H5N1亚型禽流感病毒NS第263～277位核苷酸缺失提高病毒对鸡的致病力

为研究2000年以来绝大多数H5N1亚型禽流感病毒分离株在非结构基因的第263~277位发生15个碱基缺失现象的生物学意义,构建H5N1 A/D/SD/04株HA、NA、NS的全基因表达/转录载体,以及NS的删除突变载体(m248),A/D/YZ/04株的NS基因表达/转录载体(848)和其补加15个核苷酸的NS突变载体(m848)。构建的载体分别与编码WSN(H1N1)内部基因载体进行组合转染,拯救获得4个具不同NS的重组的H5N1亚型流感病毒:RWSN-848和RWSN-m248在263~277位缺失15个碱基,RWSN-m848和RWSN-248则在相同位置不发生缺失。4个重组病毒的平均鸡胚繁殖效价(HA)、鸡胚的平均死亡时间(MDT)和鸡胚半数感染量(EID50)均无显著差异;但RWSN-848和RWSN-m248对6周龄SPF鸡的致病力明显高于RWSN-m848和RWSN-248。结果说明H5N1的NS基因在263~277位核苷酸发生缺失后,不影响重组H5N1在鸡胚中的繁殖性能,但提高了病毒对鸡的致病力。     

禽流感病毒NS第263～277位核苷酸缺失降低其抗干扰素能力

2000年以来,多数H5N1亚型禽流感病毒在NS基因的263～277位发牛15个碱基的缺失。为了研究此缺失在流感病毒进化中的生物学意义,构建H5N1亚型流感病毒A／SD／04株的HA、NA、NS的全基因表达载体,以及NS基因263～277位删除的突变载体。通过反向遗传学技术,与编码WSN的其他内部基因（PB2,PB1,PA,NP和M）的表达载体进行组合转染,获得在NS基因的263～277位缺失和不缺失的2个重组H5N1亚型流感病毒（RWSN—m248和RWSN-248）。此两个重组病毒在无干扰素产生的Vero细胞上的繁殖滴度相似,在能产生干扰素的细胞MDCK和COS-1细胞上的繁殖滴度有明显差异。两个重组病毒在鸡胚中的繁殖滴,IVPI,MDT和EID50均无显著差异。说明NS基因的263～277位核苷酸的缺失不影响病毒的整体毒力,但降低了H5N1的抗干扰素能力。     

禽流感病毒NS第263~277位核苷酸缺失降低其抗干扰素能力*

2000年以来,多数H5N1亚型禽流感病毒在NS基因的263~277位发生15个碱基的缺失。为了研究此缺失在流感病毒进化中的生物学意义,构建H5N1亚型流感病毒A/SD/04株的HA、NA、NS的全基因表达载体,以及NS基因263~277位删除的突变载体。通过反向遗传学技术,与编码WSN的其他内部基因(PB2,PB1,PA,NP和M)的表达载体进行组合转染,获得在NS基因的263~277位缺失和不缺失的2个重组H5N1亚型流感病毒(RWSN-m248和RWSN-248)。此两个重组病毒在无干扰素产生的Ve     

H5N1亚型禽流感病毒拯救体系的建立

选择鸡胚高产的鸭源H5N1亚型禽流感病毒A/Duck/Shandong/093/2004株作为骨架病毒,在完成了全基因组序列测定基础上,设计合成的11对引物对病毒的8个基因分11段进行扩增。通过与转录载体PHW2000连接,构建A/SD/04的8个基因的拯救载体,经测序获得序列准确的拯救质粒:2412、42、243、244、245、246、247和248。A/SD/04的8质粒与PR8(H1N1)进行不同组合的拯救,获得8个均含A/SD/04 HA基因的H5重组流感病毒。鸡胚尿囊液中重组病毒的血凝效价在28~210,EID50在10-8.5~10-9之间,MDT在34~46h之间,均与野生A/SD/04(wt A/SD/04)相似。重组病毒对6周龄的SPF鸡的静脉接种指数(IVPI)与wt A/SD/04却有明显的差异,说明不同组合的内部基因影响病毒对鸡的致病力,但不影响病毒的鸡胚致死能力、对鸡胚的感染能力和病毒在鸡胚中的繁殖能力。构建的A/SD/04的8个质粒拯救系统,为H5N1的基因功能研究和新型疫苗开发奠定基础。     

一株H5N1亚型禽流感病毒的分离与鉴定

2004年1月湖北宜昌某鸡场暴发疫病,从该鸡场濒死鸡肺组织中分离到了一株病毒,电镜切片观察到典型的禽流感病毒粒子;采用ELISA检测禽流感抗原为阳性;RT-PCR扩增HA、NA基因并测序,经BLAST分析,HA基因与A／Goose／Guangdong／1／96(H5N1)HA基因同源性为97％;NA基因与A／Goose／Guangdong／1／96(H5N1)NA基因同源性为96％,确定该分离株为禽流感病毒H5N1亚型(A／Chicken／Yichang／Lung-1／04(H5N1))。     

利用反向遗传学技术构建H5亚型禽流感高产疫苗株

采用RT-PCR技术分别扩增了鹅源高产禽流感病毒的6条内部基因片段,近期分离的H5N1亚型禽流感病毒的血凝素基因以及N3亚型参考毒株的神经氨酸酶基因,分别构建了8个基因的转录与表达载体,利用反向遗传学技术拯救出了全部基因都源于禽源的重组流感病毒疫苗株rH5N3。通过对血凝素蛋白HA1和HA2连接肽处的5个碱性氨基酸(R-R-R-K-K)基因缺失与修饰,从而消除了病毒基因的毒力相关序列,拯救的rH5N3疫苗株对鸡和鸡胚均无致病性,病毒在鸡胚尿囊液和细胞培养上清的HA效价得到极大提高,分别为12048和1512。制备的禽流感疫苗免疫动物后4~5周即可诱导产生高效价的HI抗体,鸡免疫后18周依然保持高水平的HI抗体。重组疫苗不论是对于国内早期分离的禽流感病毒A/Goose/Guangdong/1/96还是近期分离的A/Goose/HLJ/QFY/04都能够产生完全的免疫保护作用,免疫鸡攻毒后不发病、不排毒、不死亡。带有N3鉴别诊断标记禽流感疫苗株的研制为H5N1高致病性禽流感的防治提供了新的技术保障。     

抗体选择压作用下H9N2亚型禽流感病毒HA基因的变异

摘要：【目的】了解H9N2亚型禽流感病毒(AIV)在抗体选择压作用下的遗传变异。【方法】将制备疫苗用的LG1株H9N2亚型AIV分别接种含有母源抗体鸡胚(A组)和不含有母源抗体的SPF鸡胚(B组),并连续传代。其中A组再分为4 个独立的传代系列A1-4,B组再分为2 个独立传代系列B1-2。在每个传代系列,分别对第10,20,30,40,50 代病毒的HA基因进行扩增克隆测序,并与原始病毒的序列比较。【结果】LG1株H9N2在没有抗体的鸡胚的传代过程中,仅发生少数碱基的不稳定随机变异,且多为无义突变。在2 个传代系列的10 个代次病毒,共出现了29 个位点变异,有义突变(NS)与无义突变(S)比值NS/S为1.42 。但在有抗体的鸡胚的传代过程中,发生了多个呈现稳定遗传的有义突变。在4 个传代系列的20 个代次病毒,共出现了45 个位点变异,有义突变(NS)与无义突变(S)比值NS/S为3.46。【结论】在鸡胚传代过程中母源抗体提供的免疫选择压确实能影响H9N2的HA基因的变异。同时表明,带有母源抗体的鸡胚是实验室条件下研究免疫选择压对病毒抗原性变异影响的一种有效的实验模型。     

H3N2亚型猪流感病毒的拯救及HA与NA基因的替换

摘要：【目的】为研究流感病毒突破种间屏障分子机制,筛选流感基因工程疫苗株。【方法】本实验以猪流感病毒A/Swine/Henan/S4/01(H3N2)为亲本株,利用反向遗传学操作技术,采用RT-PCR技术对该病毒的8个基因片段分段进行扩增,通过与双向转录载体pHW2000连接, 重组质粒转染293T和MDCK共培养细胞,拯救出全部基因均来自于亲本株的猪流感病毒rgH3N2,并分别以人流感病毒A/ PR/8/34(H1N1)、禽流感病毒A/Duck/Nanchang /4-165/2000 (H4N6 )、马流感病毒A/Equine/Fuyun/2008/(H3N8)的HA和NA基因替换A/Swine/Henan/S4/01的相应基因,【结果】生物学实验结果表明rgH3N2在鸡胚半数感染量、组织培养半数感染量、稳定性试验等方面都与亲本株保持一致。rgH3N2经鸡胚多次传代后血凝价最高可达到1:256,接种MDCK细胞60 h后,血凝价可以达到1:64。基因替换后成功拯救出的重组病毒rgH1N1、rgH4N6和rgH3N8在鸡胚和细胞上均具有较高的增殖能力。【结论】病毒的成功拯救为流感病毒突破种间屏障分子机制,HA、NA基因在流感病毒跨种属传播中所扮演角色的研究和流感基因工程疫苗株的筛选奠定了基础。     

用8质粒病毒拯救系统产生H9N2/WSN重组A型流行性感冒病毒

把禽流行性感冒(流感)病毒A／Chicken／Shanghai／F／98(H9N2)的血凝素(HA)和神经氨酸酶(NA)基因cDNA克隆至polⅠ-pol Ⅱ双向转录和表达载体pHW2000,用这两种质粒与8质粒病毒拯救系统中流感病毒A／WSN／33(H1N1)6个内部基因cDNA的质粒组合(6 2重排),共转染COS-1细胞,产生了能在鸡胚中高滴度增殖的H9N2／、WSN重组病毒。用A／WSN／33的8个基因cDNA质粒作对照,也产生了转染子病毒。经过EID50测定和MDCK感染实验,新基因型H9N2／WSN病毒感染鸡胚的能力强(EID50为10^-11／0．2m1),而且对鸡胚的毒力弱,在不加胰酶的情况下不使MDCK细胞产牛病变。经电镜观察,两个转染子病毒的形态与野生型流感病毒相似。反向遗传操作技术的建立,为对禽流感病毒基因功能和疫苗构建等方面的研究提供了新的手段。     

一株H5N1亚型禽流感病毒的分离与鉴定

2004年1月湖北宜昌某鸡场暴发疫病,从该鸡场濒死鸡肺组织中分离到了一株病毒,电镜切片观察到典型的禽流感病毒粒子;采用ELISA检测禽流感抗原为阳性;RT-PCR扩增HA、NA基因并测序,经BLAST分析,HA基因与A/Goose/Guangdong/1/96(H5N1)HA基因同源性为97%;NA基因与A/Goose/Guangdong/1/96(H5N1)NA基因同源性为96%,确定该分离株为禽流感病毒H5N1亚型(A/Chicken/Yichang/Lung-1/04(H5N1)).     

Highly pathogenic avian influenza viruses do not inhibit interferon synthesis in infected chickens but can override the interferon-induced antiviral state

From infection studies with cultured chicken cells and experimental mammalian hosts, it is well known that influenza viruses use the nonstructural protein 1 (NS1) to suppress the synthesis of interferon (IFN). However, our current knowledge regarding the in vivo role of virus-encoded NS1 in chickens is much more limited. Here, we report that highly pathogenic avian influenza viruses of subtypes H5N1 and H7N7 lacking fully functional NS1 genes were attenuated in 5-week-old chickens. Surprisingly, in diseased birds infected with NS1 mutants, the IFN levels were not higher than in diseased birds infected with wild-type virus, suggesting that NS1 cannot suppress IFN gene expression in at least one cell population of infected chickens that produces large amounts of the cytokine in vivo. To address the question of why influenza viruses are highly pathogenic in chickens although they strongly activate the innate immune system, we determined whether recombinant chicken alpha interferon (IFN-α) can inhibit the growth of highly pathogenic avian influenza viruses in cultured chicken cells and whether it can ameliorate virus-induced disease in 5-week-old birds. We found that IFN treatment failed to confer substantial protection against challenge with highly pathogenic viruses, although it was effective against viruses with low pathogenic potential. Taken together, our data demonstrate that preventing the synthesis of IFN is not the primary role of the viral NS1 protein during infection of chickens. Our results further suggest that virus-induced IFN does not contribute substantially to resistance of chickens against highly pathogenic influenza viruses.     

The Surface Glycoproteins of H5 Influenza Viruses Isolated from Humans, Chickens, and Wild Aquatic Birds Have Distinguishable Properties

In 1997, 18 confirmed cases of human influenza arising from multiple independent transmissions of H5N1 viruses from infected chickens were reported from Hong Kong. To identify possible phenotypic changes in the hemagglutinin (HA) and neuraminidase (NA) of the H5 viruses during interspecies transfer, we compared the receptor-binding properties and NA activities of the human and chicken H5N1 isolates from Hong Kong and of H5N3 and H5N1 viruses from wild aquatic birds. All H5N1 viruses, including the human isolate bound to Sia2-3Gal-containing receptors but not to Sia2-6Gal-containing receptors. This finding formally demonstrates for the first time that receptor specificity of avian influenza viruses may not restrict initial avian-to-human transmission. The H5N1 chicken viruses differed from H5 viruses of wild aquatic birds by a 19-amino-acid deletion in the stalk of the NA and the presence of a carbohydrate at the globular head of the HA. We found that a deletion in the NA decreased its ability to release the virus from cells, whereas carbohydrate at the HA head decreased the affinity of the virus for cell receptors. Comparison of amino acid sequences from GenBank of the HAs and NAs from different avian species revealed that additional glycosylation of the HA and a shortened NA stalk are characteristic features of the H5 and H7 chicken viruses. This finding indicates that changes in both HA and NA may be required for the adaptation of influenza viruses from wild aquatic birds to domestic chickens and raises the possibility that chickens may be a possible intermediate host in zoonotic transmission.     

A naturally occurring deletion in its NS gene contributes to the attenuation of an H5N1 swine influenza virus in chickens

In 2001 and 2003, we isolated two H5N1 viruses, A/swine/Fujian/1/01 (SW/FJ/01) and A/swine/Fujian/1/03 (SW/FJ/03), from pigs in Fujian Province, southern China. Genetically, these two viruses are similar, although the NS gene of the SW/FJ/03 virus has a 15-nucleotide deletion at coding positions 612 to 626. The SW/FJ/01 virus is highly lethal for chickens, whereas the SW/FJ/03 virus is nonpathogenic for chickens when administrated intravenously or intranasally. To understand the molecular basis for the difference in virulence, we used reverse genetics to create a series of single-gene recombinants of both viruses. We found that a recombinant virus containing the mutated NS gene from the SW/FJ/03 virus in the SW/FJ/01 virus background was completely attenuated in chickens. We also found that viruses expressing the mutant NS1 protein of SW/FJ/03 did not antagonize the induction of interferon (IFN) protein. Conversely, only the recombinant virus containing the wild-type SW/FJ/01 NS gene in the SW/FJ/03 background was lethal in chickens and antagonized IFN protein levels. Further, we proved that the NS1 genes of the two viruses differ in their stabilities in the host cells and in their abilities to interact with the chicken cleavage and polyadenylation specificity factor. These results indicate that the deletion of amino acids 191 to 195 of the NS1 protein is critical for the attenuation of the SW/FJ/03 virus in chickens and that this deletion affects the ability of the virus to antagonize IFN induction in host cells.     

Emergence of avian influenza viruses with enhanced transcription activity by a single amino acid substitution in the nucleoprotein during replication in chicken brains

To explore the genetic basis of the pathogenesis and adaptation of avian influenza viruses (AIVs) to chickens, the A/duck/Yokohama/aq10/2003 (H5N1) (DkYK10) virus was passaged five times in the brains of chickens. The brain-passaged DkYK10-B5 caused quick death of chickens through rapid and efficient replication in tissues, accompanied by severe apoptosis. Genome sequence comparison of two viruses identified a single amino acid substitution at position 109 in NP from isoleucine to threonine (NP (I)109(T)). By analyzing viruses constructed by the reverse-genetic method, we established that the NP (I)109(T) substitution also contributed to increased viral replication and polymerase activity in chicken embryo fibroblasts, but not in duck embryo fibroblasts. Real-time RT-PCR analysis demonstrated that the NP (I)109(T) substitution enhances mRNA synthesis quickly and then cRNA and viral RNA (vRNA) synthesis slowly. Next, to determine the mechanism underlying the appearance of the NP (I)109(T) substitution during passages, four H5N1 highly pathogenic AIVs (HPAIVs) were passaged in the lungs and brains of chicken embryos. Single-nucleotide polymorphism analysis, together with a database search, suggests that the NP (I)109(T) mutation would be induced frequently during replication of HPAIVs in brains, but not in lungs. These results demonstrate that the amino acid at position 109 in NP enhances viral RNA synthesis and the pathogenicity of highly pathogenic avian influenza viruses in chickens and that the NP mutation emerges quickly during replication of the viruses in chicken brains.     

H9N2亚型禽流感病毒NS1基因的进化分析

利用RT-PCR方法,扩增了1998～2005年间分离的9株H9N2亚型禽流感病毒的NS1基因,对其进行了序列测定和进化分析.序列分析表明,9株AIV NS1基因完整的阅读框均为654bp,编码217个氨基酸,其核苷酸和推导的氨基酸同源性分别为95.4%～99.8%和93.6%～100%;9株病毒的NS1蛋白的C端均有13个氨基酸的缺失;进化分析表明,9株AIV属于A群,且形成一个独立分支,在该分支中,只有Ck/HN/A3/98株属于Ck/HK/Y280/97-like亚类,且与Ck/BJ/8/98的进化关系最近,其余8株属于Ck/SH/F/98-like亚类,说明Ck/SH/F/98-like亚类的H9N2亚型AIV在中国大陆的鸡群中广泛存在.NS1基因的进化及其编码产物的特性分析,为AIV的毒力变异、致病机制、药物靶位点的设计及鉴别诊断的研究奠定了基础.     

Characterization of a human H5N1 influenza A virus isolated in 2003

In 2003, H5N1 avian influenza virus infections were diagnosed in two Hong Kong residents who had visited the Fujian province in mainland China, affording us the opportunity to characterize one of the viral isolates, A/Hong Kong/213/03 (HK213; H5N1). In contrast to H5N1 viruses isolated from humans during the 1997 outbreak in Hong Kong, HK213 retained several features of aquatic bird viruses, including the lack of a deletion in the neuraminidase stalk and the absence of additional oligosaccharide chains at the globular head of the hemagglutinin molecule. It demonstrated weak pathogenicity in mice and ferrets but caused lethal infection in chickens. The original isolate failed to produce disease in ducks but became more pathogenic after five passages. Taken together, these findings portray the HK213 isolate as an aquatic avian influenza A virus without the molecular changes associated with the replication of H5N1 avian viruses in land-based poultry such as chickens. This case challenges the view that adaptation to land-based poultry is a prerequisite for the replication of aquatic avian influenza A viruses in humans.     

Role of position 627 of PB2 and the multibasic cleavage site of the hemagglutinin in the virulence of H5N1 avian influenza virus in chickens and ducks

Highly pathogenic H5N1 avian influenza viruses have caused major disease outbreaks in domestic and free-living birds with transmission to humans resulting in 59% mortality amongst 564 cases. The mutation of the amino acid at position 627 of the viral polymerase basic-2 protein (PB2) from glutamic acid (E) in avian isolates to lysine (K) in human isolates is frequently found, but it is not known if this change affects the fitness and pathogenicity of the virus in birds. We show here that horizontal transmission of A/Vietnam/1203/2004 H5N1 (VN/1203) virus in chickens and ducks was not affected by the change of K to E at PB2-627. All chickens died between 21 to 48 hours post infection (pi), while 70% of the ducks survived infection. Virus replication was detected in chickens within 12 hours pi and reached peak titers in spleen, lung and brain between 18 to 24 hours for both viruses. Viral antigen in chickens was predominantly in the endothelium, while in ducks it was present in multiple cell types, including neurons, myocardium, skeletal muscle and connective tissues. Virus replicated to a high titer in chicken thrombocytes and caused upregulation of TLR3 and several cell adhesion molecules, which may explain the rapid virus dissemination and location of viral antigen in endothelium. Virus replication in ducks reached peak values between 2 and 4 days pi in spleen, lung and brain tissues and in contrast to infection in chickens, thrombocytes were not involved. In addition, infection of chickens with low pathogenic VN/1203 caused neuropathology, with E at position PB2-627 causing significantly higher infection rates than K, indicating that it enhances virulence in chickens.     

Identification of a new genotype of Torque Teno Mini virus

Background

Since we were able to isolate viable virus from brain and lung of H7N1 low pathogenic avian influenza virus (LPAIV) infected chickens, we here examined the distribution of different LPAIV strains in chickens by measuring the viral AI RNA load in multiple organs. Subtypes of H5 (H5N1, H5N2), H7 (H7N1, H7N7) and H9 (H9N2), of chicken (H5N2, H7N1, H7N7, H9N2), or mallard (H5N1) origin were tested. The actual presence of viable virus was evaluated with virus isolation in organs of H7N7 inoculated chickens.

Findings

Viral RNA was found by PCR in lung, brain, intestine, peripheral blood mononuclear cells, heart, liver, kidney and spleen from chickens infected with chicken isolated LPAIV H5N2, H7N1, H7N7 or H9N2. H7N7 virus could be isolated from lung, ileum, heart, liver, kidney and spleen, but not from brain, which was in agreement with the data from the PCR. Infection with mallard isolated H5N1 LPAIV resulted in viral RNA detection in lung and peripheral blood mononuclear cells only.

Conclusion

We speculate that chicken isolated LPAI viruses are spreading systemically in chicken, independently of the strain.