A sandwich ELISA for detecting the hemagglutinin of avian influenza A (H10N8) virus

Novel influenza A virus (H10N8) infected human with fatality in China during 2013-2014. It is important to detect such nonprevalent subtype influenza A virus in clinic and regular surveillance in the early stage for effective control and prevention from the potential pandemic. Unavailability of convenient rapid diagnosis for this subtype virus in resources-limited setting is an obstacle for timely recognizing human case. In the present study, a panel of mouse H10 specific monoclonal antibodies (mAbs) was generated, two of which were used to develop a sandwich enzyme-linked immunosorbent assay (ELISA) for detecting the hemagglutinin of avian influenza A (H10N8) virus. ELISA results showed high sensitivity with the lowest detection limit of 0.5HAU/50 μL for live virus, which laid a foundation for clinic use as a promising diagnostic methodology.     

Generation and characterisation of monoclonal antibodies specific to avian influenza H7N9 haemagglutinin protein

Introduction: Emerging virulent strains of influenza virus pose a serious public health threat with potential pandemic consequences. A novel avian influenza virus, H7N9, breached the species barrier from infected domestic poultry to humans in 2013 in China. Since then, it has caused numerous infections in humans with a close contact to poultry. Materials and Methods: In this study, we describe the preliminary characterisation of five murine monoclonal antibodies (MAbs) developed against recombinant haemagglutinin (rHA) protein of avian H7N9 A/Anhui/1/2013 virus by their Western blot and enzyme-linked immunosorbent assay (ELISA) reactivity and binding affinity. Results: Of the five MAbs, four were highly specific to H7N9 HA and did not show any cross-reactivity in ELISA with rHA protein from pandemic as well as seasonal H1N1, H2N2, H3N2, H5N1 and influenza virus B (B/Brisbane/60/2008). However, one of the MAbs, MA-24, in addition to HA protein of H7N9 also reacted strongly with HA protein of H3N2 and weakly with HA of pandemic and seasonal H1N1 and H2N2. All the five MAbs also reacted with H7N9 rHA in Western blot. The MAbs bound H7N9 rHA with an equilibrium dissociation constant (K_D) ranging between 0.14 and 25.20 nM, indicating their high affinity to HA. Conclusions: These antibodies may be useful in developing diagnostic tools for the detection of influenza H7N9 virus infections.     

A cross-sectional serological survey of the Dutch commercial poultry population for the presence of low pathogenic avian influenza virus infections.

After the discovery of poultry infected with highly pathogenic avian influenza (HPAI) virus of subtype H7N7 in the central area of The Netherlands on 28 February 2003, the hypothesis was put forward that an outbreak of the low pathogenic (LP) variant of H7N7 had preceded, unnoticed, the occurrence of the HPAI virus. Consequently, a cross-sectional serological survey of the Dutch poultry population was executed in the second week of March 2003. The basic requirements set were detection of a 5% prevalence of flocks exposed to LPAI virus with 95% confidence within the production type stratification level within each province in The Netherlands. Because of supposed higher risk of avian influenza infections in ducks, turkeys and free-range poultry, all the commercial flocks of these production types present in The Netherlands were sampled. The serological screening of 28018 sera from 1193 randomly selected poultry farms, located outside surveillance zones showed that LPAI H7 virus infections had occurred on three neighbouring farms all located in the southwest of The Netherlands. No antibodies against the neuraminidase N7 subtype were detected in the sera of these farms, indicating that the subtype was different from the HPAI H7N7 subtype that caused the avian influenza epidemic in 2003. In addition, evidence of infections with non-H5 or non-H7 subtypes of influenza A virus were obtained in two other farms located in the northeast and the southeast of The Netherlands. It was concluded that the HPAI subtype H7N7 outbreak was most likely not preceded by a significant circulation of a LPAI subtype H7N7 virus. Based on the Dutch experience, recommendations are made to detect avian influenza infections faster in the future.     

Single-step multiplex reverse transcription-polymerase chain reaction (RT-PCR) for influenza A virus subtype H5N1 detection

Influenza A virus subtype H5N1 causes a rapidly fatal systemic disease in domestic poultry and spreads directly from poultry to humans. The aim of this study was to develop a rapid, cost-saving and effective method for influenza A virus subtype H5N1 detection. The selected primer set was used in single-step RT-PCR for simultaneous detection in multiplex format of the 276-, 189-, and 131-bp fragments, corresponding to sequences specific for M, H5 and N1. The amplified DNA fragments were clearly separated by agarose gel electrophoresis. The sensitivity of this assay was about 10(3) copies/microL. Moreover, this method can be applied to detect not only avian but also human influenza A virus subtype H5N1. In conclusion, the highlights of this particular method are its rapidity and cost-effectiveness, thus rendering it feasible and attractive for large-scale screening at times of influenza A virus subtype H5N1 outbreak.     

The first lack of evidence of H7N9 avian influenza virus infections among pigs in Eastern China

In this study, we sought to examine whether evidence existed suggesting that pigs were being infected with the novel H7N9 avian influenza virus. From November 2012 to November 2013, blood was drawn from 1560 pigs from 100 large farms in 4 provinces of eastern China. Many of these pigs were in close proximity to wild birds or poultry. Swine sera were studied using hemagglutinin inhibition (HI) assays and enzyme-linked immunosorbent assays (ELISAs) against the H7 antigen derived from the emergent H7N9 avian influenza virus (AIV). Only 29 of the 1560 samples had HI titers of 1:20 when using the H7N9 AIV antigens, and none of the 29 (H7N9 AIV) HI-positive samples were positive when using ELISA, indicating that no samples were positive for H7N9. The negative results were also verified using a novel competitive HA-ELISA. As pigs have been shown to be infected with other avian influenza viruses and as the prevalence of novel influenza A viruses (e.g., H7N9 AIV) may be increasing among poultry in China, similar seroepidemiological studies of pigs should be periodically conducted in the future.     

广州地区禽H9N2亚型流感病毒的发现及感染人调查

目的 了解广州地区禽流感病毒在家禽中的流行及感染人的情况，防止香港H5N1禽流感在广州地区流行。方法 对广州地区的主要鸡场和农贸市场的家禽和密切接触家禽的职业人群进行病原学和血清学的检测。病毒分离同时采用MDCK细胞和鸡胚双腔接种法；采用微量血凝抑制半致敏法进行血清学检测。结果 从54份鸡咽拭液中分离到1株H9N2亚型流感病毒；鸡及职业人群血对分离的H9N2毒株的血抑抗体阳性率分别为12．8％和15．1％。结论 广州地区鸡群中有H9N2，亚型流感病毒存在，禽H9N2亚型流感病毒能感染人。     

Full-genome analysis of avian influenza virus H9N2 from Bangladesh reveals internal gene reassortments with two distinct highly pathogenic avian influenza viruses

Low-pathogenic avian influenza viruses (LPAIVs) of subtype H9N2 have become widespread in poultry in many Asian countries with relevance to respiratory diseases of multifactorial origin. In Bangladesh, LPAIVs of subtype H9N2 co-circulate simultaneously with highly pathogenic avian influenza viruses (HPAIVs) of subtype H5N1 in commercial and backyard poultry. The aim of this study was to characterize LPAIVs of subtype H9N2 currently circulating in Bangladesh. The selected isolate A/Chicken/Bangladesh/VP01/2006 (H9N2) was propagated in chicken embryos. All eight gene segments were amplified by RT-PCR, cloned, and subjected to full-length sequencing. The sequence data obtained were compared with reference strains available in GenBank. Phylogenetic analysis of LPAIV H9N2 from Bangladesh revealed a close relationship to Indian, Pakistani and Middle Eastern isolates and identified an ancestor relationship to LPAIV H9N2 Quail/HK/G1/1997. The internal genes M and NP belong to lineage G1, whereas NS, PA, PB1 and PB2 belong to the prototype virus A/Chicken/Korea/38349-p96323/96. The internal genes showed high sequence homology to an HPAIV of subtype H7N3 from Pakistan, whereas the PB1 gene showed similarly high nucleotide homologies to recently circulating HPAIV H5N1 from Bangladesh, revealing two independent reassortment events. Examination of the hemagglutinin cleavage site of LPAIV H9N2 confirmed its low pathogenicity. The receptor-binding sites indicated a binding preference for human-type receptors. Several mutations in internal proteins are associated with increased virulence and altered host range, while other amino acids were found to be highly conserved among LPAIV H9N2 isolates.     

Complete genome characterization of avian influenza virus subtype H9N2 from a commercial quail flock in Egypt

The suspected presence of avian influenza virus subtype H9N2 in poultry in Egypt is a major concern since this subtype is widely distributed in different countries in the Middle East, here we describe the full genetic characterization of an avian influenza A virus (Qa/Egypt/11; H9N2) of subtype H9N2 that was previously isolated from a clinically normal quail flock in Giza, Egypt in May 2011. The nucleotide sequence analysis of the hemagglutinin gene of the isolated Egyptian virus showed the highest similarity with one group of recent Israeli strains (97?%) circulating from 2006-2010. Sequence homology and phylogenetic analysis indicated that the Qa/Egypt/11 isolate belonged to the A/quail/Hong Kong/G1/1997-like lineage with new mutations identified in all viral proteins. The phylogenetic analysis for the eight genes indicated placement of the Egyptian virus within the same lineage of H9N2 viruses that circulated in the region from 2006, especially with one group of recent Israeli strains. However, phylogenetic analysis of the internal genes like PB2, NP, and PA genes identified possible reassortment events for these genes with singular Israeli strains. This study indicates progressive evolution of this subtype in the Middle East region and possible mechanism of virus adaptation in land-based poultry like in quails.     

流感/禽流感病毒及其致病力鉴别的基因芯片技术研究

目的 建立流感/禽流感病毒及其致病力鉴别的基因芯片检测技术.方法 以血凝素(HA)、神经氨酸酶(NA)、核蛋白(NP)基冈作为靶片段,设计病毒检测和致病力特异性鉴别探针,建立基因芯片鉴别检测技术,采用单引物扩增法(SPA)处理样本核酸,分别对此芯片进行特异性、敏感性和符合率评价.结果 此芯片能够特异性的检测H1N1、H3N2、B型流感病毒及H5N1、H9N2禽流感病毒,敏感性分别为8HAU、16HAU、32HAU及8HAU、8HAU.致病力鉴别探针敏感性为32HAU.同RT-PCR方法比较,检测灵敏度为83.9%.结论 建立的常见流感病毒检测基因芯片特异性高、敏感性高、灵敏度高,更能够对致病力进行有效甄别,可作为临床诊断、传染病防控等方面的有益补充.     

Comparison of commercial influenza A virus assays in detecting avian influenza H7N9 among poultry cloacal swabs,China

BackgroundAvian H7N9 virus emerged in China in February 2013 and has since spread widely among China's poultry, causing numerous human infections.ObjectivesTo compare World Health Organization (WHO) and US commercial influenza assays in detecting avian H7N9 virus in poultry cloacal specimens.Study designBetween April 6 and July 15, 2013, 261 cloacal swabs were collected from commercial poultry in Nanjing and Wuxi City, Jiangsu Province, China. Swabs were screened with the WHO's influenza A and H7N9 real-time RT-PCR (qRT-PCR) assays. A blinded panel of 97 specimens (27 H7N9-positive and 70 influenza A-negative) was then used to compare 3 antigen based commercial assays (Remel Xpect Flu A&B, Quidel Quickvue influenza, and Quidel Sofia Influenza A + B), and 2 molecular commercial assays (Quidel Molecular Influenza A + B assay and Life Technologies VetMAX™-Gold SIV Detection Kit). None of these commercial assays were approved for use with poultry specimens.ResultsConsidering the WHO H7N9 qRT-PCR assay as the gold standard, all assays except the Quidel Quickvue influenza assay had high specificity (ranging from 96 to 99%). Regarding sensitivity, the Life Technologies VetMAX™-Gold SIV Detection Kit (100%; 95% CI 87–100%) and the Quidel Molecular Influenza A + B assay (85%; 95% CI 66–96%) performed the best. The sensitivities of the non-molecular antigen detection assays were either unable to detect small amounts of H7N9 viral RNA or were inhibited by specimen type.ConclusionsThe Life Technologies VetMAX™-Gold SIV Detection Kit and the Quidel Molecular Influenza A + B assay are comparable in performance to the WHO H7N9 qRT-PCR assay in detecting H7N9 from poultry cloacal specimens.     

Effects of closing and reopening live poultry markets on the epidemic of human infection with avian influenza A virus

Live poultry markets (LPMs) are crucial places for human infection of influenza A (H7N9 virus). In Yangtze River Delta, LPMs were closed after the outbreak of human infection with avian influenza A (H7N9) virus, and then reopened when no case was found. Our purpose was to quantify the effect of LPMs’ operations in this region on the transmission of influenza A (H7N9) virus. We obtained information about dates of symptom onset and locations for all human influenza A (H7N9) cases reported from Shanghai, Jiangsu and Zhejiang provinces by May 31, 2014, and acquired dates of closures and reopening of LPMs from official media. A two-phase Bayesian model was fitted by Markov Chain Monte Carlo methods to process the spatial and temporal influence of human cases. A total of 235 cases of influenza A (H7N9) were confirmed in Shanghai, Jiangsu and Zhejiang by May 31, 2014. Using these data, our analysis showed that, after LPM closures, the influenza A (H7N9) outbreak disappeared within two weeks in Shanghai, one week in Jiangsu, and one week in Zhejiang, respectively. Local authorities reopened LPMs when there was no outbreak of influenza A (H7N9), which did not lead to reemergence of human influenza A (H7N9). LPM closures were effective in controlling the H7N9 outbreak. Reopening of LPM in summer did not increase the risk of human infection with H7N9. Our findings showed that LPMs should be closed immediately in areas where the H7N9 virus is confirmed in LPM. When there is no outbreak of H7N9 virus, LPMs can be reopened to satisfy the Chinese traditional culture of buying live poultry. In the long term, local authorities should take a cautious attitude in permanent LPM closure.     

H5N1流感病毒M1蛋白免疫制备的单克隆抗体的研究

目的 制备抗人流感病毒H5N1株M1蛋白的单克隆抗体,为流感的快速诊断和研究提供新的工具.方法 应用在大肠埃希菌中表达的人H5N1亚型禽流感病毒(A/Anhui/1/2005)株M1蛋白,以纯化的表达产物免疫BALB/c小鼠,取脾细胞与sp2/0细胞系作细胞融合后,间接ELISA法筛选阳性的杂交瘤细胞,并应用间接免疫荧光法对抗体的特异性进行鉴定.结果 获得3株能稳定分泌抗禽流感病毒M1抗原的McAb杂交瘤细胞株,交叉反应试验及间接免疫荧光检测表明,三株McAb具有型特异性.结论 用H5N1禽流感病毒M1蛋白免疫制备的单克隆抗体,具有一定的交叉反应性,可用于多种亚型甲型流感病毒的检测.     

Generation and characterisation of monoclonal antibodies against influenza virus A, subtype H5N1

The emergence of highly pathogenic avian influenza A virus (HPAIV) subtype H5N1 in 1997 has since resulted in large outbreaks in poultry and in transmission from poultry to humans, mostly in southeast Asia, but also in several European countries. Effective diagnosis and control measures are essential for the management of HPAIV infections. To develop a rapid diagnostic test, a panel of murine monoclonal antibodies (mAbs) against influenza virus A subtype H5 was generated. Eleven mAbs were produced and characterised according to their reactivity by indirect and sandwich ELISA and western blotting against different H5 subtypes representing past and viruses currently circulating. Ten out of 11 mAbs reacted strongly with the haemagglutinin (HA) protein of H5 viruses, whereas one mAb reacted with the M1 protein. Targeted HA protein epitopes seemed to be conformational. One hybridoma clone binds to a linear epitope of the M1 protein. One specific mAb reacts with HPAIV H5 in the immunofluorescence test, and two antibodies neutralised H5 viruses. On the basis of the results, the set of seven mAbs is appropriate for developing diagnostic tests. With the generated mAbs, a sandwich ELISA was developed recognising all H5N1 strains tested but no other influenza viruses. With this ELISA, as little as 0.005 HA units or 0.1 ng/ml H5N1 was detected, surpassing other ELISA tests. The novel reagents have the potential to improve significantly available rapid antigen detection systems.     

Immunohistochemical detection of Influenza virus infection in formalin-fixed tissues with anti-H5 monoclonal antibody recognizing FFWTILKP

The worldwide outbreak of avian influenza among poultry species and humans is associated with the H5N1 subtype of avian influenza A virus (AIV). This highlighted the need to develop safe H5 AIV diagnostic methods. 7H10, an H5-specific monoclonal antibody (Mab), can be used for immunohistochemical (IHC) staining for formalin-fixed tissue. An assortment of H5N1 tissue specimens infected naturally in paraffin sections from Asia, between years 2002-2006, including one human specimen, were tested. 7H10 detected H5 infection in all of these tissue samples infected naturally. In addition, 24 different human H5N1 isolates from Indonesia, 5 avian H5 isolates and 3 non-H5 isolates from Asia were inoculated into BALB/C mice and chicken embryos. Among these influenza viruses, 7H10 detected 28 of the 29 H5 virus strains by immunohistochemical staining, while none of non-H5 strains used in this study could be detected by 7H10, confirming its specificity to H5. Further, the eight-residue-long linear epitope, "FFWTILKP", identified through epitope mapping, enables 7H10 to detect >98.3% of H5 subtype viruses reported worldwide before 2007. This study describes a specific H5 diagnostic system with minimal possibility of exposure to live virus based on immunochemical staining.     

Development of an antigen-capture ELISA for detection of H7 subtype avian influenza from experimentally infected chickens

Emergence of highly pathogenic avian influenza H7N1 was due to mutation of low pathogenic avian influenza H7N1 strain, which caused outbreaks in Italy between 1999 and 2000, and resulted in complete mortality of infected poultry. This outbreak places increased importance on the early detection of H7N1 AIV. Here we describe the development of a detection method for H7N1 virus from infected chickens using a specific antigen-capture-ELISA (AC-ELISA). A panel of mAbs was developed against the surface antigen HA of H7N1 AIV strain A/chicken/Singapore/94. The mAbs were screened by immunofluorescence assays, ELISA and immunoblotting. Selected mAbs 5E5 and 8F10 were of isotypes IgM and IgG and were conformation- or linear epitope-specific, respectively. These mAbs were used as capture antibodies for AC-ELISA development. The detection limit was as little as 10(2)-10(3) TCID(50) units of virus derived from tissue culture supernatants. Virus from the tracheal swab samples of experimentally infected chickens was detected from days 3 to 7 post-infection using the AC-ELISA, with results being confirmed by RT-PCR. AIV subtypes H4N1, H5N3 H9N2 and H10N5 did not react in the AC-ELISA but were RT-PCR positive, indicating that this AC-ELISA is specific for H7N1 strains.     

Genetic diversity of early (1998) and recent (2010) avian influenza H9N2 virus strains isolated from poultry in Iran

Infection with avian influenza H9N2 virus is widespread in the Asian poultry industry, resulting in great economic losses due to mortality and a severe decline in egg production. To obtain more-comprehensive genomic data from circulating H9N2 viruses in Iran, we sequenced the whole genomes of early (Ck/IR/ZMT-101/98) and recent (Ck/IR/EBGV-88/10) isolates of this virus in Iran. The M and NS genes of Ck/IR/EBGV-88/10 shared a high level of similarity with a highly pathogenic H7N3 virus isolated from Pakistan. The cleavage site within the HA protein of these viruses contained two different motifs, RSSR and KSSR, which are similar to those found in low-pathogenic viruses. The deduced amino acid sequence of the new isolate contained the mutation Q226L, which is a characteristic of human-type sialic acid influenza receptor binding. An analysis of the viral amino acid sequence of the M2 protein of the recent strain revealed a V27A mutation, which is associated with amantadine resistance in avian influenza virus. The present results emphasize the need for continuous surveillance of H9N2 viruses in poultry and the human population to obtain more information about the nature and evolution of future pandemic influenza viruses.     

A study of family clustering in two young girls with novel avian influenza A (H7N9) in Dongyang,Zhejiang Province,in 2014

BackgroundThe avian influenza A H7N9 virus, previously unknown in humans, has infected humans in many areas of China since February 2013. Here we report on a clustering case of H7N9 in two little girls in one family in Dongyang city, Jinhua area, Zhejiang Province.ObjectivesTo determine (1) whether the infections were due to person-to-person transmission or to co-exposure to poultry and (2) the prevalence of this novel H7N9 virus in Dongyang inferred by this family clustering case.Study designSamples were collected from patients and environment. We undertook detailed epidemiological investigations and laboratory work. Phylogenetic analyses were done based on the sequenced genomes. The concentration of cytokines and chemokines in the serum was detected by cytometric bead array analyses.ResultsA mixture of H7 and H9 was detected from the environmental sample. The three H7N9 viruses shared one infection source. The index patient who had significantly higher levels of IL-4, IL-8 and IL-10 suffered severe infection.ConclusionsBased on a comparison with previous isolations of the virus in 2013, H7N9 has evolved different lineages through recombination with local H9N2 viruses. Determining whether it was human-to-human transmission or exposure to the same live poultry, since both patients had identical exposure histories, was ambiguous. The results from the cytokine analyses agreed with the conclusion that H7N9 severity is associated with a higher level of cytokines/chemokines. Long term influenza surveillance remains essential to allow for early warning of potential transmission events.     

An outbreak of avian influenza subtype H9N8 among chickens in South Korea.

Low pathogenic avian influenza subtype H9N8 was diagnosed on a Korean native chicken farm in Gyeonggi province, South Korea, in late April 2004. Clinical signs included moderate respiratory distress, depression, mild diarrhoea, loss of appetite and a slightly elevated mortality (1.4% in 5 days). Pathologically, mucopurulent tracheitis and air sacculitis were prominently found with urate renal deposition. The isolated A/chicken/Kr/164/04 (H9N8) had an Ala-Ser-Gly-Arg (A/S/G/R) motif at the cleavage site of haemagglutinin, which has been commonly found in H9N2 isolated from Korean poultry. Phylogenetic analysis of the haemagglutinin and neuraminidase genes of the H9N8 avian influenza virus (AIV) isolate showed that reassortment had occurred. Its haemagglutinin gene was similar to that of Korean H9N2 AIVs, but its neuraminidase gene was closely related to that of A/WBF/Kr/KCA16/03 (H3N8) isolated from the faeces of wild birds in Korea. The pathogenicity of the isolate was tested on 6-week-old specific pathogen free chickens. The inoculated virus (H9N8) was recovered from most tested organs, including the trachea, lung, kidney, spleen, and caecal tonsil. This is the first report of an outbreak of low pathogenic avian influenza in chickens caused by AIV subtype H9N8.