidemic and predict the future status of dengue in China.
文摘Using two pairs of primer based on conserved sequences of the NP gene of influenza A virus, a RT-nested PCR assay was developed to detect Swine influenza virus in clinical samples. No cross reactions with three other swine pathogens of respiratory disease such as Porcine respiratory and reproductive syndrome virus, Porcine circovirus type 2 and Mycoplasma suipneumoniae. With agarose electrophoresis detection, the RT-nested PCR was 100 times more sensitive compared to that of non-nested RT-PCR. 35 clinical samples including 4 blood samples and 31 lung tissues from Southern China were tested by RT-nested PCR, and 28 (80%) out of 35 sample were positive. By propagated in 9-to 10-day-old SPF embryonated chicken eggs, 23 strains of Swine influenza virus were isolated from 28 positive samples. The result of RT-nested PCR was 82% in agreement with that of virus isolation, and the RT-nested PCR were more sensitive than the virus isolation. No Swine influenza virus was isolated from the all RT-nested PCR negative samples. On the basis of characterization of HA gene sequences, 5 out of 23 strains had H3 and H9 subtypes. It suggested that the RT-nested PCR assay was rapid,specific and sensitive, and it was is advantageous to detect influenza virus in latent infections.
