禽流感病毒分型基因芯片的研制

[目的]禽流感病毒是一种全球重要的人和动物呼吸道病病原,快速确定其不同亚型对于全球流感监测具有重要的意义.本研究意在研制一种可同时鉴定禽流感病毒所有亚型的方法.[方法]根据GenBank上已发表的禽流感病毒不同亚型(16个HA亚型和9个NA亚型)的基因序列,设计合成了25对特异性引物和1对通用引物,然后以各亚型病毒的参考株RNA作为模板,建立扩增不同亚型的多重RT-PCR方法.参考各亚型病毒靶cDNAs区域的保守序列设计了52条亚型特异的探针,进而利用扩增的各亚型病毒的靶cDNAs对其特异性进行评价.在此基础上,将设计好的探针点制到处理好的玻片上,制备了禽流感病毒分型鉴定基因芯片,结合所建立的扩增不同亚型的多重RT-PCR方法,开发了禽流感病毒亚型鉴定基因芯片试剂.利用收集自49个地区的2653份标本对其特异性和敏感性进行了初步评价.[结果]用于评价的各亚型参考毒株均出现良好的特异性杂交信号,检测的敏感度可达2.47 PFU/mL或2.5 ng靶DNA片段,而且与禽类常见的IBV、NDV等6种病毒均无交叉反应.[结论]证明该病毒分型基因芯片具有良好的特异性、敏感性.     

利用基因芯片技术区分禽流感病毒主要亚型

[目的]研制可同时区分AIV的H5、H7、H9血凝素亚型及N1、N2神经氨酸酶亚型的基因诊断芯片.[方法]分别克隆了禽流感病毒的M基因,H5、H7、H9亚型HA基因,N1、N2亚型NA基因以及看家基因GAPDH的重组质粒.以重组质粒为模板,用PCR方法扩增制备探针,纯化后点于氨基修饰的片基上,制备基因芯片.在PCR过程中对待检样品进行标记,然后与芯片杂交,洗涤,扫描并进行结果分析.[结果]结果显示检测探针可特异性的与相应的标记样品进行杂交,呈现较强的杂交信号,且无交叉杂交.同时用RT-PCR、鸡胚接种和基因芯片方法对H1-H15亚型AIV参考毒株、30份人工感染样品、21份现地疑似样品进行检测,结果发现,对人工感染样品芯片检测方法与鸡胚接种和RT-PCR的符合率分别为100%和96%,现地样品符合率为100%.[结论]研究表明该方法可用于同步鉴别部分主要流行的禽流感亚型,是一种有效的新方法.     

The Genetic Diversity of Influenza A Viruses in Wild Birds in Peru

Our understanding of the global ecology of avian influenza A viruses (AIVs) is impeded by historically low levels of viral surveillance in Latin America. Through sampling and whole-genome sequencing of 31 AIVs from wild birds in Peru, we identified 10 HA subtypes (H1-H4, H6-H7, H10-H13) and 8 NA subtypes (N1-N3, N5-N9). The majority of Peruvian AIVs were closely related to AIVs found in North America. However, unusual reassortants, including a H13 virus containing a PA segment related to extremely divergent Argentinian viruses, suggest that substantial AIV diversity circulates undetected throughout South America.     

Comparative Pathogenesis of an Avian H5N2 and a Swine H1N1 Influenza Virus in Pigs

Pigs are considered intermediate hosts for the transmission of avian influenza viruses (AIVs) to humans but the basic organ pathogenesis of AIVs in pigs has been barely studied. We have used 42 four-week-old influenza naive pigs and two different inoculation routes (intranasal and intratracheal) to compare the pathogenesis of a low pathogenic (LP) H5N2 AIV with that of an H1N1 swine influenza virus. The respiratory tract and selected extra-respiratory tissues were examined for virus replication by titration, immunofluorescence and RT-PCR throughout the course of infection. Both viruses caused a productive infection of the entire respiratory tract and epithelial cells in the lungs were the major target. Compared to the swine virus, the AIV produced lower virus titers and fewer antigen positive cells at all levels of the respiratory tract. The respiratory part of the nasal mucosa in particular showed only rare AIV positive cells and this was associated with reduced nasal shedding of the avian compared to the swine virus. The titers and distribution of the AIV varied extremely between individual pigs and were strongly affected by the route of inoculation. Gross lung lesions and clinical signs were milder with the avian than with the swine virus, corresponding with lower viral loads in the lungs. The brainstem was the single extra-respiratory tissue found positive for virus and viral RNA with both viruses. Our data do not reject the theory of the pig as an intermediate host for AIVs, but they suggest that AIVs need to undergo genetic changes to establish full replication potential in pigs. From a biomedical perspective, experimental LP H5 AIV infection of pigs may be useful to examine heterologous protection provided by H5 vaccines or other immunization strategies, as well as for further studies on the molecular pathogenesis and neurotropism of AIVs in mammals.     

1998～2008年中国中部H9N2亚型AIV分离毒株HA基因的进化分析

从过去10年由中国中部分离的具有不同致病力的25株H9N2亚型禽流感选出6株(3#、12#、25#、14#、4#、22#)代表性毒株,利用RT-PCR扩增它们的HA基因,并比较分析该基因的序列,旨在探讨HA基因的变异对AIV毒力、抗原性变化的影响。结果表明:6株H9N2 AIV亚型分离株的HA基因在HA1和HA2的氨基酸裂解位点上没有出现高致病性禽流感病毒所特有的R-X-R/K-R模式,它们均为弱毒力毒株。HA上潜在糖基化位点除了3#和12#分离株多出一个之外,其余均为8个。3#和12#所表现出较强的致病性可能与其在HA的头部(HA1)的A抗原位点上多了一个糖基化位点(145~147aa),改变了HA基因空间构型有关,空间构型的改变导致抗HA抗体作用位点的变异或缺失并影响其较近的受体结合位点,从而改变该毒株的抗原性。研究结果提示需要持续跟踪H9N2 AIV在中国鸡群中的传播和进化,以便及时掌握疫情,有效防控禽流感。     

H5亚型禽流感病毒一步法RT-PCR检测方法的建立

针对家禽中流行较为广泛、危害相对大的H5亚型禽流感病毒的血凝素（HA）基因,通过分析流感数据库221个HA序列,在保守区内用Oligo6.0软件设计并合成了一对引物,建立了用于快速诊断H5亚型禽流感病毒的一步法RT-PCR方法,其扩增的目的片段大小为372bp。通过对H5亚型禽流感病毒尿囊液和棉拭子浸出液进行不同稀释倍数检测,结果表明病毒尿囊液最低检出量为10-4稀释;阳性棉拭子最低检出量为8倍稀释。用病毒分离和该方法同时检测不同脏器、口咽及泄殖腔棉拭子样品,结果表明该方法检测灵敏度比病毒分离低10～100倍。用该方法检测H1～H15亚型禽流感病毒和鸡新城疫病毒等其他14种禽病病原,仅有H5亚型禽流感病毒扩增出特异性目的条带。该方法具有方便快捷、特异性强、敏感性高等特点,为我国禽流感的快速诊断和分子流行病学调查提供了技术支撑。     

Influenza A viruses in American White Pelican (Pelecanus erythrorhynchos)

The role of many wild waterbird species in the ecology and epidemiology of avian influenza viruses (AIV) remains unclear. We report the first isolation of AIV from American White Pelicans (Pelecanus erythrorhynchos; Pelecaniformes) in North America. Two H13N9 AIVs were isolated from hatchling birds in breeding colonies in Minnesota, USA, during 2007 and 2008. Based on molecular sequencing of the hemagglutinin and neuraminidase genes, the 2008 virus was genetically related to AIVs previously isolated from gulls and shorebirds in North America. The 2007 isolate was most related to AIVs from Eurasian gulls and North American ducks, reflecting both global movement of these viruses and reassortment between viruses associated with duck and gull reservoirs.     

Genetic and biological properties of H7N9 avian influenza viruses detected after application of the H7N9 poultry vaccine in China

The H7N9 avian influenza virus (AIV) that emerged in China have caused five waves of human infection. Further human cases have been successfully prevented since September 2017 through the use of an H7N9 vaccine in poultry. However, the H7N9 AIV has not been eradicated from poultry in China, and its evolution remains largely unexplored. In this study, we isolated 19 H7N9 AIVs during surveillance and diagnosis from February 2018 to December 2019, and genetic analysis showed that these viruses have formed two different genotypes. Animal studies indicated that the H7N9 viruses are highly lethal to chicken, cause mild infection in ducks, but have distinct pathotypes in mice. The viruses bound to avian-type receptors with high affinity, but gradually lost their ability to bind to human-type receptors. Importantly, we found that H7N9 AIVs isolated in 2019 were antigenically different from the H7N9 vaccine strain that was used for H7N9 influenza control in poultry, and that replication of these viruses cannot, therefore, be completely prevented in vaccinated chickens. We further revealed that two amino acid mutations at positions 135 and 160 in the HA protein added two glycosylation sites and facilitated the escape of the H7N9 viruses from the vaccine-induced immunity. Our study provides important insights into H7N9 virus evolution and control.     

Subtyping animal influenza virus with general multiplex RT-PCR and Liquichip high throughput (GMPLex)

This study developed a multiplex RT-PCR integrated with luminex technology to rapidly subtype simultaneously multiple influenza viruses. Primers and probes were designed to amplify NS and M genes of influenza A viruses HA gene of H1, H3, H5, H7, H9 subtypes, and NA gene of the N1 and N2 subtypes. Universal super primers were introduced to establish a multiplex RT-PCR (GM RT-PCR). It included three stages of RT-PCR amplification, and then the RT-PCR products were further tested by LiquiChip probe, combined to give an influenza virus (IV) rapid high throughput subtyping test, designated as GMPLex. The IV GMPLex rapid high throughput subtyping test presents the following features: high throughput, able to determine the subtypes of 9 target genes in H1, H3, H5, H7, H9, N1, and N2 subtypes of the influenza A virus at one time; rapid, completing the influenza subtyping within 6 hours; high specificity, ensured the specificity of the different subtypes by using two nested degenerate primers and one probe, no cross reaction occurring between the subtypes, no non-specific reactions with other pathogens and high sensitivity. When used separately to detect the product of single GM RT-PCR for single H5 or N1 gene, the GMPLex test showed a sensitivity of 10⁻⁵(= 280ELD₅₀) forboth tests and the Luminex qualitative ratio results were 3.08 and 3.12, respectively. When used to detect the product of GM RT-PCR for H5N1 strain at the same time, both showed a sensitivity of 10⁻⁴(=2800 ELD₅₀). The GMPLex rapid high throughput subtyping test can satisfy the needs of influenza rapid testing.Key words: Influenza Virus, General multiplex RT-PCR, Iuminex assay, Subtyping, HA and NA genes     

Development of multiplex rt-PCR assays for rapid detection and subtyping of influenza type A viruses from clinical specimens

We developed multiplex RT-PCR assays that can detect and identify 12 hemagglutinin (H1-H12) and 9 neuraminidase (N1-N9) subtypes that are commonly isolated from avian, swine, and human influenza A viruses. RT-PCR products with unique sizes characteristic of each subtype were amplified by multiplex RT-PCRs, and sequence analysis of each amplicon was demonstrated to be specific for each subtype with 24 reference viruses. The specificity was demonstrated further with DNA or cDNA templates from 7 viruses, 5 bacteria, and 50 influenza A virus negative specimens. Furthermore, the assays could detect and subtype up to 105 dilution of each of the reference viruses that had an original infectivity titer of 106 EID50/ml. Of 188 virus isolates, the multiplex RT-PCR results agreed completely with individual RT-PCR subtyping results and with results obtained from virus isolations. Furthermore, the multiplex RT-PCR methods efficiently detected mixed infections with at least two different subtypes of influenza viruses in one host. Therefore, these methods could facilitate rapid and accurate subtyping of influenza A viruses directly from field specimens.     

Genetic Structure of Avian Influenza Viruses from Ducks of the Atlantic Flyway of North America

Wild birds, including waterfowl such as ducks, are reservoir hosts of influenza A viruses. Despite the increased number of avian influenza virus (AIV) genome sequences available, our understanding of AIV genetic structure and transmission through space and time in waterfowl in North America is still limited. In particular, AIVs in ducks of the Atlantic flyway of North America have not been thoroughly investigated. To begin to address this gap, we analyzed 109 AIV genome sequences from ducks in the Atlantic flyway to determine their genetic structure and to document the extent of gene flow in the context of sequences from other locations and other avian and mammalian host groups. The analyses included 25 AIVs from ducks from Newfoundland, Canada, from 2008–2011 and 84 available reference duck AIVs from the Atlantic flyway from 2006–2011. A vast diversity of viral genes and genomes was identified in the 109 viruses. The genetic structure differed amongst the 8 viral segments with predominant single lineages found for the PB2, PB1 and M segments, increased diversity found for the PA, NP and NS segments (2, 3 and 3 lineages, respectively), and the highest diversity found for the HA and NA segments (12 and 9 lineages, respectively). Identification of inter-hemispheric transmissions was rare with only 2% of the genes of Eurasian origin. Virus transmission between ducks and other bird groups was investigated, with 57.3% of the genes having highly similar (≥99% nucleotide identity) genes detected in birds other than ducks. Transmission between North American flyways has been frequent and 75.8% of the genes were highly similar to genes found in other North American flyways. However, the duck AIV genes did display spatial distribution bias, which was demonstrated by the different population sizes of specific viral genes in one or two neighbouring flyways compared to more distant flyways.     

H7亚型禽流感病毒一步法RT-PCR检测方法的建立

通过分析流感数据库45个H7亚型禽流感病毒的HA序列,在保守区内设计并合成引物,建立了一步法RT-PCR检测方法,扩增片段大小为501bp。通过对H7亚型禽流感病毒尿囊液和棉拭子浸出液不同滴度检测,证实病毒尿囊液最低检出量为105.5EID50/mL;阳性棉拭子最低检出量为103EID50/mL。用该方法检测H1～H15亚型禽流感病毒和鸡新城疫病毒等其他14种禽病病原进行检测,仅有H7亚型AIV有特异性目的条带,与其他均无交叉反应。从脏器及咽喉、泄殖腔棉拭子样品的病毒分离和RT-PCR方法比较,表明在10-1的样品浓度下,两者可以达到相同的检出量。表明该一步法RT-PCR方法具有特异性强、敏感性高和准确率高的特点。     

改良逆转录环介导等温扩增检测技术快速实时检测H9亚型禽流感病毒

利用改良逆转录环介导等温扩增(RT-LAMP)技术建立一种快速、灵敏的检测方法用于H9亚型禽流感病毒检测。根据H9亚型禽流感病毒血凝素(HA)基因保守区序列中的8个区段设计6条特异性引物,在恒温条件下进行核酸扩增反应,并以琼脂糖凝胶电泳和目视检查绿色荧光两种方法对扩增结果进行判定。结果表明,RT-LAMP的最小检测限为100 fg,灵敏度比PT-PCR高100倍,且与H5亚型、H7亚型禽流感病毒,新城疫病毒(NDV)无交叉反应。目视检查绿色荧光与常规琼脂糖凝胶电泳的判定结果一致。整个扩增检测过程在35 min内即可完成。利用临床样本对RT-LAMP法进行验证,结果与RT-PCR一致。由实时浊度分析得到的标准曲线,计算出临床样本中的病毒质粒拷贝数均在2×107-2×104之间。因此,本研究建立的RT-LAMP方法快速、灵敏、特异性强,是H9亚型禽流感病毒的一种高效检测方法。     

PrimerHunter: a primer design tool for PCR-based virus subtype identification

Rapid and reliable virus subtype identification is critical for accurate diagnosis of human infections, effective response to epidemic outbreaks and global-scale surveillance of highly pathogenic viral subtypes such as avian influenza H5N1. The polymerase chain reaction (PCR) has become the method of choice for virus subtype identification. However, designing subtype-specific PCR primer pairs is a very challenging task: on one hand, selected primer pairs must result in robust amplification in the presence of a significant degree of sequence heterogeneity within subtypes, on the other, they must discriminate between the subtype of interest and closely related subtypes. In this article, we present a new tool, called PrimerHunter, that can be used to select highly sensitive and specific primers for virus subtyping. Our tool takes as input sets of both target and nontarget sequences. Primers are selected such that they efficiently amplify any one of the target sequences, and none of the nontarget sequences. PrimerHunter ensures the desired amplification properties by using accurate estimates of melting temperature with mismatches, computed based on the nearest neighbor model via an efficient fractional programming algorithm. Validation experiments with three avian influenza HA subtypes confirm that primers selected by PrimerHunter have high sensitivity and specificity for target sequences.     

Investigation of Avian Influenza Infections in Wild Birds,Poultry and Humans in Eastern Dongting Lake,China

We investigated avian influenza infections in wild birds, poultry, and humans at Eastern Dongting Lake, China. We analyzed 6,621 environmental samples, including fresh fecal and water samples, from wild birds and domestic ducks that were collected from the Eastern Dongting Lake area from November 2011 to April 2012. We also conducted two cross-sectional serological studies in November 2011 and April 2012, with 1,050 serum samples collected from people exposed to wild birds and/or domestic ducks. Environmental samples were tested for the presence of avian influenza virus (AIV) using quantitative PCR assays and virus isolation techniques. Hemagglutination inhibition assays were used to detect antibodies against AIV H5N1, and microneutralization assays were used to confirm these results. Among the environmental samples from wild birds and domestic ducks, AIV prevalence was 5.19 and 5.32%, respectively. We isolated 39 and 5 AIVs from the fecal samples of wild birds and domestic ducks, respectively. Our analysis indicated 12 subtypes of AIV were present, suggesting that wild birds in the Eastern Dongting Lake area carried a diverse array of AIVs with low pathogenicity. We were unable to detect any antibodies against AIV H5N1 in humans, suggesting that human infection with H5N1 was rare in this region.     

Epidemiology,Evolution, and Recent Outbreaks of Avian Influenza Virus in China

Novel reassortants of H7N9, H10N8, and H5N6 avian influenza viruses (AIVs) are currently circulating in China''s poultry flocks, occasionally infecting humans and other mammals. Combined with the sometimes enzootic H5N1 and H9N2 strains, this cauldron of genetically diverse AIVs pose significant risks to public health. Here, we review the epidemiology, evolution, and recent outbreaks of AIVs in China, discuss reasons behind the recent increase in the emergence of novel AIVs, and identify warning signs which may point to the emergence of a potentially virulent and highly transmissible AIV to humans. This review will be useful to authorities who consider options for the detection and control of AIV transmission in animals and humans, with the goal of preventing future epidemics and pandemics.     

H9与H5亚型禽流感病毒血凝素基因的快速鉴别

建立一步法RT-PCR检测方法,对禽流感病毒(Avian influenza virus,AIV)的血凝素(Hemagglutinin,HA)分型进行了研究。参照AIV的HA基因序列设计1对引物,对H9和H5亚型AIV进行了扩增,产物大小分别为579bp和177bp。经测试,该引物不与新城疫病毒等鸡的其它传染性病原及鸡肌肉组织的核酸发生交叉反应。敏感性分析发现,从50pg的AIV总RNA中亦能扩增到目的条带。结果表明,此次利用1对引物建立的一步法RT-PCR方法简便适用,可以在一次反应中同时将H9和H5亚型AIV进行快速检测和分型。另外,两个亚型的扩增产物均包含了HA裂解位点在内的基因序列,可通过测序推导氨基酸顺序以预测H5或H9亚型禽流感病毒的潜在毒力。     

Homologous recombination as an evolutionary force in the avian influenza A virus

Avian influenza A viruses (AIVs), including the H5N1, H9N2,and H7N7 subtypes, have been directly transmitted to humans,raising concerns over the possibility of a new influenza pandemic.To prevent a future avian influenza pandemic, it is very importantto fully understand the molecular basis driving the change inAIV virulence and host tropism. Although virulent variants ofother viruses have been generated by homologous recombination,the occurrence of homologous recombination within AIV segmentsis controversial and far from proven. This study reports threecirculating H9N2 AIVs with similar mosaic PA genes descendedfrom H9N2 and H5N1. Additionally, many homologous recombinantsare also found deposited in GenBank. Recombination events canoccur in PB2, PB1, PA, HA, and NP segments and between lineagesof the same/different serotype. These results collectively demonstratethat intragenic recombination plays a role in driving the evolutionof AIVs, potentially resulting in effects on AIV virulence andhost tropism changes.