Avian Influenza Virus Subtypes H5 Research Articles

A novel epitope-blocking ELISA for specific and sensitive detection of antibodies against H5-subtype influenza virus hemagglutinin

BackgroundH5-subtype highly pathogenic (HP) avian influenza viruses (AIVs) cause high mortality in domestic birds and sporadic infections in humans with a frequently fatal outcome, while H5N1 viruses have pandemic potential. Due to veterinary and public health significance, these HPAIVs, as well as low pathogenicity (LP) H5-subtype AIVs having a propensity to mutate into HP viruses, are under epidemiologic surveillance and must be reported to the World Organization for Animal Health (OIE). Our previous work provided a unique panel of 6 different monoclonal antibodies (mAbs) against H5 hemagglutinin (HA), which meets the demand for high-specificity tools for monitoring AIV infection and vaccination in poultry. In this study, we selected one of these mAbs to develop an epitope-blocking (EB) ELISA for detecting H5 subtype-specific antibodies in chicken sera (H5 EB-ELISA).MethodsIn the H5 EB-ELISA, H5 HA protein produced in a baculovirus-expression vector system was employed as a coating antigen, and the G-7-27-18 mAb was employed as a blocking antibody. The performance characteristics of the assay were evaluated by testing 358 sera from nonimmunized chickens and chickens immunized with AIVs of the H1–H16 subtypes or recombinant H5 HA antigen to obtain the reference and experimental antisera, respectively. The samples were classified as anti-H5 HA positive or negative based on the results of the hemagglutination inhibition (HI) assay, the gold standard in subtype-specific serodiagnosis.ResultsThe H5 EB-ELISA correctly discriminated between the anti-H5 HA negative sera, including those against the non-H5 subtype AIVs, and sera positive for antibodies against the various-origin H5 HAs. Preliminary validation showed 100% analytical and 97.6% diagnostic specificities of the assay and 98.0% and 99.1% diagnostic sensitivities when applied to detect the anti-H5 HA antibodies in the reference and experimental antisera, respectively.ConclusionsThe H5 EB-ELISA performed well in terms of diagnostic estimates. Thus, further optimization and validation work using a larger set of chicken sera and receiver operating characteristic (ROC) analysis are warranted. Moreover, the present assay provides a valuable basis for developing multispecies screening tests for birds or diagnostic tests for humans.

Development of an immunochromatographic strip for rapid detection of H7 subtype avian influenza viruses

BackgroundH7N9 avian influenza virus (AIV) including highly and low pathogenic viruses have been detected in China since 2013. H7N9 AIV has a high mortality rate after infection in humans, and most human cases have close contacted with poultry in the live poultry market. Therefore, it is necessary to develop a rapid point-of-care testing (POCT) technique for H7N9 AIV detection.MethodsThe H7N9 AIV was inactivated and purified, and was used as the antigen to immunize BALB/c. Twelve H7-HA specific monoclonal antibodies (McAbs) were produced through the hybridoma technique. The McAb 10A8 was conjugated with colloid gold as detecting antibody; McAb 9B6 was dispensed on the nitrocellulose membran as the capture test line and the Goat-anti mouse IgG antibody was dispensed as control line respectively. The immunochromatographic strip was prepared.ResultsThe analysis of ELISA and virus neutralization test showed that the obtained McAbs specifically recognized H7 HA. Based on the prepared strip, the detection of H7 AIV was achieved within 10 min. No cross-reaction occurred between H7 AIVs and other tested viruses. The detection limit of the strip for H7 was 2.4 log10EID50/0.1 mL for chicken swab samples.ConclusionThe McAbs were specific for H7 and the immunochromatographic strip developed in this study was convenient, rapid and reliable for the detection of H7 AIV. The strip could provide an effective method for the rapid and early detection of H7 AIV.

Differential mitochondrial proteomic analysis of A549 cells infected with avian influenza virus subtypes H5 and H9

BackgroundBoth the highly pathogenic avian influenza (HPAI) H5N1 and low pathogenic avian influenza (LPAI) H9N2 viruses have been reported to cross species barriers to infect humans. H5N1 viruses can cause severe damage and are associated with a high mortality rate, but H9N2 viruses do not cause such outcomes. Our purpose was to use proteomics technology to study the differential expression of mitochondrial-related proteins related to H5N1 and H9N2 virus infections.MethodsAccording to the determined viral infection titer, A549 cells were infected with 1 multiplicity of infection virus, and the mitochondria were extracted after 24 h of incubation. The protein from lysed mitochondria was analyzed by the BCA method to determine the protein concentration, as well as SDS-PAGE (preliminary analysis), two-dimensional gel electrophoresis, and mass spectrometry. Differential protein spots were selected, and Western blotting was performed to verify the proteomics results. The identified proteins were subjected to GO analysis for subcellular localization, KEGG analysis for functional classification and signaling pathways assessment, and STRING analysis for functional protein association network construction.ResultsIn the 2-D gel electrophoresis analysis, 227 protein spots were detected in the H5N1-infected group, and 169 protein spots were detected in the H9N2-infected group. Protein spots were further subjected to mass spectrometry identification and removal of redundancy, and 32 differentially expressed proteins were identified. Compared with the H9N2 group, the H5N1-infected group had 16 upregulated mitochondrial proteins and 16 downregulated proteins. The differential expression of 70-kDa heat shock protein analogs, short-chain enoyl-CoA hydratase, malate dehydrogenase, and ATP synthase was verified by Western blot, and the results were consistent with the proteomics findings. Functional analysis indicated that these differentially expressed proteins were primarily involved in apoptosis and metabolism.ConclusionsCompared with their expression in the H9N2 group, the differential expression of eight mitochondrial proteins in the H5N1 group led to host T cell activation, antigen presentation, stress response, ATP synthesis and cell apoptosis reduction, leading to higher pathogenicity of H5N1 than H9N2.

H7N7 Avian Influenza Virus Mutation from Low to High Pathogenicity on a Layer Chicken Farm in the UK.

Avian influenza virus (AIV) subtypes H5 and H7 are capable of mutating from low to high pathogenicity strains, causing high mortality in poultry with significant economic losses globally. During 2015, two outbreaks of H7N7 low pathogenicity AIV (LPAIV) in Germany, and one each in the United Kingdom (UK) and The Netherlands occurred, as well as single outbreaks of H7N7 high pathogenicity AIV (HPAIV) in Germany and the UK. Both HPAIV outbreaks were linked to precursor H7N7 LPAIV outbreaks on the same or adjacent premises. Herein, we describe the clinical, epidemiological, and virological investigations for the H7N7 UK HPAIV outbreak on a farm with layer chickens in mixed free-range and caged units. H7N7 HPAIV was identified and isolated from clinical samples, as well as H7N7 LPAIV, which could not be isolated. Using serological and molecular evidence, we postulate how the viruses spread throughout the premises, indicating potential points of incursion and possible locations for the mutation event. Serological and mortality data suggested that the LPAIV infection preceded the HPAIV infection and afforded some clinical protection against the HPAIV. These results document the identification of a LPAIV to HPAIV mutation in nature, providing insights into factors that drive its manifestation during outbreaks.

Non-competitive fluorescence polarization immunoassay for detection of H5 avian influenza virus using a portable analyzer.

Nowadays, the diagnosis of viral infections is receiving broad attention. We have developed a non-competitive fluorescence polarization immunoassay (NC-FPIA), which is a separation-free immunoassay, for a virus detection. H5 subtype avian influenza virus (H5-AIV) was used as a model virus for the proof of concept. The fluorescein-labeled Fab fragment that binds to H5 hemagglutinin was used for NC-FPIA. The purified H5-AIV which has H5 hemagglutinin was mixed with the fluorescein-labeled Fab fragment. After that, the degree of fluorescence polarization was measured with a portable FPIA analyzer. H5-AIV was successfully detected with an incubation time of 15 min. In addition, the portable FPIA analyzer enables performance of on-site NC-FPIA with a sample volume of 20 μL or less. This is the first research of detecting a virus particle by FPIA. This NC-FPIA can be applied to rapid on-site diagnosis of various viruses.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00216-021-03193-y.

Active Surveillance and Risk Assessment of Avian Influenza Virus Subtype H9 from Non-Vaccinated Commercial Broilers of Pakistan

A cross-sectional study was conducted to investigate seroprevalence and virus prevalence of the H9 subtype of avian influenza virus in non-vaccinated broiler farms of dense poultry-populated districts, Lahore and Sheikhupura of Punjab-Pakistan. A convenient sampling method was adopted for collection of blood (n=500) and oropharyngeal swab (n=500) samples from 25 broiler farms of each district for hemagglutination inhibition assay and RT-PCR test, respectively. Proportional estimates were calculated using R software and overall seroprevalence of H9 was estimated at 36.3% (95% CI 33.3-39), with no significant difference (p>0.05) between Lahore (37.2 %, 95% CI=31.2-39.59) and Sheikhupura (35.4%, 95% CI= 29.64-39.76). RT-PCR identified 2% (4/200) pool level viral prevalence. None of the farms from Lahore districts were RT-PCR positive for H9. Simple logistic regression followed by multivariable analysis, identified the presence of foot bath/dipping area at the entrance (OR=0.7, 95% CI=0.52-0.93) and availability of rubber shoes for visitors (OR=0.36, 95% CI 0.26-0.48) as protective factors. History of respiratory signs (OR=1.51, 95%=CI 1.12-2.04), history of sudden death in past flocks (OR=3.26, 95% CI=2.41-4.41), and birds previously infected with avian influenza virus (OR=1.33, 95% CI=1-1.76) were significant risk factors. Negligence in preventive measures at farms level was associated with the spread of H9 infection between the farms. To control future outbreaks, biosecurity and continuous monitoring of non-vaccinated flocks are suggested.

Epidemiological and Genetic Characteristics of the H3 Subtype Avian Influenza Viruses in China.

Epidemiological and Genetic Characteristics of the H3 Subtype Avian Influenza Viruses in China.

Seroprevalence of Avian Influenza Virus Subtype H5 among Poultry Workers of Central Traditional Markets in Indonesia

Seroprevalence of Avian Influenza Virus Subtype H5 among Poultry Workers of Central Traditional Markets in Indonesia

Development of reverse transcription loop-mediated isothermal amplification assays for point-of-care testing of avian influenza virus subtype H5 and H9.

Avian influenza (AIV) outbreaks can induce fatal human pulmonary infections in addition to economic losses to the poultry industry. In this study, we aimed to develop a rapid and sensitive point-of-care AIV test using loop-mediated isothermal amplification (LAMP) technology. We designed three sets of reverse transcription LAMP (RT-LAMP) primers targeting the matrix (M) and hemagglutinin (HA) genes of the H5 and H9 subtypes. RT-LAMP targeting the universal M gene was designed to screen for the presence of AIV and RT-LAMP assays targeting H5-HA and H9-HA were designed to discriminate between the H5 and H9 subtypes. All three RT-LAMP assays showed specific amplification results without nonspecific reactions. In terms of sensitivity, the detection limits of our RT-LAMP assays were 100 to 1,000 RNA copies per reaction, which were 10 times more sensitive than the detection limits of the reference reverse‒transcription polymerase chain reaction (RT-PCR) (1,000 to 10,000 RNA copies per reaction). The reaction time of our RT-LAMP assays was less than 30 min, which was approximately four times quicker than that of conventional RT-PCR. Altogether, these assays successfully detected the existence of AIV and discriminated between the H5 or H9 subtypes with higher sensitivity and less time than the conventional RT-PCR assay.

Evolution and pathogenicity of H6 avian influenza viruses isolated from Southern China during 2011 to 2017 in mice and chickens

H6 subtype avian influenza viruses spread widely in birds and pose potential threats to poultry and mammals, even to human beings. In this study, the evolution and pathogenicity of H6 AIVs isolated in live poultry markets from 2011 to 2017 were investigated. These H6 isolates were reassortant with other subtypes of influenza virus with increasing genomic diversity. However, no predominant genotype was found during this period. All of the H6N2 and most of the H6N6 isolates replicated efficiently in lungs of inoculated mice without prior adaptation. All of the H6N2 and two H6N6 isolates replicated efficiently in nasal turbinates of inoculated mice, which suggested the H6N2 viruses were more adaptive to the upper respiratory tract of mice than the H6N6 viruses. One of H6N2 virus caused systemic infection in one out of three inoculated mice, which indicated that H6 avian influenza virus, especially the H6N2 viruses posed a potential threat to mammals. Five H6 strains selected from different genotypes caused no clinical signs to inoculated chickens, and their replication were limited in chickens since the viruses have been detected only from a few tissues or swabs at low titers. Our study strongly suggests that the H6 avian influenza virus isolated from live poultry markets pose potential threat to mammals.

Quick and improved immune responses to inactivated H9N2 avian influenza vaccine by purified active fraction of Albizia julibrissin saponins

BackgroundH9N2 Low pathogenic avian influenza virus (LPAIV) raises public health concerns and its eradication in poultry becomes even more important in preventing influenza. AJSAF is a purified active saponin fraction from the stem bark of Albizzia julibrissin. In this study, AJSAF was evaluated for the adjuvant potentials on immune responses to inactivated H9N2 avian influenza virus vaccine (IH9V) in mice and chicken in comparison with commercially oil-adjuvant.ResultsAJSAF significantly induced faster and higher H9 subtype avian influenza virus antigen (H9–Ag)-specific IgG, IgG1, IgG2a and IgG2b antibody titers in mice and haemagglutination inhibition (HI) and IgY antibody levels in chicken immunized with IH9V. AJSAF also markedly promoted Con A-, LPS- and H9–Ag-stimulated splenocyte proliferation and natural killer cell activity. Furthermore, AJSAF significantly induced the production of both Th1 (IL-2 and IFN-γ) and Th2 (IL-10) cytokines, and up-regulated the mRNA expression levels of Th1 and Th2 cytokines and transcription factors in splenocytes from the IH9V-immunized mice. Although oil-formulated inactivated H9N2 avian influenza vaccine (CH9V) also elicited higher H9–Ag-specific IgG and IgG1 in mice and HI antibody titer in chicken, this robust humoral response was later produced. Moreover, serum IgG2a and IgG2b antibody titers in CH9V-immunized mice were significantly lower than those of IH9V alone group.ConclusionsAJSAF could improve antigen-specific humoral and cellular immune responses, and simultaneously trigger a Th1/Th2 response to IH9V. AJSAF might be a safe and efficacious adjuvant candidate for H9N2 avian influenza vaccine.

Antibody Response of an H9 Subtype Avian Influenza Poultry Vaccine on Three Kinds of Wild Birds in Shanghai Zoo.

A semiannual immunization with a commercial inactivated H9 subtype avian influenza virus (AIV) vaccine developed for poultry has been used to prevent and control the avian influenza (AI) infections among captured wild birds in Shanghai Zoo. However, the overall safety and effectiveness of the poultry vaccine for housed birds in the zoo remain unclear. To verify the safety and efficacy of the commercial inactivated H9 AI vaccine on zoo birds and to explore a more reasonable and effective immunization procedure, 48 zoo birds, including 11 Oriental white storks, 25 peafowl, and 12 silver pheasants, were administered the AI vaccine developed for poultry use. Then, the clinical signs of the immunized birds were observed for 2 weeks, and the antibodies against H9 AI were determined via the hemagglutination inhibition test. Results showed that no harmful effects related to the vaccination were observed, and the antibody titers of the Oriental white stork, peafowl, and silver pheasants were all higher than 7 log 2 at 21 days, 30 days, 60 days, 120 days, and 180 days postimmunization. For further study, the H9 AIV titers of 11 peafowls and 6 Oriental storks, which were raised in the nursing ground, were continuously monitored for 15 months. All of their antibody titers were above the national standards of China (5 log 2; GB/T18936-2003), even at 12 months and 15 months postimmunization. We concluded that the commercial inactivated H9 AI vaccine used at the present time in Shanghai Zoo can induce high and prolonged immune responses in vaccinated birds.

Serological evidence of avian influenza virus subtype H5 and H9 in live bird market, Myanmar

Avian Influenza (AI), caused by Alphainfluenzaviruses (AIVs), is a contagious respiratory disease in birds and mammals. AIVs have been reported in poultry worldwide and the impact of AIVs on human health is immense. In this study, a serological survey of AIV subtype H5 and H9 was conducted in a live bird market (LBM) in Yangon, Myanmar during February 2016 to September 2016. A total of 621 serum samples were collected from chickens (n = 489) and ducks (n = 132) from 48 vendors in the LBM. The samples were examined for antibodies against influenza viruses by using NP-ELISA and specific antibodies against AIV-H5N1 (Clade 2.3.4) and AIV-H9N2 (Clade 9.4.2) by using Hemagglutination Inhibition (HI) assay. The result of NP-ELISA assay showed that 12.88 % (80/621) of poultry in LBM was positive for AIV antibodies. In detail, 38.06 % (51/134) of layers, 7.08 % (8/113) of backyard chicken, 2.07 % (5/242) of broilers and 12.12 % (16/132) of ducks were AIV positive. The HI test for specific antibodies against AIV-H5N1 and AIV-H9N2 were 1.77 % (11/621) and 4.51 % (28/621), respectively. Our findings revealed the evidence of AIV-H5N1 and AIV-H9N2 exposure in both chicken and ducks in the LBM in Yangon, Myanmar. Risks of influenza infections and transmission among poultry and humans in the LBMs could not be ignored.

Characterization of H3 subtype avian influenza viruses isolated from poultry in Vietnam.

To date, avian influenza viruses (AIVs) have persisted in domestic poultry in wet markets in East Asian countries. We have performed ongoing virus surveillance in poultry populations in Vietnam since 2011, with the goal of controlling avian influenza. Throughout this study, 110 H3 AIVs were isolated from 2760 swab samples of poultry in markets and duck farms. H3 hemagglutinin (HA) genes of the isolates were phylogenetically classified into eight groups (I-VIII). Genetic diversity was also observed in the other seven gene segments. Groups I-IV also included AIVs from wild waterbirds. The epidemic strains in poultry switched from groups I-III and VI to groups I, IV, V, and VIII around 2013. H3 AIVs in groups I and V were maintained in poultry until at least 2016, which likely accompanied their dissemination from the northern to the southern regions of Vietnam. Groups VI-VIII AIVs were antigenically distinct from the other groups. Some H3 AIV isolates had similar N6 neuraminidase and matrix genes as H5 highly pathogenic avian influenza viruses (HPAIVs). These results reveal that genetically and antigenically different H3 AIVs have been co-circulating in poultry in Vietnam. Poultry is usually reared outside in this country and is at risk of infection with wild waterbird-originating AIVs. In poultry flocks, the intruded H3 AIVs must have experienced antigenic drift/shift and genetic reassortment, which could contribute to the emergence of H5 HPAIVs with novel gene constellations.

Genetic and Molecular Characterization of H9N2 Avian Influenza Viruses Isolated from Live Poultry Markets in Hubei Province, Central China, 2013-2017.

H9N2 subtype avian influenza virus (AIV) is an influenza A virus that is widely spread throughout Asia, where it jeopardizes the poultry industry and provides genetic material for emerging human pathogens. To better understand the epidemicity and genetics of H9 subtype AIVs, we conducted active surveillance in live poultry markets (LPMs) in Hubei Province from 2013 to 2017.A total of 4798 samples were collected from apparent healthy poultry and environment. Real-time RT-PCR revealed that the positivity rate of influenza A was 26.6% (1275/4798), of which the H9 subtype accounted for 50.3% (641/1275) of the positive samples. Of the 132 H9N2 viral strains isolated, 48 representative strains were subjected to evolutionary analysis and genotyping. Phylogenetic analysis revealed that all H9N2 viral genes had 91.1%-100% nucleotide homology, clustered with genotype 57, and had high homology with human H9N2 viruses isolated from 2013 to 2017 in China. Using a nucleotide divergence cutoff of 95%, we identified ten distinct H9N2 genotypes that continued to change over time. Molecular analysis demonstrated that six H9N2 isolates had additional potential glycosylation sites at position 218 in the hemagglutinin protein, and all isolates had I155T and Q226L mutations. Moreover, 44 strains had A558V mutations in the PB2 protein and four had E627V mutations, along with H9N2 human infection strains A/Beijing/1/2016 and A/Beijing/1/2017. These results emphasize that the H9N2 influenza virus in Hubei continues to mutate and undergo mammalian adaptation changes, indicating the necessity of strengthening the surveillance of the AIV H9N2 subtype in LPMs.

Epidemiological and genetic characteristics of H5 subtype avian influenza virus in Guangzhou, 2014-2019

Objective: To analyze the characteristics of spread and genetic evolution of H5 subtype avian influenza virus in Guangzhou from 2014 to 2019. Methods: H5 subtype virus was detected by fluorescence quantitative RT-PCR from the environmental samples in Guangzhou poultry markets. The genes of HA and NA of 48 isolates randomly selected were sequenced, including 46 isolates from environmental samples and 2 isolates from cases. The characteristics of molecular variation and genetic evolution were analyzed by using bioinformatics software. Results: A total of 1 094 strains of H5 subtype avian influenza virus were isolated from 52 284 samples (2.09%). All the strains belonged to Clade 2.3.4.4.C. NA gene belonged to H6N6 of Eurasian lineage. The cleavage sites of all the strains showed the characteristics of highly pathogenicity. Receptor binding sites were avian-derived receptors. However, mutations of S123P, S133A and T156A occurred, which implied that these strains could tend to bind to human receptors. There was an additional glycosylation site at 140 in strains isolated after 2017. The variation of antigen loci mainly occurred in B and E regions. Conclusions: H5 subtype avian influenza virus spread in Guangzhou from 2014 to 2019 with annual increased proportion of positive rate, and the sequencing results indicated that it belonged to Clade 2.3.4.4.C of H5N6 highly pathogenic virus, and genetic evolution and mutation continued, especially the common mutations which could enhance the binding capacity to human receptors. It is necessary to strengthen the surveillance.

Molecular Identification of Some Respiratory Pathogens from Broiler Chickens in Sharkia Governorate, Egypt

In this study, we investigated the prevelance of avian influenza virus (AIV) (subtypes H5 and H9), infectious bronchitis virus (IBV), Newcastle disease virus (ND) and Mycoplasma gallisepticum (MG) incriminated in respiratory troubles among chicken broilers at different localities in Sharkia Governorate during 2013-2017 outbreaks. Out of 53 broiler flocks, 225 chickens showing respiratory manifestations were examined. Viral isolation from respiratory tissues, serological and molecular identification were done. Based on hemagglutination inhibition (HI) test, for 42 positive haemagglutinating allantoic fluids, the prevalence of NDV and AIV (H9 and H5 subtypes) was 64.3%, 45.3%and 2,4% respectively. Two diagnostic polymerase chain reaction (PCR) assays were used. Based on TaqMan probe base real-time PCR, 17/19, 10/42 and 1/42 flocks were confirmed to be infected with IBV, AIV subtype H9 and H5 respectively. Moreover, conventional PCR amplification for detection of Mycoplasma gallisepticum in 25 flocks was performed and revealed 56% (14/25) positive flocks. Subsequently, amplified products of three M. gallisepticum representative isolates were sequenced and phylogenitically analyzed with other strains on GenBank. The results revealed that the three examined isolates were wild type (field isolates).We could conclude that there were continuous circulation of different respiratory agents (viral (NDV, AIVH9 & H5, and IBV) and bacterial (MG) in broiler flocks in Sharkia Governorate either as single or mixed infection although wide using of different vaccination regimes and antibiotics so early diagnosis of these pathogens is considered the most effective tool in prevention and control of diseases.

Co-circulation of multiple reassortant H6 subtype avian influenza viruses in wild birds in eastern China, 2016\u20132017

BackgroundH6 subtype influenza viruses were prevalent in domestic poultry and wild birds, which also could pose potential threat to humans. However, little is known about the prevalence of H6 subtype viruses in wild birds in eastern China, a crucial stopover or wintering site for migratory wild birds along the East Asian-Australasian Flyway.MethodsDuring the routine surveillance in 2016–2017, H6 subtype AIVs positive samples were identified, and the representative strains were selected for further sequence and phylogenetic analysis and the pathogenicity in mice were evaluated.ResultsAmong the 30 H6 positive samples, there were at least four subtypes H6N1, H6N2, H6N5 and H6N8 co-circulated in Shanghai, China. Genetic analysis showed the 8 representative isolates shared homology with different AIV sub-lineages isolated from domestic ducks or wild birds in different countries along the East Asian-Australasian flyways, and were classified into 7 new genotypes. The pathogenicity to mice showed that these H6 viruses could replicate efficiently in the lungs without prior adaptation, but could not cause mice death.ConclusionsEight novel strains belonged to H6N1, H6N2, H6N5 and H6N8 subtypes were isolated. Phylogenetic analyses revealed multiple origins of internal genes indicative of robust reassortment events and frequent wild birds-poultry interaction encouraging the evolution and emergence of new genotypes. The pathogenicity to mammals should be closely monitored to prevent the emergence of novel pandemic viruses.

Rapid detection of anti-H5 avian influenza virus antibody by fluorescence polarization immunoassay using a portable fluorescence polarization analyzer

A rapid, facile and selective detection of anti-H5 subtype avian influenza virus (AIV) antibody in serum by fluorescence polarization immunoassay (FPIA) was achieved. A fragment of recombinant H5 subtype AIV hemagglutinin was produced and labeled with fluorescein to use it as a labeled antigen in FPIA. This labeled antigen was mixed with anti-AIV sera (H1–H16 subtypes) and FP of the mixture was measured using a portable FP analyzer on a microdevice. It was found that FP increased in proportion to the concentration of anti-H5 AIV antibody (serum) and was significantly higher than FP obtained with the other sera. The selective detection of anti-H5 subtype AIV antibody was confirmed. The required volume of original sample was 2 μL and analysis time was within 20 min. This detection system realizes an efficient on-site diagnosis and surveillance of AIV.

Development of an antigen-capture enzyme-linked immunosorbent assay and immunochromatographic strip based on monoclonal antibodies for detection of H6 avian influenza viruses.

Continuous surveillance has shown that H6 subtype avian influenza viruses (AIVs) are prevalent in poultry and occasionally break the species barrier to infect humans. It is therefore necessary to establish a specific, rapid and sensitive method to screen H6 AIVs. In this study, a panel of monoclonal antibodies (mAbs) against the hemagglutinin (HA) of an H6 AIV isolate was produced. The purified mAbs have high affinity and specificity for H6 AIVs. An antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) and immunochromatographic strip were developed based on two mAbs (1D7 and 1F12). The AC-ELISA results showed high sensitivity with a limit of detection (LOD) of 3.9 ng/ml for H6 HA protein and 0.5 HAU (HA units)/100µl for live H6 subtype AIVs. The average recovery of the AC-ELISA with allantoic fluid, respiratory specimens, and cloacal swabs was 91.907 ± 1.559%, 82.977 ± 1.497% and 73.791 ± 2.588%, respectively. The intra- and inter-assay coefficient of variation was less than 10%. The LOD of immunochromatographic strip was 1 HAU when evaluated by the naked eye, and the detection time was less than 10 min without any equipment. Storage at room temperature or 4°C for 30days or 60days had no effect on sensitivity and specificity of the strip. Thus, the AC-ELISA and immunochromatographic strips described here could be a secondary method to diagnose H6 AIV infections with high specificity, sensitivity, and stability.