Reverse Transcription Recombinase-aided Amplification Assay for H5 Subtype avian Influenza Virus

Background: The H5 subtype avian influenza virus (AIV) has caused huge economic losses to the poultry industry and is a threat to human health. A rapid and simple test is needed to confirm infection in suspected cases during disease outbreaks. Methods: In this study, we developed a reverse transcription recombinase-aided amplification (RT-RAA) assay for the detection of H5 subtype AIV. Assays were performed at a single temperature (39°C), and the results were obtained within 20 min. Results: The assay showed no cross-detection with Newcastle disease virus or infectious bronchitis virus. The analytical sensitivity was 103 RNA copies/μL at a 95% confidence interval according to probit regression analysis, with 100% specificity. Compared with published reverse transcription quantitative real-time polymerase chain reaction assays, the κ value of the RT-RAA assay in 420 avian clinical samples was 0.983 (p < 0.001). The sensitivity for avian clinical sample detection was 97.26% (95% CI, 89.56–99.52%), and the specificity was 100% (95% CI, 98.64–100%). Conclusions: These results indicated that our RT-RAA assay may be a valuable tool for detecting H5 subtype AIV.

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

Background H5-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). Methods In 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. Results The 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. Conclusions The 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.

Global dynamic analysis of H5 subtype avian influenza virus

In order to study the comprehensive influence of factors such as contact between resident birds and poultry, poultry recruitment, environment and other factors on the transmission and control of H5 subtype avian influenza virus, a dynamic model of resident birds and poultry was developed was established. Firstly, the basic reproduction number R0 was obtained. When R0 > 1, the dynamic model has a unique positive equilibrium and the disease persists. Secondly, the Lyapunov functions was constructed to determine the global stability of the disease-free equilibrium and the endemic equilibrium. The results of numerical simulation show that regular disinfection and sterilization can increase the mortality of virus and effectively prevent the occurrence of epidemic situation. Although closing the live poultry trading market is not the main measure to control the epidemic, but it can control the epidemic to a lower level. Therefore, the regular closure of trading markets and sterilization can prevent and control the spread of the epidemic.

A Novel Epitope-blocking ELISA for Specific and Sensitive Detection of Antibodies against H5-Subtype Influenza Virus Hemagglutinin

Background: H5-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).Methods: In 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.Results: The 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.Conclusions: The 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.

Reverse-transcription recombinase-aided amplification assay for H5 subtype avian influenza virus

The H5 subtype Avian Influenza Virus has caused huge economic losses to the poultry industry and is a threat to human health. A rapid and simple test is needed to confirm infection in suspected cases during disease outbreaks. In this study, we developed a reverse-transcription recombinase-aided amplification assay for the detection of H5 subtype avian influenza virus. Assays were performed at a single temperature (39°C), and the results were obtained within 20 min. The assay showed no cross-detection with Newcastle disease virus or infectious bronchitis virus. The analytical sensitivity was 103 RNA copies per reaction at a 95% confidence interval according to probit regression analysis, with 100% specificity. Compared with published reverse-transcription quantitative real-time polymerase chain reaction assays, the κ value of the reverse transcription recombinase-aided amplification assay in 365 avian clinical samples was 0.970 (p < 0.001). The sensitivity for avian clinical sample detection was 94.44% (95%CI, 70.63% - 99.71%), and the specificity was 100% (95%CI, 98.64% - 100%). These results indicated that our reverse-transcription recombinase-aided amplification assay may be a valuable tool for detecting H5 subtype avian influenza virus.

A Novel Epitope-Blocking ELISA for Specific and Sensitive Detection of Antibodies Against H5-Subtype Influenza Virus Hemagglutinin

Background: H5-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).Methods: In 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.Results: The 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.Conclusions: The 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.

A fluorescence polarization immunoassay for the rapid detection of antibody against influenza A virus in chicken and goat sera

Highly pathogenic influenza A viruses (IAVs) cause substantial damage to the poultry industry. A simple and quick testing method is required for strict control of this infectious agent. The fluorescence polarization immunoassay (FPIA) is a rapid test based on antigen–antibody binding, which can detect antigen-specific antibody in the infected animal samples within a few minutes. FPIA is a one-step reaction assay that does not require a secondary antibody and complicated steps. We evaluated the usefulness of FPIA for the detection of anti-IAV antibodies, including those against internal proteins and H5 subtype HA, in sera. In the FPIA using fluorescent peptides of internal NP and M1 proteins, millipolarization units (MPUs), which increase depending on the amount of antibody, were higher in antibody-positive sera than in antibody-negative sera. Moreover, in FPIA using fluorescent recombinant H5 subtype HA proteins, anti-H5 serum gave the highest MPUs among the antisera raised in goats against individual H1–H15 subtype IAVs. Our results support the utility of FPIA for the detection of anti-IAV antibodies, especially the anti-H5 antibody.