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Long-lasting chemiluminescence (CL) emissions are necessary for improving the detection accuracy and expanding the application scope. Here, we have synthesized three oil-in-water (O/W) multicolor protein capsules (LCBA, F/LCBA, and RB/F/LCBA) using a simple ultrasound method and have engineered specific target-triggered catalytic hairpin assembly on their surface and chemiluminescence resonance energy transfer inside. Consequently, three multicolor capsules exhibit excellent structural stability, generate blue-, green-, and red-colored emissions when reacting with H 2 O 2 , have long-lasting CL emission over 1 h, and successfully achieve the accurate multiple visualization detection of avian influenza virus subtype targets. Without the need for complex instruments and analysis procedures, the CL imaging assays can be carried out and recorded with a common smartphone. The detection limits for visualizing H1N1, H7N9, and H5N1 are 5.5, 7.6, and 9.0 pM, respectively. There is a linear range between 20.0 and 625 pM and excellent selectivity against interfering DNA. Furthermore, visualization detection has been successfully applied for the detection of H1N1, H7N9, and H5N1 in healthy human serum samples. With these merits, this facile, ultrasensitive, and multiple visualization sensor has potential applications in point-of-care testing and early diagnosis.

A one-step logical analysis of multiple targets remains challenging. Herein, we report a one-pot and intelligent DNA logical analysis platform for the diagnosis of avian influenza virus (AIV) biomarkers based on chemiluminescence resonance energy transfer (CRET) and logic operations. On the surface of Lum/PEI/CaCO 3 microparticles, the excited state of luminol underwent CRET with fluorescein isothiocyanate or rhodamine B isothiocyanate, producing three well-separated light emissions at 425, 530, and 590 nm, respectively. Taking advantage of the close distance between fluorophores aligned by the catalytic hairpin assembly reaction, the CRET efficiency was greatly enhanced (53.1%). H1N1, H7N9, and H5N1 were detected with limits of detection values as low as 15, 34, and 58 pM, respectively. Three-input logic circuits were simultaneously conducted on the surface of Lum/PEI/CaCO 3 microparticles, enabling the rapid and accurate discrimination of multiple AIV biomarkers in one solution. In terms of peak positions and the normalized value of the total peak intensity, three biomarkers can be simultaneously discriminated without any other complex operations. In summary, the CRET-based multiple analytical assay was developed as an intelligent biosensor for identifying AIV biomarkers, having promising application prospects in the field of multiple analysis and precise disease diagnosis.

The H5N1 avian influenza virus seriously affects the health of poultry and humans. Once infected, the mortality rate is very high. Therefore, accurate and timely detection of the H5N1 avian influenza virus is beneficial for controlling its spread. This article establishes a dual gene detection method based on dual RPA for simultaneously detecting the HA and M2 genes of H5N1 avian influenza virus, for the detection of H5N1 avian influenza virus. Design specific primers for the conserved regions of the HA and M2 genes. The sensitivity of the dual RT-RPA detection method for HA and M2 genes is 1 × 10 -7 ng/μL. The optimal primer ratio is 1:1, the optimal reaction temperature is 40 °C, and the optimal reaction time is 20 min. Dual RT-RPA was used to detect 72 samples, and compared with RT-qPCR detection, the Kappa value was 1 ( p value < 0.05), and the clinical sample detection sensitivity and specificity were both 100%. The dual RT-RPA method is used for the first time to simultaneously detect two genes of the H5N1 avian influenza virus. As an accurate and convenient diagnostic tool, it can be used to diagnose the H5N1 avian influenza virus., Competing Interests: The authors declare no conflict of interest.

Avian influenza is caused by avian influenza virus infection; the H5N1 avian influenza virus is a highly pathogenic subtype, affecting poultry and human health. Since the discovery of the highly pathogenic subtype of the H5N1 avian influenza virus, it has caused enormous losses to the poultry farming industry. It was recently found that the H5N1 avian influenza virus tends to spread among mammals. Therefore, early rapid detection methods are highly significant for effectively preventing the spread of H5N1. This paper discusses the detection technologies used in the detection of the H5N1 avian influenza virus, including serological detection technology, immunological detection technology, molecular biology detection technology, genetic detection technology, and biosensors. Comparisons of these detection technologies were analyzed, aiming to provide some recommendations for the detection of the H5N1 avian influenza virus.

Objectives: Interpreting real-time reverse transcription-polymerase chain reaction (rRT-PCR) results for human avian influenza A virus (AIV) detection in contaminated settings like live bird markets (LBMs) without serology or viral culture poses a challenge., Methods: During February-March 2012 and November 2012-February 2013, we screened workers at nine LBMs in Dhaka, Bangladesh, to confirm molecular detections of AIV RNA in respiratory specimens with serology. We tested nasopharyngeal (NP) and throat swabs from workers with influenza-like illness (ILI) and NP, throat, and arm swabs from asymptomatic workers for influenza virus by rRT-PCR and sera for seroconversion and antibodies against HPAI A(H5N1) and A(H9N2) viruses., Results: Among 1273 ILI cases, 34 (2.6%) had A(H5), 56 (4%) had A(H9), and six (0.4%) had both A(H5) and A(H9) detected by rRT-PCR. Of 192 asymptomatic workers, A(H5) was detected in eight (4%) NP and 38 (20%) arm swabs. Of 28 ILI cases with A(H5) or A(H9) detected, none had evidence of seroconversion, but one (3.5%) and 12 (43%) were seropositive for A(H5) and A(H9), respectively., Conclusion: Detection of AIV RNA in respiratory specimens from symptomatic and asymptomatic LBM workers without evidence of seroconversion or virus isolation suggests environmental contamination, emphasizing caution in interpreting rRT-PCR results in high viral load settings., Competing Interests: Declarations of Competing Interest The authors have no competing interests to declare., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

During the early stages of the UK 2021-2022 H5N1 high-pathogenicity avian influenza virus (HPAIV) epizootic in commercial poultry, 12 infected premises (IPs) were confirmed by four real-time reverse-transcription-polymerase chain reaction (RRT)-PCRs, which identified the viral subtype and pathotype. An assessment was undertaken to evaluate whether a large sample throughput would challenge laboratory capacity during an exceptionally large epizootic; hence, assay performance across our test portfolio was investigated. Statistical analysis of RRT-PCR swab testing supported it to be focused on a three-test approach, featuring the matrix (M)-gene, H5 HPAIV-specific (H5-HP) and N1 RRT-PCRs, which was successfully assessed at 29 subsequent commercial IPs. The absence of nucleotide mismatches in the primer/probe binding regions for the M-gene and limited mismatches for the H5-HP RRT-PCR underlined their high sensitivity. Although less sensitive, the N1 RRT-PCR remained effective at flock level. The analyses also guided successful surveillance testing of apparently healthy commercial ducks from at-risk premises, with pools of five oropharyngeal swabs tested by the H5-HP RRT-PCR to exclude evidence of infection. Serological testing at anseriform H5N1 HPAIV outbreaks, together with quantitative comparisons of oropharyngeal and cloacal shedding, provided epidemiological information concerning the chronology of initial H5N1 HPAIV incursion and onward spread within an IP.

Global surveillance for Avian Influenza Virus (AIV) in birds is essential for assessing public and animal health risks and real-time polymerase chain reaction (RT-qPCR) is among the official methods recommended by the World Organisation for Animal Health (WOAH) to confirm the presence of the virus in laboratory specimens. Yet, in low-resource setting laboratories, the detection of AIV can be hampered by the need to maintain a cold chain for wet reagents. In such cases, alternatives should be ready to maximize surveillance capacities and mining of AIV. Therefore, we compared two lyophilized RT-qPCR reagents (1st - 5 × CAPITAL™ 1-Step qRT-PCR Probe Reagent, lyophilized kit, and 2nd - Qscript lyo 1-step-kit) to the WOAH recommended protocol by Nagy et al., 2020 using QuantiTect Probe RT-PCR-kit as wet reagent. The comparative study panel comprised 102 RNA samples from two AIV subtypes, i.e. H5 and H9 subtypes. Despite that the wet reagent exhibited the lowest limit of detection (LOD) compared to the two lyophilized reagents, the inter-assay agreement was substantial between the 1st lyophilized reagent and the comparator with 95.1% of shared positive results. Cohen's-kappa was fair between the 2nd lyophilized reagent and the comparator with 75.5% of shared positive results. Agreement using the statistical test Bland-Altman was good for samples with Cq-values < 25 for all reagents, revealing discrepancies when the viral load is low. This trend was especially evident while using the 2nd lyophilized reagent. Similar trends were obtained using the same lyophilized reagents but following the protocol by Heine et al., 2015 with AgPath-ID™ One-Step RT-PCR as a comparator, showing that Cq-values increase using lyophilized reagents but correlate strongly with the wet reagent. Further, inter-assay agreement between reagents improved when the protocol from Heine et al., 2015 was applied, suggesting a higher resilience to chemistry changes allowing easier reagents interchangeability., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Food and Agriculture Organization of the United Nations (FAO), The Author(s). Published by Elsevier B.V. All rights reserved.)

In autumn 2022, the Spanish Influenza National Reference Laboratory (NRL) confirmed the detection of influenza A(H5N1) in samples from two asymptomatic workers linked to an outbreak in a poultry farm in Spain. Nasopharyngeal swabs were taken according to a national screening protocol for exposed workers. Absence of symptoms, low viral load and negative serology in both workers suggested environmental contamination. These findings motivated an update of the early detection strategy specifying timing and sampling conditions in asymptomatic exposed persons.

The objective of this study was to determine the inactivation efficiency of common sample preparation reagents against highly pathogenic avian influenza A (HPAI) H5N1 virus. HPAI H5N1 virus has caused infections in humans with a mortality rate of over 50%. Due to the high mortality and the risk of aerosol transmission of that virus to humans and birds, infectious HPAI H5N1 viruses are contained in a biosafety level 3 laboratory. However, many procedures for further molecular analyses would be easier in lower biosafety conditions. To ensure the laboratory safety the successful inactivation procedures should be demonstrated before the samples are transferred to a lower containment facility. We tested the inactivation capacity of commonly used cell lysis buffer radio-immuno precipitation assay (RIPA) buffer for protein samples, cell fixatives methanol (MeOH) and paraformaldehyde (PFA) and guanidine isothiocyanate-containing lysis buffer for RNA isolation (RLT, Qiagen) in H5N1-infected cells. Based on our results RLT buffer, 90% MeOH (20 min, -20 °C) and 4% PFA (30 min, RT) all completely inactivated the HPAI H5N1 virus. However, RIPA buffer alone was not sufficient to inactivate the HPAI H5N1 virus in infected cell samples but, instead, combining RIPA lysis buffer and boiling for 10 min the samples in Laemmli buffer led to complete inactivation of the virus., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The aim of this study was to develop a microarray assay for the simultaneous detection of the H5, H7, H9, N1, N9 and N2 genes of the avian influenza virus (AIV) using a Nanogold-streptavidin and silver-stain-enhanced nucleic acid dot-blot hybridisation system. The conserved sequences of H5 genes from H5N1, H7 genes from H7N9, H9 genes from H9N2, N9 genes from H7N9 and N2 genes from H9N2 AIV were cloned, together with that of N1 obtained commercially, and were used as templates for generating the probes using biotin-labeled primers, which targeted the conserved regions of H5, H7, H9, N1, N9 and N2 genes, respectively. The oligonucleotide probes were diluted using the spotting buffer and ddH 2 O, and each probe was then spotted to each specific position on the microarray. The PCR products including biotin-labeled lambda, NP, H5, H7, H9, N1, N9 and N2 were mixed, 200 μL of which was then added to the microarray chamber after denaturing. Following a hybridization incubation at 45℃ for 120 min, the microarray was then incubated with nanogold-streptavidin about 4 μg/mL for 30 min. After the supplementary of 200 μL of silver buffer A and silver buffer B in the chamber, the hybridization results were assessed by direct visualization in the dark at room temperature. The microarray assay was optimized and its specificity, sensitivity and stability were evaluated. The optimal conditions comprised a probe concentration of 50 μmol/L, a hybridization temperature of 45℃ and a hybridization time of 2 h. The optimal concentration of nanogold-streptavidin was 4 μg/mL and the optimal staining time was 7 min. The results of specificity evaluation showed that no cross-binding of the probes with each other and no cross-hybridization with Newcastle disease virus, infectious bronchitis virus and infectious laryngotracheitis virus was observed. The optimized microarray assay was significantly more sensitivity than the reverse-transcription PCR assay. The microarray was available after storing at less 90 d at 4 ℃. The optimized microarray assay was validated on clinical specimens and the results showed that it had over 95.6 % correlation with reverse-transcription PCR method. Therefore, the microarray assay could be used for the high throughput detection of AIV infections due to H5N1, H7N9 and H9N2., (Copyright © 2021. Published by Elsevier B.V.)

Many studies have been conducted on measuring avian influenza viruses and their hemagglutinin (HA) antigens via electrochemical principles; most of these studies have used gold electrodes on ceramic, glass, or silicon substrates, and/or labeling for signal enhancement. Herein, we present a paper-based immunosensor for label-free measurement of multiple avian influenza virus (H5N1, H7N9, and H9N2) antigens using flexible screen-printed carbon nanotube-polydimethylsiloxane electrodes. These flexible electrodes on a paper substrate can complement the physical weakness of the paper-based sensors when wetted, without affecting flexibility. The relative standard deviation of the peak currents was 1.88% when the electrodes were repeatedly bent and unfolded twenty times with deionized water provided each cycle, showing the stability of the electrodes. For the detection of HA antigens, approximately 10-μl samples (concentration: 100 pg/ml-100 ng/ml) were needed to form the antigen-antibody complexes during 20-30 min incubation, and the immune responses were measured via differential pulse voltammetry. The limits of detections were 55.7 pg/ml (0.95 pM) for H5N1 HA, 99.6 pg/ml (1.69 pM) for H7N9 HA, and 54.0 pg/ml (0.72 pM) for H9N2 HA antigens in phosphate buffered saline, and the sensors showed good selectivity and reproducibility. Such paper-based sensors are economical, flexible, robust, and easy-to-manufacture, with the ability to detect several avian influenza viruses., (© 2022. The Author(s).)

Rapid and low-cost diagnosis of multiple infectious diseases is of great significance especially in densely populated or resource-constrained settings. Herein, we developed a one-step fast and label-free imaging array for multiplexed detection of trace avian influenza virus (AIV) DNA biomarkers. By designing a series of specific and efficient catalytic hairpin assembly (CHA) amplification reactions and utilizing thioflavin T, a specific G-quadruplex fluorescence probe, three subtypes of AIV DNA biomarkers (H1N1, H7N9 and H5N1) were simultaneously and quickly detected within only 20 min, which just needed a small reagent volume of 50 μL and a smartphone instead of a spectrometer. With the combination of fluorescence imaging output and grey-level analysis, the array sensor can be on-site with the limit of detection of 136 pM, 141 pM and 129 pM for H1N1, H7N9 and H5N1, respectively. The imaging array also displayed good mismatch discrimination, excellent anti-interference, and real sample application. In view of its advantages of fast detection, low cost and multiplexed analysis, the imaging array is expected to have potential applications for early infectious disease diagnosis in resource-constrained settings., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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.

The highly pathogenic avian influenza (HPAI) H5N8 virus has been detected in wild birds and poultry worldwide. The threat caused by HPAI H5N8 virus still exists with concerns for human infection. The preparedness for epidemic prevention and decreasing the agricultural and economic lost is extremely important. Hemagglutinin (HA), a surface glycoprotein of influenza viruses, is considered as the major target for detection of the influenza virus subtype in the infected samples. In this study, the recombinant H5N8 HA1 and HA2 proteins were expressed in Escherichia coli, and were utilized to generate two monoclonal antibodies, named 7H6C and YC8. 7H6C can bind the HA proteins of H5N1 and H5N8, but cannot bind the HA proteins of H1N1, H3N2, and H7N9, indicating that it has H5-subtype specificity. In contrast, YC8 can bind the HA proteins of H1N1, H5N1, and H5N8, but cannot bind the HA proteins of H3N2 and H7N9, indicating that it has H1-subtype and H5-subtype specificity. The epitope sequences recognized by 7H6C are located in the head domain of H5N8 HA, and are highly conserved in H5 subtypes. The epitope sequences recognized by YC8 are located in the stalk domain of H5N8 HA, and are highly conserved among the H1 and H5 subtypes. 7H6C and YC8 can be applied for specific detection of the HA proteins of H5N8 and H5Nx avian influenza viruses. KEY POINTS: • The mAb 7H6C or YC8 was generated by using the HA1 or HA2 of the HPAI H5N8 virus as the immunogen. • 7H6C recognized the head domain of H5N8 HA, and YC8 recognized the stalk domain of H5N8 HA. • 7H6C and YC8 can detect the HA proteins of H5Nx subtypes specifically.

A mixed-aged flock of 130 turkeys in Bangladesh reported the sudden death of 1 bird in September 2017. Highly pathogenic avian influenza A(H5N1) virus was detected in 3 turkeys, and phylogenetic analysis placed the viruses in the reassortant clade 2.3.2.1a. The birds had clinical signs of depression, diarrhea, weakness, closed eyes, and finally death. The mortality rate of the flock was 13% over the 6 d prior to the flock being euthanized. At autopsy, we observed congestion in lungs and brain, hemorrhages in the trachea, pancreas, breast muscle, coronary fat, intestine, bursa of Fabricius, and kidneys. Histopathology revealed hemorrhagic pneumonia, hemorrhages in the liver and kidneys, and hemorrhages and necrosis in the spleen and pancreas. Significant changes in the brain included gliosis, focal encephalomalacia and encephalitis, and neuronophagia.

Avian influenza (AI) has severely affected the poultry industry worldwide and has caused the deaths of millions of birds. Highly pathogenic avian influenza virus is characterized by high mortality and the ability to transmit from birds to humans. Early diagnosis is difficult because of the variation in pathogenicity and the genetic diversity between virus subtypes. Therefore, development of a sensitive and accurate diagnostic system is an urgent priority. We developed ssDNA aptamer probes to detect AI viruses. Through seven rounds of SELEX to search for a probe specific to the highly pathogenic AI virus subtype H5N1, we identified 16 binding aptamers and selected two with the highest binding frequency. These two aptamers had strong binding affinities and low detection limits. We found that they could bind more specifically to H5N1, as compared to other subtypes. Furthermore, these aptamers inhibited hemagglutination, which is caused by the virus surface protein hemagglutinin. Our results indicate that our screened aptamers are effective molecular probes for diagnosing H5N1 and can be used as therapeutic agents to inhibit viral surface proteins. Sensitive diagnosis and suppression of avian influenza will help maintain a stable and healthy livestock industry, as well as protect human health.

The H5N1 subtype of highly pathogenic avian influenza virus has been circulating in poultry in Indonesia since 2003 and vaccination has been used as a strategy to eradicate the disease. However, monitoring of vaccinated poultry flocks for H5N1 infection by serological means has been difficult, as vaccine antibodies are not readily distinguishable from those induced by field viruses. Therefore, a test that differentiates infected and vaccinated animals (DIVA) would be essential. Currently, no simple and specific DIVA test is available for screening of a large number of vaccinated chickens. Several epitopes on E29 domain of the haemagglutinin H5N1 subunit 2 (HA2) have recently been examined for their antigenicity and potential as possible markers for DIVA in chicken. In this study, the potential of E29 as an antigen for DIVA was evaluated in detail. Three different forms of full-length E29 peptide, a truncated E29 peptide (E15), and a recombinant E29 were compared for their ability to detect anti-E29 antibodies. Preliminary ELISA experiments using mono-specific chicken and rabbit E29 sera, and a mouse monoclonal antibody revealed that the linear E29 peptide was the most antigenic. Further examination of the E29 antigenicity in ELISA, using several sera from experimentally infected or vaccinated chickens, revealed that the full-length E29 peptide had the greatest discrimination power between infected and vaccinated chicken sera while providing the least non-specific reaction. This study demonstrates the usefulness of the HPAI H5N1 HA2 E29 epitope as a DIVA antigen in HPAI H5N1-vaccinated and -infected chickens. RESEARCH HIGHLIGHTS E29 (HA2 positions 488-516) epitope is antigenic in chickens.Antibodies to E29 are elicited following live H5N1 virus infection in chickens.E29 epitope is a potential DIVA antigen for use in ELISA.

Background & Objectives: Avian influenza (AI) viruses have been a major cause of public health concern. Wild migratory birds and contaminated environmental sources such as waterbodies soiled with bird droppings play a significant role in the transmission of AI viruses. The objective of the present study was to develop a sensitive and user-friendly method for the concentration and detection of AI viruses from environmental water sources., Methods: Municipal potable water, surface water from reservoirs and sea were spiked with low pathogenic AI viruses. To concentrate the viruses by precipitation, a combination of potassium aluminium sulphate with milk powder was used. Real-time reverse transcription-polymerase chain reaction was performed for virus detection, and the results were compared with a virus concentration method using erythrocytes. Drinking water specimens from poultry markets were also tested for the presence of AI viruses., Results: A minimum of 10 1.0 EID 50 (50% egg infectious dose)/ml spiked H5N1 and 10 1.7 EID 50 /ml spiked H9N2 viruses were detected from spiked potable water; 10 1.0 and 10 2.0 EID 50 /ml spiked H5N1 virus was detected from surface water and seawater samples, respectively. The present method was more sensitive than the erythrocyte-binding method as approximately 10-fold higher infectious virus titres were obtained. AI H9N2 viruses were detected and isolated from water from local poultry markets, using this method., Interpretation & Conclusions: Viability and recovery of the spiked viruses were not affected by precipitation. The present method may be suitable for the detection of AI viruses from different environmental water sources and can also be applied during outbreak investigations., Competing Interests: None

In the present study, we fabricated a label-free avian influenza (AIV H5N1) detection biosensor composed of a multi-functional DNA 3 way-Junction (3 W J) on a hollow Au spike-like nanoparticle (hAuSN) using a localized surface plasmon resonance (LSPR) method. To construct the multi-functional DNA (MF-DNA) as a bioprobe, the 3 W J was introduced. The proposed AIV detection bioprobe should contain three functionalities: target recognition, signal amplification, and connection to substrate. To achieve this goal, each piece of the DNA 3 W J was tailored to a hemagglutinin (HA) binding aptamer, FAM dye and thiol group, respectively. The assembly of each DNA 3 W J functional fragment was then confirmed by TBM-Native PAGE. Moreover, the hAuSN was immobilized on the indium-tin-oxide (ITO) substrate for LSPR measurement. The DNA 3 W J was immobilized onto the hAuSN electrode through the thiol-group of DNA 3 W J. The fabricated DNA 3 W J/hAuSN heterolayer on the ITO substrate was investigated by field emission scanning electron microscopy (FE-SEM) and atomic force microscopy (AFM). LSPR experiments were conducted to confirm HA protein binding to the DNA 3 W J/ hAuSN -modified electrode. The proposed biosensor can detected the HA protein in PBS buffer (LOD: 1 pM) as well as in the diluted chicken serum (LOD: 1 pM). The present study details a label-free, simple fabrication method consisted of DNA 3 W J/ hAuSN heterolayer that uses easy-to-tailor elements to detect not only AIV but also various viruses detection platform easily., (Copyright © 2019 Elsevier B.V. All rights reserved.)

H5 avian influenza virus (AIV) and velogenic Newcastle disease virus (v-NDV) are pathogens listed in the OIE Terrestrial Animal Health Code and are considered key pathogens to be eliminated in poultry production. Molecular techniques for rapid detection of H5 AIV and v-NDV are required to investigate their transmission characteristics and to guide prevention. Traditional virus isolation, using embryonated chicken eggs, is time-consuming and cannot be used as a rapid diagnostic technology. In this study, a multiplex real-time RT-PCR (RRT-PCR) detection method for six H5 AIV clades, three v-NDV subtypes, and one mesogenic NDV subtype was successfully established. The detection limit of our multiplex NDV and H5 AIV RRT-PCR was five copies per reaction for each pathogen, with good linearity and efficiency (y = -3.194x + 38.427 for H5 AIV and y = -3.32x + 38.042 for NDV). Multiplex PCR showed good intra- and inter-assay reproducibility, with coefficient of variance (CV) less than 1%. Furthermore, using the RRT-PCR method, H5 AIV and NDV detection rates in clinical samples were higher overall than those obtained using the traditional virus isolation method. Therefore, our method provides a promising technique for surveillance of various H5 AIV clades and multiple velogenic and mesogenic NDV subtypes in live-poultry markets.

In Bangladesh, the poultry industry is an economically and socially important sector, but it is persistently threatened by the effects of H5N1 highly pathogenic avian influenza. Thus, identifying the optimal control policy in response to an emerging disease outbreak is a key challenge for policy-makers. To inform this aim, a common approach is to carry out simulation studies comparing plausible strategies, while accounting for known capacity restrictions. In this study we perform simulations of a previously developed H5N1 influenza transmission model framework, fitted to two separate historical outbreaks, to assess specific control objectives related to the burden or duration of H5N1 outbreaks among poultry farms in the Dhaka division of Bangladesh. In particular, we explore the optimal implementation of ring culling, ring vaccination and active surveillance measures when presuming disease transmission predominately occurs from premises-to-premises, versus a setting requiring the inclusion of external factors. Additionally, we determine the sensitivity of the management actions under consideration to differing levels of capacity constraints and outbreaks with disparate transmission dynamics. While we find that reactive culling and vaccination policies should pay close attention to these factors to ensure intervention targeting is optimised, across multiple settings the top performing control action amongst those under consideration were targeted proactive surveillance schemes. Our findings may advise the type of control measure, plus its intensity, that could potentially be applied in the event of a developing outbreak of H5N1 amongst originally H5N1 virus-free commercially-reared poultry in the Dhaka division of Bangladesh., Competing Interests: The authors have declared that no competing interests exist.

Rapid and sensitive on-site detection of avian influenza virus (AIV) is the key for achieving near real-time surveillance of AIV and reducing the risk of dissemination. However, unlike the laboratory-prepared transparent buffer solutions containing a single type of influenza virus, distinction between real- and false- positive outputs and detection of low concentrations of AIV in stool specimens or cloacal swabs are difficult. Here, we developed a rapid and background-free lateral flow immunoassay (LFA) platform that utilizes near-infrared (NIR)-to-NIR upconversion nanoparticles (UCNPs) to yield a sensor that detects AIV nucleoproteins (NPs) from clinical samples within 20 min. Ca 2+ as a heterogeneous dopant ion in the shell enhanced the NIR-to-NIR upconversion photoluminescence (PL) emission without inducing significant changes in the morphology of the UCNPs. In a mixture of opaque stool samples and gold nanoparticles (GNPs), which are components of commercial AIV LFA, the background signal of the stool samples masked the absorption peak of GNPs. However, UCNPs dispersed in the stool samples still show strong emission centered at 800 nm when excited at 980 nm, which enables the NIR-to-NIR upconversion nanoparticle-based lateral flow immunoassay (NNLFA) platform to detect 10-times lower viral load than a commercial GNP-based AIV LFA. The detection limit of NNLFA for LPAI H5N2 and HPAI H5N6 viruses was 10 2 and 10 3.5 EID 50 /mL, respectively. Moreover, the viruses were successfully detected within dark brown-colored samples using the NNLFA but not the commercial AIV LFA. Therefore, the rapid and background-free NNLFA platform can be used for sensitive on-site detection of AIV., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Highly pathogenic avian influenza (HPAI) virus H5N1 spread throughout Nigeria between 2006 and 2007. Bird samples collected across the country were submitted through the free-of-charge (FOC) program to the National Veterinary Research Institute, Vom (NVRI-Vom) laboratory. The present article describes the spatial distributions and evaluated clustering of the FOC submissions from poultry farms at the global, local, and focal levels between 2006 and 2007 epidemic in Nigeria. Spatial statistics evaluating clustering of the FOC submissions were implemented using the Moran's I test, the purely spatial cluster analysis with the SaTScan Poisson model, and the Bithell's linear score test. A significant global clustering of the FOC submissions was observed. Significant local clusters of submissions were observed in the North-East, North-Central, and South-West zones. There was significant decline in FOC submissions with increasing distance from NVRI-Vom. These results indicated that the geographic area of influence of the FOC submission program in Nigeria was limited to regions closer to the diagnostic laboratory. This work provides a detailed insight into the surveillance activities during the HPAI outbreaks in Nigeria, and should assist policy-makers and field veterinarians to improve the effectiveness of national eradication plans in the face of any outbreak of animal diseases.

In this paper, we present a phase-intensity surface plasmon resonance (SPR) biosensor and demonstrate its use for avian influenza A (H5N1) antibody biomarker detection. The sensor probes the intensity variation produced by the steep phase response at surface plasmon excitation. The prism sensor head is fixed between a pair of polarizers with a perpendicular orientation angle and a forbidden transmission path. At SPR, a steep phase change is introduced between the p- and s-polarized light, and this rotates the polarization ellipse of the transmission beam. This allows the light at resonance to be transmitted and a corresponding intensity change to be detected. Neither time-consuming interference fringe analysis nor a phase extraction process is required. In refractive index sensing experiments, the sensor resolution was determined to be 6.3 × 10 -6 refractive index values (RIU). The sensor has been further applied for H5N1 antibody biomarker detection, and the sensor resolution was determined to be 193.3 ng mL -1 , compared to 1 μg mL -1 and 0.5 μg mL -1 , as reported in literature for influenza antibody detection using commercial Biacore systems. It represents a 517.3% and 258.7% improvement in detection limit, respectively. With the unique features of label-free, real-time, and sensitive detection, the phase-intensity SPR biosensor has promising potential applications in influenza detection.

Current methods to detect avian influenza viruses (AIV) are time consuming and lo inw sensitivity, necessitating a faster and more sensitive sensor for on-site epidemic detection in poultry farms and urban population centers. This study reports a field effect transistor (FET) based AIV sensor that detects nucleoproteins (NP) within 30 minutes, down to an LOD of 10 3 EID 50 mL -1 from a live animal cloacal swab. Previously reported FET sensors for AIV detection have not targeted NPs, an internal protein shared across multiple strains, due to the difficulty of field-effect sensing in a highly ionic lysis buffer. The AIV sensor overcomes the sensitivity limit with an FET-based platform enhanced with a disposable well gate (DWG) that is readily replaceable after each measurement. In a single procedure, the virus-containing sample is immersed in a lysis buffer mixture to expose NPs to the DWG surface. In comparison with commercial AIV rapid kits, the AIV sensor is proved to be highly sensitive, fast, and compact, proving its potential effectiveness as a portable biosensor., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Highly pathogenic avian influenza virus H5N1 is a continuous threat to public health and poultry industry. The recurrence of the H5N1 led us to develop a robust, specific, and rapid detection method for the virus. In this study, an impedance aptasensor was developed for the virus detection using specific H5N1 aptamer and a gold interdigitated microelectrode. Streptavidin was immobilized on the microelectrode surface and biotin labeled H5N1 aptamer was bound to the immobilized streptavidin. The microelectrode was blocked with the polyethylene glycol and the bound aptamer captured the virus. The impedance change caused by the captured virus was measured using an impedance analyzer. To enhance impedance signal, a nanoparticle-based amplifier was designed and implemented by forming a network-like gold nanoparticles/H5N1-aptamer/thiocyanuric acid. The detection limit of the impedance aptasensor was 0.25 HAU for the pure virus and 1 HAU for the tracheal chicken swab samples spiked with the H5N1 virus. The detection time of aptasensor without employing the amplifier was less than an hour. The amplifier increased impedance by a 57-fold for the 1 HAU samples. Only negligible impedance change was observed for non-target viruses such as H5N2, H5N3, H7N2, H1N1, and H2N2. This aptasensor provides a foundation for the development of a portable aptasensor instrument., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Oropharyngeal and cloacal swabs have been widely used for the detection of H5N1 highly pathogenic avian Influenza A virus (HPAI virus) in birds. Previous studies have shown that the feather calamus is a site of H5N1 virus replication and therefore has potential for diagnosis of avian influenza. However, studies characterizing the value of feathers for this purpose are not available, to our knowledge; herein we present a study investigating feathers for detection of H5N1 virus. Ducks and chickens were experimentally infected with H5N1 HPAI virus belonging to 1 of 3 clades (Indonesian clades 2.1.1 and 2.1.3, Vietnamese clade 1). Different types of feathers and oropharyngeal and cloacal swab samples were compared by virus isolation. In chickens, virus was detected from all sample types: oral and cloacal swabs, and immature pectorosternal, flight, and tail feathers. During clinical disease, the viral titers were higher in feathers than swabs. In ducks, the proportion of virus-positive samples was variable depending on viral strain and time from challenge; cloacal swabs and mature pectorosternal feathers were clearly inferior to oral swabs and immature pectorosternal, tail, and flight feathers. In ducks infected with Indonesian strains, in which most birds did not develop clinical signs, all sampling methods gave intermittent positive results; 3-23% of immature pectorosternal feathers were positive during the acute infection period; oropharyngeal swabs had slightly higher positivity during early infection, while feathers performed better during late infection. Our results indicate that immature feathers are an alternative sample for the diagnosis of HPAI in chickens and ducks., (© 2015 The Author(s).)

A new colorimetric method for monitoring of rolling circle amplification was developed. At first H5N1 target hybrids with padlock probe (PLP) and then PLP is circularized upon the action of T4 ligase enzyme. Subsequently, the circular probe is served as a template for hyperbranched rolling circle amplification (HRCA) by utilizing Bst DNA polymerase enzyme. By improving the reaction, pyrophosphate is produced via DNA polymerization and chelates the Mg(2+) in the buffer solution. This causes change in solution color in the presence of hydroxy naphthol blue (HNB) as a metal indicator. By using pH shock instead of heat shock and isothermal RCA reaction not only the procedure becomes easier, but also application of HNB for colorimetric detection of RCA reaction further simplifies the assay. The responses of the biosensor toward H5N1 were linear in the concentration range from 0.16 to 1.20 pM with a detection limit of 28 fM., (Copyright © 2015 Elsevier B.V. All rights reserved.)

This paper concerns the development of genosensors based on redox-active monolayers incorporating (dipyrromethene)2Cu(II) and (dipyrromethene)2Co(II) complexes formed step by step on a gold electrode surface. They were applied for electrochemical determination of oligonucleotide sequences related to avian influenza virus (AIV) type H5N1. A 20-mer probe (NH2-NC3) was covalently attached to the gold electrode surface via a reaction performed in the presence of ethyl(dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide (EDC/NHS) between the amine group present in the probe and carboxylic groups present on the surface of the redox-active layer. Each modification step has been controlled with Osteryoung square-wave voltammetry. The genosensor incorporating the (dipyrromethene)2Cu(II) complex was able to detect a fully complementary single-stranded DNA target with a detection limit of 1.39 pM. A linear dynamic range was observed from 1 to 10 pM. This genosensor displays good discrimination between three single-stranded DNA targets studied: fully complementary, partially complementary (with only six complementary bases), and totally noncomplementary to the probe. When the (dipyrromethene)2Co(II) complex was applied, a detection limit of 1.28 pM for the fully complementary target was obtained. However, this genosensor was not able to discriminate partially complementary and totally noncomplementary oligonucleotide sequences to the probe. Electrochemical measurements, using both types of genosensors in the presence of different supporting electrolytes, were performed in order to elaborate a new mechanism of analytical signal generation based on an ion barrier "switch-off" system.

This paper describes the development of a biosensor for the detection of anti-hemagglutinin antibodies against the influenza virus hemagglutinin. The steps of biosensor fabrications are as follows: (i) creation of a mixed layer containing the thiol derivative of dipyrromethene and 4-mercapto-1-butanol, (ii) complexation of Cu(II) ions, (iii) oriented immobilization of the recombinant histidine-tagged hemagglutinin, and (iv) filling free spaces with bovine serum albumin. The interactions between recombinants hemagglutinin from the highly pathogenic avian influenza virus type H5N1 and anti-hemagglutinin H5 monoclonal antibodies were explored with Osteryoung square-wave voltammetry. The biosensor displayed a good detection limit of 2.4 pg/mL, quantification limit of 7.2 pg/mL, and dynamic range from 4.0 to 100.0 pg/mL in buffer. In addition, this analytical device was applied for the detection of antibodies in hen sera from individuals vaccinated and non-vaccinated against the avian influenza virus type H5N1. The limit of detection for the assay was the dilution of sera 1: 7 × 10(6), which is about 200 times better than the enzyme-linked immunosorbent assay.

The 2006 outbreaks of H5N1 avian influenza in Egypt interrupted poultry production and caused staggering economic damage. In addition, H5N1 avian influenza viruses represent a significant threat to public health. Therefore, the rapid detection of H5 viruses is very important in order to control the disease. In this study, a qualitative reverse transcription recombinase polymerase amplification (RT-RPA) assay for the detection of hemagglutinin gene of H5 subtype influenza viruses was developed. The results were compared to the real-time reverse transcription polymerase chain reaction (RT-PCR). An in vitro transcribed RNA standard of 970 nucleotides of the hemagglutinin gene was developed and used to determine the assay sensitivity. The developed H5 RT-RPA assay was able to detect one RNA molecule within 7 min, while in real-time RT-PCR, at least 90 min was required. H5 RT-RPA assay did not detect nucleic acid extracted from H5 negative samples or from other pathogens producing respiratory manifestation in poultry. The clinical performance of the H5 RT-RPA assay was tested in 30 samples collected between 2014 and 2015; the sensitivity of H5 RT-RPA and real-time RT-PCR was 100%. In conclusion, H5 RT-RPA was faster than real-time RT-PCR and easily operable in a portable device. Moreover, it had an equivalent sensitivity and specificity., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Early screening of suspected cases is the key to control the spread of avian influenza (AI) H5N1. In our previous studies, an impedance biosensor with an interdigitated array microelectrode based biochip was developed and validated with pure AI H5 virus, but had limitations in cost and reliability of the biochip, specificity of the antibody against Asian in-field H5N1 virus and detection of H5N1 virus in real samples. The purpose of this study is to develop a low-cost impedance immunosensor for rapid detection of Asian in-field AI H5N1 virus in chicken swabs within 1h and validate it with the H5N1 virus. Specific monoclonal antibodies against AI H5N1 virus were developed by fusion of mouse myeloma cells with spleen cells isolated from an H5N1-virus-immunized mouse. Dot-ELISA analysis demonstrated that the developed antibodies had good affinity and specificity with the H5N1 virus. The microelectrodes were redesigned with compact size, fabricated using an improved wet-etching micro-fabrication process with a higher qualified production rate of 70-80%, and modified with the antibodies by the Protein A method. Equivalent circuit analysis indicated that electron transfer resistor was effective with the increase in impedance after capturing of the H5N1 viruses. Linear relationship between impedance change and logarithmic value of H5N1 virus at the concentrations from 2(-1) to 2(4) HAU/50 μl was found and the lower limit of detection was 2(-1) HAU/50 μl. No obvious interferences from non-target viruses such as H6N2, H9N2, Newcastle disease virus, and infectious bronchitis virus were found. Chicken swab tests showed that the impedance immunosensor had a comparable accuracy with real-time RT-PCR compared to viral isolation., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Highly pathogenic avian influenza virus (HPAIV) H5N1 has been endemic in Egypt since 2006, and there is increasing concern for its potential to become highly transmissible among humans. Infection by HPAIV H5N1 has been described in experimentally challenged birds. However, the pathogenicity of the H5N1 isolated in Egypt has never been reported in naturally infected chickens and ducks. Here we report a 2013 outbreak of HPAIV H5N1 in commercial poultry farms and backyards in Sharkia Province, Egypt. The main symptoms were ecchymosis on the shanks and feet, cyanosis of the comb and wattles, subcutaneous edema of the head and neck for chickens, and nervous signs (torticollis) for ducks. Within 48-72 hrs of the onset of illness, the average mortality rates were 22.8-30% and 28.5-40% in vaccinated chickens and non-vaccinated ducks, respectively. Tissue samples of chickens and ducks were collected for analyses with cross-section immunohistochemistry and real-time RT-PCR for specific viral RNA transcripts. While viral RNA was detected in nearly all tissues and sera collected, viral nucleoprotein was detected almost ubiquitously in all tissues, including testis. Interestingly, viral antigen was also observed in endothelial cells of most organs in chickens, and clearly detected in the trachea and brain in particular. Viral nucleoprotein was also detected in mononuclear cells of various organs, especially pulmonary tissue. We performed phylogenetic analyses and compared the genomic sequences of the hemagglutinin (HA) and nonstructural proteins (NS) among the isolated viruses, the HPAIV circulated in Egypt in the past and currently, and some available vaccine strains. Further analysis of deduced amino acids of both HA and NS1 revealed that our isolates carried molecular determinants of HPAIV, including the multibasic amino acids (PQGERRRK/KR*GLF) in the cleavage site in HA and glutamate at position 92 (D92E) in NS1. This is the first report of the pathogenicity of the HPAIVH5N1 strain currently circulating in naturally infected poultry in Egypt, which may provide unique insights into the viral pathogenesis in HPAIV-infected chickens and ducks.

A distinct cluster of highly pathogenic avian influenzaviruses of subtype A(H5N1) has been found to emergewithin clade 2.2.1.2 in poultry in Egypt since summer2014 and appears to have quickly become predominant.Viruses of this cluster may be associated withincreased incidence of human influenza A(H5N1) infectionsin Egypt over the last months.

This paper concerns the development of a redox-active monolayer and its application for the construction of an electrochemical genosensor designed for the detection of specific DNA and RNA oligonucleotide sequences related to the avian influenza virus (AIV) type H5N1. This new redox layer was created on a gold electrode surface step by step. Cyclic Voltammetry, Osteryoung Square-Wave Voltammetry and Differential Pulse Voltammetry were used for its characterization. This new redox-active layer was applied for the construction of the DNA biosensor. The NH2-NC3 probe (20-mer) was covalently attached to the gold electrode surface via a "click" reaction between the amine and an epoxide group. The hybridization process was monitored using the Osteryoung Square-Wave Voltammetry. The 20-mer DNA and ca. 280-mer RNA oligonucleotides were used as the targets. The constructed genosensor was capable to determine complementary oligonucleotide sequences with a detection limit in the pM range. It is able to distinguish the different position of the part RNA complementary to the DNA probe. The genosensor was very selective. The 20-mer DNA as well as the 280-mer RNA oligonucleotides without a complementary sequence generated a weak signal., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Surveillance for highly pathogenic avian influenza viruses (HPAIV) in wild birds is logistically demanding due to the very low rates of virus detection. Serological approaches may be more cost effective as they require smaller sample sizes to identify exposed populations. We hypothesized that antigenic differences between classical Eurasian H5 subtype viruses (which have low pathogenicity in chickens) and H5N1 viruses of the Goose/Guangdong/96 H5 lineage (which are HPAIV) may be used to differentiate populations where HPAIVs have been circulating, from those where they have not. To test this we performed hemagglutination inhibition assays to compare the reactivity of serum samples from wild birds in Mongolia (where HPAIV has been circulating, n = 1,832) and Europe (where HPAIV has been rare or absent, n = 497) to a panel of reference viruses including classical Eurasian H5 (of low pathogenicity), and five HPAIV H5N1 antigens of the Asian lineage A/Goose/Guangdong/1/96. Antibody titres were detected against at least one of the test antigens for 182 Mongolian serum samples (total seroprevalence of 0.10, n = 1,832, 95% adjusted Wald confidence limits of 0.09-0.11) and 25 of the European sera tested (total seroprevalence of 0.05, n = 497, 95% adjusted Wald confidence limits of 0.03-0.07). A bias in antibody titres to HPAIV antigens was found in the Mongolian sample set (22/182) that was absent in the European sera (0/25). Although the interpretation of serological data from wild birds is complicated by the possibility of exposure to multiple strains, and variability in the timing of exposure, these findings suggest that a proportion of the Mongolian population had survived exposure to HPAIV, and that serological assays may enhance the targeting of traditional HPAIV surveillance toward populations where isolation of HPAIV is more likely.

The continued spread of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 among poultry in Vietnam poses a potential threat to animals and public health. To evaluate the pathogenicity of a 2012 H5N1 HPAIV isolate and to assess the utility of conjunctival swabs for viral detection and isolation in surveillance, an experimental infection with HPAIV subtype H5N1 was carried out in domestic ducks. Ducks were infected with 10(7.2) TCID50 of A/duck/Vietnam/QB1207/2012 (H5N1), which was isolated from a moribund domestic duck. In the infected ducks, clinical signs of disease, including neurological disorder, were observed. Ducks started to die at 3 days-post-infection (dpi), and the study mortality reached 67%. Viruses were recovered from oropharyngeal and conjunctival swabs until 7 dpi and from cloacal swabs until 4 dpi. In the ducks that died or were sacrificed on 3, 5, or 6 dpi, viruses were recovered from lung, brain, heart, pancreas and intestine, among which the highest virus titers were in the lung, brain or heart. Results of virus titration were confirmed by real-time RT-PCR. Genetic and phylogenetic analysis of the HA gene revealed that the isolate belongs to clade 2.3.2.1 similarly to the H5N1 viruses isolated in Vietnam in 2012. The present study demonstrated that this recent HPAI H5N1 virus of clade 2.3.2.1 could replicate efficiently in the systemic organs, including the brain, and cause severe disease with neurological symptoms in domestic ducks. Therefore, this HPAI H5N1 virus seems to retain the neurotrophic feature and has further developed properties of shedding virus from the oropharynx and conjunctiva in addition to the cloaca, potentially posing a higher risk of virus spread through cross-contact and/or environmental transmission. Continued surveillance and diagnostic programs using conjunctival swabs in the field would further verify the apparent reliability of conjunctival samples for the detection of AIV., (Copyright © 2013 Elsevier B.V. All rights reserved.)

In this work, we report on oligonucleotide probes bearing metallacarborane [3-iron bis(dicarbollide)] redox label, deposited on gold electrode for electrochemical determination of DNA sequence derived from Avian Influenza Virus (AIV), type H5N1. The oligonucleotide probes containing 5'-terminal NH2 group were covalently attached to the electrode, via NHS/EDC coupling to 3-mercaptopropionic acid SAM, previously deposited on the surface of gold. The changes in redox activity of Fe(III) centre of the metallacarborane complex before and after hybridization process was used as analytical signal. The signals generated upon hybridization with targets such as complementary or non-complementary 20-mer ssDNA or various PCR products consisting of 180-190 bp (dsDNA) were recorded by Osteryoung square-wave voltammetry (OSWV). The developed system was very sensitive towards targets containing sequence complementary to the probe with the detection limit estimated as 0.03 fM (S/N=3.0) and 0.08 fM (S/N=3.0) for 20-mer ssDNA and for dsDNA (PCR product), respectively. The non-complementary targets generated very weak responses. Furthermore, the proposed genosensor was suitable for discrimination of PCR products with different location of the complementarity region., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The present study was carried out with an aim to develop anti-nucleoprotein (anti-NP) monoclonal antibodies (MAbs) for use in immunodiagnostic testing for detection of avian influenza virus (AIV) antigen or antibodies. The NP gene of AIV, cloned in pET vector, was expressed in Escherichia coli BL 21 strain to produce a 6x-His tagged recombinant NP (rNP) antigen of ∼61 kDa molecular weight as soluble fraction. The rNP antigen was detected in soluble fraction of bacterial cell lysate with anti-His HRPO conjugate and reacted with the reference AIV antibody positive serum in immunoblotting. The rNP was used to immunize BALB/c mice to produce hybridoma secreting anti-NP MAbs. Out of 11 anti-NP MAbs produced, 8D2-H5, 8D2-H9, and 6D11-A7 were of IgM isotype and 5D10-C9 and 5D10-F11 were of IgG2b type, while 3F3-D2, 7D2-C9, 7D2-G7, and 7D2-G8 were of IgG1 isotype. The MAbs 3F3-D2 and 7D2-G8 showed high intensity positive reaction with rNP and a low intensity reaction with H5N1 virus in Western blot analysis. The anti-NP MAbs produced in the present work may be valuable in developing a competitive ELISA or immunochromatographic strip test-based assays for the rapid diagnosis of avian influenza.

H5 subtype avian influenza (AIV-H5) is a major causative agent of animalloimia a rapid and sensitive molecular biological diagnosis is crucial to the control program of AIV-H5. AIV-H5 real-time fluorescent reverse transcription loop-mediated isothermal amplification (qRT-LAMP) was established by means of heat treatment of the samples. The sensitivity, specificity and repeatability of this method were assessed and the performance of Calcein,SYBR Green I,HNB,SYTO 81 in colorimetric detection was comparatively analyzed to screen the optimum dye. The results showed the sensitivity of this method was 100 times higher than that of standard real-time fluorescent RT-PCR, and the detection limit was one copy of the gene per reaction. This method had no cross-reactivity with other common avian respiratory tract infectious disease-related pathogens such as IBV and NDV. The present study suggested Calcein was the optimum dye. Small-scale tests suggested this method was reliable for survey monitoring of AIV-H5 on the spot, indicating its potential applications in field investigation.

Previous studies have reported the detection of H5N1 HPAI virus in feathers from ducks naturally and experimentally infected and suggested that feather calami (FC) could be used as diagnostic samples for the early detection of H5N1 HPAI infections. Ducks are readily infected with H5N1 HPAI viruses although the development of clinical signs and deaths were reported as age-related with younger birds being more susceptible. The correlation between age and virus localisation in FC of infected ducks has not been studied to date. In the present study juvenile (4-week-old) and adult (24-week-old) Pekin ducks (Anas platyrhynchos var. domestica) were infected experimentally with a clade 2.2 H5N1 HPAI virus (A/duck/Nigeria/1071-23/2007). Tracheal (Tr) and cloacal (Cl) swabs and FC were collected at 3, 5, 7 and 10 days post infection and tested by RRT-PCR and a double antibody sandwich-ELISA (DAS-ELISA) developed in house. Virus was detected in swabs and FC of challenged ducks with a higher rate of detection in juvenile ducks. In this age group virus was detected over a longer period of time in FC compared to swabs. Our study showed that FC samples collected from young ducks are a valid diagnostic specimen for H5N1 HPAI virus detection. The DAS-ELISA on FC proved to be a suitable alternative diagnostic test when molecular and/or virus isolation techniques are not available therefore it could be useful in the diagnosis of H5N1 HPAI infections in under-resourced countries., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Aptamers are artificial oligonucleotides (DNA or RNA) that can bind to a broad range of targets. In diagnostic and detection assays, aptamers represent an alternative to antibodies as recognition agents. The objective of this study was to select and characterize DNA aptamers that can specifically bind to avian influenza virus (AIV) H5N1 based on Systematic Evolution of Ligands by EXponential enrichment (SELEX) and surface plasmon resonance (SPR). The selection was started with an ssDNA (single-stranded DNA) library of 10¹⁴ molecules randomized at central 74 t. For the first four selection cycles, purified hemagglutinin (HA) from AIV H5N1 was used as the target protein, and starting from the fifth cycle, entire H5N1 virus was applied in order to improve the specificity. After 13 rounds of selection, DNA aptamers that bind to the H5N1 were isolated and three aptamer sequences were characterized further by sequencing and affinity binding. Dot blot analysis was employed for monitoring the SELEX process and conducting the preliminary tests on the affinity and specificity of aptamers. With the increasing number of selection cycles, a steady increase in the color density was observed, indicating that the aptamers with good binding affinity to the target were enriched. The best aptamer candidate had a dissociation constant (KD) of 4.65 M as determined by SPR, showing a strong binding between the HA and the selected aptamer. The specificity was determined by testing non-target AIV H5N2, H5N3, H5N9, H9N2 and H7N2. Negligible cross-reactivity confirmed the high specificity of selected aptamers. The developed aptamer was then applied for detection of AIV H5N1 in spiked poultry swab samples. The obtained aptamers could open up possibilities for the development of aptamer-based medical diagnostics and detection assays for AIV H5N1. (The H5N1 used in this study was inactivated virus.)., (Copyright © 2013 Elsevier B.V. All rights reserved.)

As continuous outbreak of avian influenza (AI) has become a threat to human health, economic development and social stability, it is urgently necessary to detect the highly pathogenic avian influenza H5N1 virus quickly. In this study, we fabricated indium-tin-oxide thin-film transistors (ITO TFTs) on a glass substrate for the detecting of AI H5N1. The ITO TFT is fabricated by a one-shadow-mask process in which a channel layer can be simultaneously self-assembled between ITO source/drain electrodes during magnetron sputtering deposition. Monoclonal anti-H5N1 antibodies specific for AI H5N1 virus were covalently immobilized on the ITO channel by (3-glycidoxypropyl)trimethoxysilane. The introduction of target AI H5N1 virus affected the electronic properties of the ITO TFT, which caused a change in the resultant threshold voltage (VT) and field-effect mobility. The changes of ID-VG curves were consistent with an n-type field effect transistor behavior affected by nearby negatively charged AI H5N1 viruses. The transistor based sensor demonstrated high selectivity and stability for AI H5N1 virus sensing. The sensor showed linear response to AI H5N1 in the concentrations range from 5×10(-9) g mL(-1) to 5×10(-6) g mL(-1) with a detection limit of 0.8×10(-10) g mL(-1). Moreover, the ITO TFT biosensors can be repeatedly used through the washing processes. With its excellent electric properties and the potential for mass commercial production, ITO TFTs can be promising candidates for the development of label-free biosensors., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The objective of this study was to develop a quartz crystal microbalance (QCM) aptasensor based on ssDNA crosslinked polymeric hydrogel for rapid, sensitive and specific detection of avian influenza virus (AIV) H5N1. A selected aptamer with high affinity and specificity against AIV H5N1 surface protein was used, and hybridization between the aptamer and ssDNA formed the crosslinker in the polymer hydrogel. The aptamer hydrogel was immobilized on the gold surface of QCM sensor using a self-assembled monolayer method. The hydrogel remained in the state of shrink if no H5N1 virus was present in the sample because of the crosslinking between the aptamer and ssDNA in the polymer network. When it exposed to target virus, the binding reaction between the aptamer and H5N1 virus caused the dissolution of the linkage between the aptamer and ssDNA, resulting in the abrupt swelling of the hydrogel. The swollen hydrogel was monitored by the QCM sensor in terms of decreased frequency. Three polymeric hydrogels with different ratio (100:1 hydrogel I, 10:1 hydrogel II, 1:1 hydrogel III) of acrylamide and the aptamer monomer were synthesized, respectively, and then were used as the QCM sensor coating material. The results showed that the developed hydrogel QCM aptasensor was capable of detecting target H5N1 virus, and among the three developed aptamer hydrogels, hydrogel III coated QCM aptasensor achieved the highest sensitivity with the detection limit of 0.0128 HAU (HA unit). The total detection time from sampling to detection was only 30 min. In comparison with the anti-H5 antibody coated QCM immunosensor, the hydrogel QCM aptasensor lowered the detection limit and reduced the detection time., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Outbreaks of avian influenza A virus infection, particularly the H5N1 strains that have affected birds and some humans for the past 15 years, have highlighted the need for increased surveillance and disease control. Such measures require diagnostic tests to detect and characterize the different subtypes of influenza virus. In the current study, a simple method for producing reference avian influenza virus antisera to be used in diagnostic tests was developed. Antisera of nine avian influenza A virus neuraminidases (NA) used for NA subtyping were produced using a recombinant baculovirus. The recombinant NA (rNA) proteins were expressed in Sf9 insect cells and inoculated intramuscularly into specific-pathogen-free chickens with the ISA70 adjuvant. The NA inhibition antibody titers of the rNA antiserum were in the ranges of 5 to 8 and 6 to 9 log(2) units after the primary and boost immunizations, respectively. The antisera were subtype specific, showing low cross-reactivity against every other NA subtype using the conventional thiobarbituric acid NA inhibition assay. These results suggest that this simple method for producing reference NA antisera without purification may be useful for the diagnosis and surveillance of influenza virus.

An H5N1 highly pathogenic avian influenza virus was isolated from conjunctiva of a whooper swan with neurological signs, which was captured during the latest H5N1 HPAI outbreak in Japan. The conjunctival swab contained a larger amount of the virus in comparison with the tracheal swab. This is the first report on H5N1 virus isolation from the conjunctiva of a wild bird, and the result may suggest the conjunctival swab to be a critical sample for H5N1 HPAIV detection in waterfowl. Phylogenetic analysis of the HA gene indicated that the virus falls into H5N1 clade 2.3.2.1.

Available avian influenza (AIV) serological diagnostic tests cannot distinguish vaccinated from naturally infected birds. Differentiation of vaccinated from infected animals (DIVA) is currently advocated as a means of achieving the full control of H5N1. In this study, for the first time, recombinant ectodomain of M2 protein (M2e) of avian influenza virus (H5N1 strain) was used for the DIVA serology test. M2e was cloned into pMAL-P4X vector and expressed in E. coli cells. We used Western blot to recognize the expressed M2e-MBP protein by chicken antisera produced against live H5N1 virus. Also, the specificity of M2e-MBP protein was compared to the M2e synthetic peptide via ELISA. In M2e-MBP ELISA, all sera raised against the live avian influenza viruses were positive for M2e antibodies, whereas sera from killed virus vaccination were negative. Furthermore, M2e-MBP ELISA of the field sera obtained from vaccinated and non-vaccinated chickens showed negative results, while challenged vaccinated chickens demonstrated strong positive reactions. H5N1-originated recombinant M2e protein induced broad-spectrum response and successfully reacted with antibodies against other AIV strains such as H5N2, H9N2, H7N7, and H11N6. The application of the recombinant protein instead of synthetic peptide has the advantages of continues access to an inexpensive reagent for performing a large scale screening. Moreover, recombinant proteins provide the possibility of testing the DIVA results with an additional technique such a Western blotting which is not possible in the case of synthetic proteins. All together, the results of the present investigation show that recombinant M2e-MBP can be used as a robust and inexpensive solution for DIVA test.