Comparative assessment of lyophilized and wet reagents for the molecular detection of H5N1 high pathogenic avian influenza virus and H9N2 low pathogenic avian influenza virus.

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.

Health monitoring in birds using bio-loggers and whole blood transcriptomics.

Monitoring and early detection of emerging infectious diseases in wild animals is of crucial global importance, yet reliable ways to measure immune status and responses are lacking for animals in the wild. Here we assess the usefulness of bio-loggers for detecting disease outbreaks in free-living birds and confirm detailed responses using leukocyte composition and large-scale transcriptomics. We simulated natural infections by viral and bacterial pathogens in captive mallards (Anas platyrhynchos), an important natural vector for avian influenza virus. We show that body temperature, heart rate and leukocyte composition change reliably during an acute phase immune response. Using genome-wide gene expression profiling of whole blood across time points we confirm that immunostimulants activate pathogen-specific gene regulatory networks. By reporting immune response related changes in physiological and behavioural traits that can be studied in free-ranging populations, we provide baseline information with importance to the global monitoring of zoonotic diseases.

Simple Strategy for Rapid and Sensitive Detection of Avian Influenza A H7N9 Virus Based on Intensity-Modulated SPR Biosensor and New Generated Antibody.

In 2013 a new reassortant avian influenza A H7N9 virus emerged in China, causing human infection with high mortality. An accurate and timely diagnosis is crucial for controlling the outbreaks of the disease. We therefore propose a simple strategy for rapidly and sensitively detecting the H7N9 virus using an intensity-modulated surface plasmon resonance (IM-SPR) biosensor integrated with a new generated monoclonal antibody. The novel antibody exhibits significant specificity to recognize H7N9 virus compared with other clinical human influenza isolates (p < 0.01). Experimentally, the detection limit of the proposed approach for H7N9 virus detection is estimated to be 144 copies/mL, which is a 20-fold increase in sensitivity compared with homemade target-captured ELISA using the identical antibody. For the measurement of mimic clinical specimens containing the H7N9 virus mixed with nasal mucosa from flu-like syndrome patients, the detection limit is calculated to be 402 copies/mL, which is better than conventional influenza detection assays; quantitative reverse transcription polymerase chain reaction (qRT-PCR) and rapid influenza diagnostic test (RIDT). Most importantly, the assay time took less than 10 min. Combined, the results of this study indicate that the proposed simple strategy demonstrates high sensitivity and time-saving in H7N9 virus detection. By incorporating a high specific recognizer, the proposed technique has the potential to be used in applications and development of other emerging or re-emerging microbe detection platforms.

Diagnostics-in-a-Suitcase: Development of a portable and rapid assay for the detection of the emerging avian influenza A (H7N9) virus.

BACKGROUND: In developing countries, equipment necessary for diagnosis is only available in few central laboratories, which are less accessible and of limited capacity to test large numbers of incoming samples. Moreover, the transport conditions of samples are inadequate, therefore leading to unreliable results. OBJECTIVES: The development of a rapid, inexpensive, and simple test would allow mobile detection of viruses. STUDY DESIGN: A suitcase laboratory "Diagnostics-in-a-Suitcase" (56cm×45.5cm×26.5cm) containing all reagents and devices necessary for performing a reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed. As an example, two RT-RPA assays were established for the detection of hemagglutinin (H) and neuraminidase (N) genes of the novel avian influenza (H7N9) virus. RESULTS: The sensitivities of the H7 and the N9 RT-RPA assays were 10 and 100 RNA molecules, respectively. The assays were performed at a single temperature (42°C). The results were obtained within 2-7min. The H7N9 RT-RPA assays did not show a cross-detection either of any other respiratory viruses affecting humans and/or birds or of the human or chicken genomes. All reagents were used, stored, and transported at ambient temperature, that is, cold chain independent. In addition, the Diagnostics-in-a-Suitcase was operated by a solar-powered battery. CONCLUSIONS: The developed assay protocol and mobile setup performed well. Moreover, it can be easily implemented to perform diagnoses at airports, quarantine stations, or farms for rapid on-site viral nucleic acid detection.

A novel immunochromatographic system for easy-to-use detection of group 1 avian influenza viruses with acquired human-type receptor binding specificity.

A switch of viral hemagglutinin receptor binding specificity from bird-type α2,3- to human-type α2,6-linked sialic acid is necessary for an avian influenza virus to become a pandemic virus. In this study, an easy-to-use strip test to detect receptor binding specificity of influenza virus was developed. A biotinylated anti-hemagglutinin antibody that bound a broad range of group 1 influenza A viruses and latex-conjugated α2,3 (blue) and α2,6 (red) sialylglycopolymers were used in an immunochromatographic strip test, with avidin and lectin immobilized on a nitrocellulose membrane at test and control lines, respectively. Accumulation of a sialylglycopolymer-virus-antibody complex at the test line was visualized by eye. The strip test could be completed in 30min and did not require special equipment or skills, thereby avoiding some disadvantages of current methods for analyzing receptor binding specificity of influenza virus. The strip test could detect the receptor binding specificity of a wide range of influenza viruses, as well as small increases in the binding affinity of variant H5N1 viruses to α2,6 sialylglycans at viral titers >128 hemagglutination units. The strip test results were in agreement with those of ELISA virus binding assays, with correlations >0.95. In conclusion, the immunochromatographic strip test developed in this study should be useful for monitoring potential changes in the receptor binding specificity of group 1 influenza A viruses in the field.

An impedance immunosensor based on low-cost microelectrodes and specific monoclonal antibodies for rapid detection of avian influenza virus H5N1 in chicken swabs.

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.

Avaliação situacional de influenza pós pandemia de 2009 - uma breve revisão / Situational assessment of influenza post 2009 Pandemic - a brief review

Os vírus influenza são responsáveis por epidemias anuais com gravidade da doença variável. Causam infecção respiratória aguda com elevada transmissibilidade devido sua alta variabilidade genética, capacidade de adaptação e rápida disseminação. Os vírus influenza apresentam genoma fragmentado,o que ocasiona variações antigênicas frequentes, e consequentemente pode induzir o aparecimento de subtipos mais virulentos, como ocorreu em 2009,quando foi registrada pandemia por um novo vírus Influenza A H1N1. A Organização Mundial de Saúde(OMS) estima que a gripe acometa 5 a 15% da população,ocasionando 3 a 5 milhões de casos graves e 250.000 a 500.000 mortes anualmente. As epidemias anuais de gripe e o risco de novas pandemias tornamo monitoramento epidemiológico do vírus influenza fundamental e, para isto, a OMS coordena a Rede Mundial de Vigilância da Influenza com a finalidade de fornecer informações necessárias para a escolha das variantes virais que farão parte da composição anual da vacina, visto que a vacinação é uma das medidas mais efetivas para prevenção da gripe e suas complicações. Além disso, a rede constitui uma vigilância rápida para identificações de vírus influenza emergentes com potencial epidêmico ou pandêmico.Esta vigilância é viabilizada pelos resultados dos testes laboratoriais que são ferramentas importantes para a Saúde Pública, sendo fundamentais para a contenção e prevenção dos vírus circulantes. O objetivo deste estudo foi apresentar informações relacionadas ao vírus influenza e a doença, como são realizados o diagnóstico e monitoramento pelas redes de vigilâncias mundiais pós-pandemia e, ainda, quais os novos desafios que se apresentam.

Influenza viruses are responsible for annual epidemics with patients presenting variable degrees of diseases everity. These virus can cause acute respiratory infection with a high transmissibility due to its high genetic variability, adaptability and rapid spread. Influenza viruses have fragmented genome which causes frequent antigenic variation, which can result in more virulent subtypes emergence, as occurred in 2009 when it was described a new pandemic influenza virus H1N1. WHO estimates that flu affects 5-15% of the population and it causes 3 to 5 million of severe cases and 250.000 to 500.000 deaths annually. The annual influenza epidemics and the new pandemics risk high lights the importance of Influenza virus epidemiological monitoring. Based in this concern WHO created and coordinates the Global Influenza Surveillance and Response System in order to provide necessary information for viral variants selection that will be part of vaccine annual composition, since that, vaccination is one of the most effective measures for influenza prevention and its complications. In addition, the network is a rapid surveillance of emerging influenza virus identifications with potential to cause epidemic or pandemic situations. The surveillance isenable due to laboratory tests results which are important tools for public health, with essential role for circulating viruses containment and prevention. The aim of this study was to present information related to influenza virus and flu disease, how the diagnosis and monitoring are performed by global surveillance networks at post pandemic time and, also,the new challenges facing.

Viral lung infections: epidemiology, virology, clinical features, and management of avian influenza A(H7N9).

PURPOSE OF REVIEW: The avian influenza A(H7N9) virus has jumped species barrier and caused severe human infections. Here, we present the virological features relevant to clinical practice, and summarize the epidemiology, clinical findings, diagnosis, treatment, and preventive strategies of A(H7N9) infection. RECENT FINDINGS: As of 18 February 2014, A(H7N9) virus has caused 354 infections in mainland China, Taiwan, and Hong Kong with a case-fatality rate of 32%. Elderly men were most affected. Most patients acquired the infection from direct contact with poultry or from a contaminated environment, although person-to-person transmission has likely occurred. A(H7N9) infection has usually presented with severe pneumonia, often complicated by acute respiratory distress syndrome and multiorgan failure. Mild infections have been reported in children and young adults. Nasopharyngeal aspirate and sputum samples should be collected for diagnosis, preferably using reverse transcriptase-PCR. Early treatment with neuraminidase inhibitors improved survival, but the efficacy of antivirals was hampered by resistant mutants. The closure of live poultry markets in affected areas has significantly contributed to the decline in the incidence of human cases. SUMMARY: The emergence of A(H7N9) virus represents a significant health threat. High vigilance is necessary so that appropriate treatment can be instituted for the patient and preventive measures can be implemented.

Indium-tin-oxide thin film transistor biosensors for label-free detection of avian influenza virus H5N1.

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.

Saving resources: avian influenza surveillance using pooled swab samples and reduced reaction volumes in real-time RT-PCR.

The occurrence of highly pathogenic (HP) avian influenza (AI) H5N1 in Asia and its spread to Africa and Europe prompted costly monitoring programs of wild birds and domestic poultry. AI virus excretion is tested by examining avian swab samples by real-time reverse transcription PCR (RT-qPCR). In this study, pools of swab samples and a reagents volume reduction per RT-qPCR were evaluated as measures of economization. Viral transport medium and faecal matrices were spiked with different low pathogenic AI virus strains and tested for loss of target RNA during all processing steps as individual rayon swabs or in sample pools of 5, 10 and 15 swabs. Fresh faeces from Mallard ducks and other aquatic bird species as sample matrix resulted in loss of AIV RNA of about 90% compared to transport medium. Due to sample RNA dilution in pools the likelihood of detection of single positive samples is decreasing with increasing size of sample pools. However, pools of five samples containing only one positive sample consistently gave positive results. Similarly, no differences in detection rates were obtained when analyzing 1030 wild bird swab samples either individually or in pools of five. Reducing the reaction volume of influenza A virus generic as well as of subtype-specific RT-qPCRs to 12.5 µl (2.5 µl template) instead of 25 µl did not adversely affect the limit of detection of these RT-qPCRs. A significant economic benefit without impeding detection efficacy can be achieved when sample pools of five samples are analyzed by RT-qPCR using a reduction of the reaction mix to the half of the original volume.

Development and preliminary application of an indirect ELISA for detecting antibodies against Avian Influenza Virus.

Fragment of 759 bp DNA spanning the Matrix 1 (M1) gene of Avian Influenza Virus (AIV) was inserted into an expression vector pET28c to construct a recombinant plasmid pET28c-M1. The pET28c-M1 plasmid was transformed into the Escherichia coli BL21 (DE3) competent cell to produce a recombinant strain E. coli 21 (DE3). After being induced by Isopropyl-b-D-galactopyranoside (IPTG), E. coli 21 (DE3) expressed a 28-kDa fusion protein at a high level. This protein can bind anti-AIV (H5N1) positive serum by Western-blot analysis. After being denatured, renatured, and purified by Ni(2+)-column, the fusion protein was used as an antigen to develop Matrix 1 Enzyme-Linked Immunosorbent Assay (M1-ELISA) for detecting antibodies against AIV from chicken serum. We found that this indirect M1-ELISA was sensitive for differentiating antisera against AIV and antisera against other six kinds of avian viruses apart from AIV and this method is more sensitive than Hemagglutination Inhibition (HI) test. When compared with HI test and ELISA (IDEXX) in evaluating 581 serum samples from field vaccinated chickens, this assay showed 93.3% agreement ratio with the HI test, as well as 96.0% agreement ratio with ELISA (IDEXX). In a preliminary application, the assay successfully detected 19 AIVs from 51 nonvaccinated chicken lungs. It concludes that an indirect ELISA was successfully developed for detecting AIV. The assay is specific and sensitive. The application will greatly contribute to the long-term prevention and control of avian influenza in China.

Risk assessment to estimate the probability of a chicken flock infected with H5N1 highly pathogenic avian influenza virus reaching slaughter undetected.

Highly pathogenic avian influenza (HPAI) H5N1 is an infectious disease of fowl that can cause rapid and pervasive mortality resulting in complete flock loss. It has also been shown to cause death in humans. Although H5N1 HPAI virus (HPAIV) has not been identified in the United States, there are concerns about whether an infected flock could remain undetected long enough to pose a risk to consumers. This paper considers exposure from an Asian lineage H5N1 HPAIV-infected chicken flock given that no other flocks have been identified as H5N1 HPAIV positive (the index flock). A state-transition model is used to evaluate the probability of an infected flock remaining undetected until slaughter. This model describes three possible states within the flock: susceptible, infected, and dead, and the transition probabilities that predict movements between the possible states. Assuming a 20,000-bird house with 1 bird initially infected, the probability that an H5N1 HPAIV-infected flock would be detected before slaughter is approximately 94%. This is because H5N1 HPAIV spreads rapidly through a flock, and bird mortality quickly reaches high levels. It is assumed that approximately 2% or greater bird mortality due to H5N1 HPAIV would result in on-farm identification of the flock as infected. The only infected flock likely to reach slaughter undetected is one that was infected within approximately 3.5 days of shipment. In this situation, there is not enough time for high mortality to present. These results suggest that the probability of an infected undetected flock going to slaughter is low, yet such an event could occur if a flock is infected at the most opportune time.

Diagnosis and strain differentiation of avian influenza viruses by restriction fragment mass analysis.

Outbreaks of highly pathogenic avian influenza (HPAI) among poultry as well as wild birds are of continuing major public concern, not only because of high economical losses but also due to lethal infections in humans. Control of the infection relies on rapid detection and identification of the causative virus strain which is carried out currently primarily by real-time RT-PCR and DNA sequencing. In a pandemic, however, the analysis of very large numbers of samples may become necessary within a short period. A method is described for the characterisation of avian influenza virus (AIV) subtypes by restriction fragment mass fingerprint (RFMF) analysis. Amplified genomic fragments encoding the pathogenicity-determining region of the hemagglutinin gene were digested with a cocktail of restriction enzymes, and the restriction fragments were assayed by mass spectrometry. Characteristic spectra with sequence coverage ranging from 75 to 100% were obtained for a panel of 27 isolates representing 18 relevant serotypes. Three marker masses were identified that are highly specific for strains of the H5N1 virus. Within the H5N1 serotype, discrimination of individual strains was possible by detailed evaluation of the spectra. The procedure described is rapid, inexpensive and compatible with automation.

Rapid detection of H5 avian influenza virus by TaqMan-MGB real-time RT-PCR.

AIMS: Real-time reverse transcription-polymerase chain reaction (RT-PCR) assay based on a TaqMan-minor groove binder (MGB) probe was developed for the rapid detection of avian influenza virus subtype H5. METHODS AND RESULTS: Conserved regions in the haemagglutinin genes of avian influenza viruses subtype H5 served as targets for the primers and TaqMan-MGB probe design. Concentrations of primers and probe were optimized to improve the sensitivity and specificity of the reactions. A plasmid containing the haemagglutinin gene was constructed and in vitro transcribed for a quantitative assay of copy numbers of the target gene. The results revealed that the optimal concentration of primers and probe was 640 and 480 nmol l(-1), respectively. The threshold of 100 copies of target molecules could be detected. The linear range for detection was determined as 10(2) to 10(8) molecules in reaction. CONCLUSIONS: It took less than 3 h to complete the detection from viral RNA extraction, with good sensitivity and repeatability. SIGNIFICANCE AND IMPACT OF THE STUDY: Real-time RT-PCR assay with MGB probe was an effective means for quick and quantitative laboratory detection and monitoring of H5 avian influenza viruses.

Preliminary evaluation of diagnostic tests for avian influenza using the Markov Chain Monte Carlo (MCMC) Method in an emergency surveillance.

In June 2005, an outbreak of avian influenza (AI) caused by a low pathogenic H5N2 virus was identified in Japan. A serological surveillance was conducted because the infected chickens did not show any clinical signs. The Markov Chain Monte Carlo Method was used to evaluate the performances of serological HI and AGP tests because there was not enough time when the surveillance was initiated to conduct a test evaluation. The sensitivity of the AGP test (0.67) was lower than that of the HI test (0.99), while the specificities were high for both tests (0.96 for AGP and 0.90 for HI). Based on the low sensitivity of the AGP test, the HI test was used for primary screening in later stages of the epidemic.

Risk assessment applied to Spain's prevention strategy against highly pathogenic avian influenza virus H5N1.

Notifiable avian influenza (NAI) had never been reported in Spain, until July 2006 when a dead Great Crested Grebe (Podiceps cristatus) was found positive to the highly pathogenic H5N1 subtype as part of the active wild bird surveillance plan. The current program of the Spanish Ministry of Agriculture, Fisheries, and Food (MAPA)'s strategic preventive plan against NAI is divided in the following parts: identification of risk areas and risk wild bird species, increased biosecurity measures, early detection of infection with surveillance intensification and development of rapid diagnostic tests, and other policies, which include continuing education and training to ensure early detection of the disease. In 2003 an active surveillance plan was introduced for domestic fowl; the plan was extended to wild birds in 2004. A total of 18,780 samples in poultry and 3687 samples in wild birds had been analyzed through December 2005 to detect the presence and spread of avian influenza subtypes H5 and H7. In the present work we suggest some contributions to be implemented in MAPA's action plan: 1) the identification of risks because of migratory birds, within the risk assessment of the introduction of NAI virus in Spain and 2) an interactive digital simulator of the disease developed for continuing education and training.

The indirect immunofluorescence assay using cardiac tissue from chickens, quails and ducks for identification of influenza A virus during an outbreak of highly pathogenic avian influenza virus (H5N1): a rapid and simple screening tool for limited resource settings.

Here we describe the diagnostic utility of the indirect immunofluorescence assay (IFA) during a recent outbreak of highly pathogenic avian influenza (HPAI) subtype H5N1 virus in southern Thailand and demonstrate the usefulness of the cardiac tissue from infected chickens, quail, and ducks for diagnosis. The most reliable sample for IFA diagnosis of influenza A virus was cardiac tissue (83.0%; 44/53) which when divided by species (chicken, quail and duck cardiac tissues) gave respective positivity rates of 88% (22/25), 88.9% (16/18) and 60.0% (6/10). Cardiac tissue also gave the highest IFA intensity for the three species. We believe that the IFA method has wide applicability in developing countries or remote settings where clinically similar avian diseases with high morbidity and mortality such as Newcastle disease and fowl cholera are common and could be rapidly excluded thereby conserving valuable reference laboratory capacity for true HPAI outbreaks.