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Porcine bocavirus (PBoV), a newly described porcine parvovirus, has received attention because it can be commonly identified in clinically affected pigs including pigs with post-weaning multisystemic wasting syndrome (PWMS) and pigs with diarrhea. In recent years, novel PBoVs have been identified and were classified into three genogroups, but the ability to detect and classify these novel PBoVs is not comprehensive to date. In this study, a multiplex conventional PCR assay for simultaneous detection and grouping of PBoVs was developed by screening combinations of mixed primer pairs followed by optimization of the PCR conditions. This method exclusively amplifies targeted fragments of 531bp from the VP1 gene of PBoV G1, 291bp from the NP1 gene of PBoV G2, and 384bp from the NP1/VP1 gene of PBoV G3. The assay has a detection limit of 1.0×10(3)copies/μL for PBoV G1 4.5×10(3) for PBoV G2 and 3.8×10(3) for PBoV G3 based on testing mixed purified plasmid constructs containing the specific viral target fragments. The performance of the multiplex PCR assay was comparable to that of the single PCRs which used the same primer pairs. Using the newly established multiplex PCR assay, 227 field samples including faeces, serum and tissue samples from pigs were investigated. All three PBoV genogroups were detected in the clinical samples with a detection rate of 1.3%, 2.6% and 12.3%, respectively for PBoV G1, G2 and G3. Additionally, coinfections with two or more PBoV were detected in 1.7% of the samples investigated. These results indicate the multiplex PCR assay is specific, sensitive and rapid, and can be used for the detection and differentiation of single and multiple infections of the three PBoV genogroups in pigs., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Here, we describe a uracil-DNA glycosylase (UNG)-treated reverse transcription loop-mediated isothermal amplification (uRT-LAMP) for the visual detection of all subtypes of avian influenza A virus (AIV). The uRT-LAMP assay can prevent unwanted amplification by carryover contamination of the previously amplified DNA, although the detection limit of the uRT-LAMP assay is 10-fold lower than that of the RT-LAMP without a UNG treatment. To the best of our knowledge, this is the first successful application of deoxyuridine triphosphate/UNG strategy in RT-LAMP for AIV detection, and the assay can be applied for the rapid, and reliable diagnosis of AIVs, even in contaminated samples., Competing Interests: There are no conflicts of interest.

The diagnosis of influenza A virus infections in poultry or wild birds is difficult due to variations in the pathogenicity of the viruses in different avian hosts and also the antigenic and genetic diversity of the virus, particularly the recent H5 highly pathogenic avian influenza viruses. A classical standard laboratory technique is virus isolation prior to subtyping and pathotyping. This diagnostic technique is crucial for further virological analyses, particularly during an initial outbreak; however, delays in diagnosis have thwarted effective disease control in recent years. Recent developments in molecular biological techniques provide an accelerated diagnosis. Such technologies, which include real-time reverse transcriptase PCR, isothermal nucleic acid amplification, next-generation sequencing and immunochromatography, contribute to simpler and more rapid diagnosis. The advantages of each of these diagnostic techniques should be considered for effective control of avian influenza., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In January 2015, a highly pathogenic Eurasian lineage H5N8 avian influenza (AI) virus (AIV) was detected in a commercial meat turkey flock in Stanislaus County, CA. Approximately 3 wk later, a similar case was diagnosed in commercial brown layers from a different company located in Kings County, CA. Five 14-wk-old turkey hens were submitted to the California Animal Health and Food Safety Laboratory System (CAHFS), Turlock, and eleven 12-wk-old chickens were submitted to CAHFS, Tulare laboratory due to an acute increase in flock mortality. Gross lesions included enlarged and mottled pale spleens and pancreas in turkeys and chickens. Histologically, the major lesions observed in turkeys and chickens were splenitis, pancreatitis, encephalitis, and pneumonia. In both cases, diagnosis was based on real-time reverse transcriptase PCR (RRT-PCR), sequencing, and virus isolation from oropharyngeal and cloacal swabs. Confirmatory diagnosis and AIV characterization was done at the National Veterinary Services Laboratory, Ames, IA. The sequence of the AIV from both cases was 99% identical to an H5N8 AI virus (A/gyrfalcon/Washington/41088-6/2014) isolated from a captive gyrfalcon (Falco rusticolus) from Washington State in December 2014. Immunohistochemistry (IHC) performed on various tissues from both cases indicated a widespread AIV tissue distribution. Except for minor variations, the tissue distribution of the AI antigen was similar in the chickens and turkeys. There was positive IHC staining in the brain, spleen, pancreas, larynx, trachea, and lungs in both chickens and turkeys. Hearts, ovaries, and air sacs from the turkeys were also positive for the AI antigen. The liver sections from the chickens had occasional AI-positive staining in mononuclear cells, but the IHC on liver sections from the turkeys were negative. The bursa of Fabricius, small intestine, kidney, and skeletal muscle sections were negative for the AI antigen in both chickens and turkeys.

Repeated interspecies transmission of H9N2 virus from poultry to humans and human infections transmitted via aerosols highlight the need for a highly sensitive, rapid diagnostic system for the detection of this virus. However, no such test exhibiting high performance has been developed. In this study, the performance of a smartphone-based rapid fluorescent diagnostic system (SRFDS) was optimized for the diagnosis of an H9N2-virus-infected animal. To suppress the nonspecific reactivity of the bioconjugate in oropharyngeal (OP) and cloacal (CL) samples derived from chickens, different blocking reagents were tested, and a mixture of casein and sucrose was found to be optimal. To assess the performance of SRFDS, OP and CL samples were obtained from specific-pathogen-free chickens and used for comparison of this method with real-time reverse transcription PCR (rRT-PCR) at time points of three, five, and seven days postinfection (dpi). The limit of detection of SRFDS was found to be 7.5 PFU/mL, which was 138-fold higher than that of a conventional colloidal-gold-based avian influenza rapid diagnostic test. In the animal study, the presence of viral antigen was monitored with SRFDS, and the relative sensitivity (relative to rRT-PCR results) was 94.44 % (17/18) and 95.23 % (20/21) in OP and CL specimens, respectively. The specificity of SRFDS was 100 %. These results imply that the diagnostic performance of SRFDS might be comparable to that of rRT-PCR for diagnosis of H9N2 in chickens and that this test can be used as a highly sensitive rapid diagnostic method in field studies on broiler poultry and wild birds.

The persistent public health threat of animal to human transmission of influenza A virus (IAV) has stimulated interest in rapid and accurate detection of all IAV subtypes in clinical specimens of animal origin. In this study, a new set of primers and probes was designed for one-step pan-IAV reverse-transcription fluorescence resonance energy transfer (FRET)-PCR. The detection limit of one-step pan-IAV RT FRET-PCR was 10 copies of the matrix gene per reaction, and proved to be equivalent or superior to virus isolation in detecting nine IAV subtypes. Application of the pan-IAV RT FRET-PCR to oral-pharyngeal and cloacal swab specimens collected from healthy poultry in 34 live bird markets in 24 provinces of China revealed that 9.2% of the animals (169/1,839) or 6.3% of their oral-pharyngeal or cloacal swabs (233/3,678) were positive for IAV, and 56.8% of IAV-positive samples were of the H9N2 subtype. Paralleling detection of IAV in H9N2-infected SPF chickens and chickens from LBM showed that pan-IAV FRET-PCR had a higher detection limit than virus isolation in eggs while the results by FRET-PCR and virus isolation overall matched. It is expected that this strategy can be useful for facile surveillance for IAV in clinical samples from a variety of sources.

A side-polished fiber optic surface plasmon resonance (SPR) sensor was fabricated to expose the core surface and then deposited with a 40 nm thin gold film for the near surface sensing of effective refractive index changes with surface concentration or thickness of captured avian influenza virus subtype H6. The detection surface of the SPR optical fiber sensor was prepared through the plasma modification method for binding a self-assembled monolayer of isopropanol chemically on the gold surface of the optical fiber. Subsequently, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide/N-hydroxysuccinimide was activated to enable EB2-B3 monoclonal antibodies to capture A/chicken/Taiwan/2838V/00 (H6N1) through a flow injection system. The detection limit of the fabricated optical fiber sensor for A/chicken/Taiwan/2838V/00 was 5.14 × 10(5) EID50/0.1 mL, and the response time was 10 min on average. Moreover, the fiber optic sensor has the advantages of a compact size and low cost, thus rendering it suitable for online and remote sensing. The results indicated that the optical fiber sensor can be used for epidemiological surveillance and diagnosing of avian influenza subtype H6 rapidly., (Copyright © 2016 Elsevier B.V. All rights reserved.)

This study identified characteristics of poultry farming with a focus on practices that affect the detection of HPAI; and estimated the system sensitivity of passive surveillance for HPAI H5N1 in commercial and backyard chicken farms in Bayelsa-State, Nigeria. Field studies were carried out in Yenegoa and Ogbia local government areas in Bayelsa state. Willingness to report HPAI was highest in commercial poultry farms (13/13) than in Backyard farms (8/13). Poor means of dead bird disposal was common to both commercial and backyard farms. Administering some form of treatment to sick birds without prior consultation with a professional was higher in backyard farms (8/13) than in commercial farms (4/13). Consumption of sick birds was reported in 4/13 backyard farms and sale of dead birds was recorded in one commercial farm. The sensitivity of passive surveillance for HPAI was assessed using scenario tree modelling. A scenario tree model was developed and applied to estimate the sensitivity, i.e. the probability of detecting one or more infected chicken farms in Bayelsa state at different levels of disease prevalence. The model showed a median sensitivity of 100%, 67% and 23% for detecting HPAI by passive surveillance at a disease prevalence of 0.1%, a minimum of 10 and 3 infected poultry farms respectively. Passive surveillance system sensitivity at a design prevalence of 10 infected farms is increasable up to 86% when the disease detection in backyard chicken farms is enhanced., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Rapid and sensitive diagnostic approaches are of the utmost importance for the detection of humans and animals infected by specific influenza virus subtype(s). Cascade-like diagnostics starting with the use of pan-influenza assays and subsequent subtyping devices are normally used. Here, we demonstrated a novel low density array combining 32 TaqMan(®) real-time RT-PCR systems in parallel for the specific detection of the haemagglutinin (HA) and neuraminidase (NA) subtypes of avian and porcine hosts. The sensitivity of the newly developed system was compared with that of the pan-influenza assay, and the specificity of all RT-qPCRs was examined using a broad panel of 404 different influenza A virus isolates representing 45 different subtypes. Furthermore, we analysed the performance of the RT-qPCR assays with diagnostic samples obtained from wild birds and swine. Due to the open format of the array, adaptations to detect newly emerging influenza A virus strains can easily be integrated. The RITA array represents a competitive, fast and sensitive subtyping tool that requires neither new machinery nor additional training of staff in a lab where RT-qPCR is already established.

Highly pathogenic avian influenza (HPAI) virus of the H5N8 subtype was isolated from a young ostrich in South Korea in March 2014. Clinical signs characterized by anorexia, depression, and signs of nervousness were observed. The isolated A/ostrich/Korea/H829/2014 (H5N8) virus had a cleavage site motif containing multiple basic amino acids, typical of HPAI virus. The phylogenetic tree of the hemagglutinin gene of the H5 HPAI virus showed that this ostrich H5N8 virus belongs to clade 2.3.4.4 viruses together with H5N8 strains isolated from ducks and wild birds in South Korea in 2014. Pathologically, redness of pancreas, enlargement and hemorrhage of spleen, friability of brain, and hydropericardium were prominently found. Histologic legions were observed in pancreas, spleen, liver, lung, heart, and brain, and influenza A nucleoproteins were detected in the same organs by immunohistochemistry. Other ostriches farmed together in open camps were not infected with HPAI virus based on the serologic and virologic tests. The findings indicate that ostriches are susceptible to H5N8 HPAI virus, but this virus does not spread efficiently among ratites.

Development of convenient but sensitive method for influenza detection is highly important in immediate and effective clinical treatment. In this study, an ultrasensitive colorimetric approach combining the advantages of the convenience of the enzyme-induced metallization and the high specificity of enzyme-linked immunosorbent assay for the detection of influenza virus A (H7N9 as model) has been developed. Two rounds of amplification are utilized to enhance the detection sensitivity. The amplification of enzymatic reaction combines with the specific optical properties of gold nanoparticles causing the enhancing of the optical signal immensely. In addition, the increased surface area and the magnetic enrichment effect also enable the magnetic bead (MB) to catch a large number of alkaline phosphatase (ALP) and detection antibody (Ab2), thus very small amounts of the virus can be easily detected. Compared with conventional method, this approach exhibits outstanding sensitivity for ALP detection, 0.2U/L of ALP can be distinguished with a spectrometer and 2U/L with the naked eye. And as low as 25 pg/mL of H7N9 can be detected by the naked eye. This approach shows an extensive horizon for bioassays and is available in clinical diagnosis with the advances of simplification, effectiveness, low cost and sensitive readout., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Objectives: In order to develop a multiplex RT-PCR assay using the GeXP analyser for the simultaneous detection of four different NA serotypes of H5-subtype AIVs, effective to control and reduce H5 subtype of avian influenza outbreak., Design: Six pairs of primers were designed using conserved and specific sequences of the AIV subtypes H5, N1, N2, N6 and N8 in GenBank. Each gene-specific primer was fused at the 5' end to a universal sequence to generate six pairs of chimeric primers, and one pair of universal primers was used for RT-PCR, and PCR product separation and detection were performed by capillary electrophoresis using the GenomeLab GeXP genetic analysis system., Setting: Single and mixed avian pathogen cDNA/DNA templates were employed to evaluate the specificity of a multiplex assay with a GeXP analyser. Corresponding specific DNA products were amplified for each gene, revealing amplification peaks for M, H5, N1, N2, N6 and N8 genes from four different NA subtypes of influenza A H5 virus., Sample: A total of 180 cloacal swabs were collected from poultry at live bird markets., Main Outcome Measures: The multiplex PCR assay demonstrated excellent specificity, with each pair of specific primers generating only products corresponding to the target genes and without cross-amplification with other NA-subtype influenza viruses or other avian pathogens. Using various premixed ssRNAs containing known AIV target genes, the detection limit for the multiplex assay was determined to be 10(2) copies/μl. The GeXP assay was further evaluated using 180 clinical specimens and compared with RRT-PCR (real-time reverse transcriptase PCR) and virus isolation., Conclusions: This GeXP analyser-based multiplex assay for four different NA subtypes of H5 HPAI viruses is both highly specific and sensitive and can be used as a rapid and direct diagnostic assay for testing clinical samples., (© 2015 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.)

In this study, we explored the potential utility of the human-focused FilmArray® Respiratory Panel for the diagnosis of a broad range of influenza viruses of veterinary concern as compared with the standard portfolio of recommended TaqMan®-based diagnostic tests. In addition, we discuss some potential operational advantages associated with the use of such integrated sample extraction, amplification and analysis devices in the context of a future long-term, dual-role strategy for the detection of emergency diseases of both human and veterinary concern., (© 2016 Australian Veterinary Association.)

A blocking enzyme-linked immunosorbent assay (bELISA) was developed for detection of antibodies against H9N2 avian influenza viruses (AIVs) based on a monoclonal antibody specific to the hemagglutinin (HA) protein of H9N2 AIV. The specificity of the bELISA was tested using antisera against H3, H4, H5, H7, and H10 AIVs and other avian viruses. The average percent inhibition (PI) value of 116 non-immune serum samples from ducks and specific pathogen-free (SPF) chickens was 2.80%. The selected cut-off PI values for negative and positive sera were 17.6% and 25.0%, respectively. A high correlation of >96% was identified between the bELISA and hemagglutinin inhibition (HI) assay, according to the detection results of sera from infected chickens (n=30) and from chickens vaccinated with an inactivated H9N2 vaccine (n=40). Sera collected from vaccinated chickens and ducks (n=660) at different weeks post vaccination that were positive by the HI assay were confirmed with the bELISA. The results revealed a time-dependent increase in antibody levels. Therefore, the bELISA offers the potential advantage of a high throughput, rapid, sensitive, and specific method for detection of specific antibodies against H9N2., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Wild waterfowl are important reservoir hosts for influenza A virus (IAV) and a potential source of spillover infections in other hosts, including poultry and swine. The emergence of highly pathogenic avian influenza (HPAI) viruses, such as H5N1 and H5N8, and subsequent spread along migratory flyways prompted the initiation of several programs in Europe, North America, and Africa to monitor circulation of HPAI and low-pathogenicity precursor viruses (low-pathogenicity avian influenza [LPAI] viruses). Given the costs of maintaining such programs, it is essential to establish best practice for field methodologies to provide robust data for epidemiological interpretation. Here, we use long-term surveillance data from a single site to evaluate the influence of a number of parameters on virus detection and isolation of LPAI viruses. A total of 26,586 samples (oropharyngeal, fecal, and cloacal) collected from wild mallards were screened by real-time PCR, and positive samples were subjected to isolation in embryonated chicken eggs. The LPAI virus detection rate was influenced by the sample type: cloacal/fecal samples showed a consistently higher detection rate and lower cycle threshold (Ct) value than oropharyngeal samples. Molecular detection was more sensitive than isolation, and virus isolation success was proportional to the number of RNA copies in the sample. Interestingly, for a given Ct value, the isolation success was lower in samples from adult birds than in those from juveniles. Comparing the results of specific real-time reverse transcriptase (RRT)-PCRs and of isolation, it was clear that coinfections were common in the investigated birds. The effects of sample type and detection methods warrant some caution in interpretation of the surveillance data., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

We developed hemagglutinin- and neuraminidase-specific one-step reverse transcription-loop-mediated isothermal amplification assays for detecting the H10N8 virus. The detection limit of the assays was 10 copies of H10N8 virus, and the assays did not amplify nonspecific RNA. The assays can detect H10N8 virus from chicken samples with high sensitivity and specificity, and they can serve as an effective tool for detecting and monitoring H10N8 virus in live poultry markets., (Copyright © 2015, American Society for Microbiology. 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).)

To detect antibody on pen-side is a rapid way to know the avian influenza (AI) infectious status in a chicken flock. The purpose of this study was to develop an immunochromatographic strip (ICS) assay to detect the antibody against the AI virus (AIV) for field applications. The ICS was constructed by fixing an AIV strain A/chicken/Taiwan/2838V/2000 (H6N1) onto a nitrocellulose membrane as the antigen at the test line and goat anti-rabbit IgG antibody at the control line. The colloidal gold conjugated with rabbit anti-chicken IgG was used as the tracer. The present ICS was used to detect antibodies against avian influenza virus in 326 chicken serum samples from the field. Compared with HI, this ICS could detect antibodies against H5 and H6 AIVs. The hemagglutination inhibition (HI) test was used as the standard to evaluate the ICS accuracy. The results showed that the sensitivity and specificity of this ICS reached 95.2% (159/167) and 94.3% (150/159), respectively. The Kappa value of the HI and ICS was 0.896 (P < 0.001). In conclusion, this ICS could be used as a rapid test to detect antibodies against AIVs in the field.

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.

In China, a novel reassortant influenza A (H7N9) virus, which has caused 435 cases of human infection, has recently emerged. Most cases of human infections with the H7N9 virus are known to be associated with a poultry farm and live-poultry markets. In this study, a one-step duplex real-time reverse transcription polymerase chain reaction (RRT-PCR) assay was developed for the simultaneous detection of the hemagglutinin (HA) and neuraminidase (NA) genes of the H7N9 virus for effective surveillance and early diagnosis of cases from clinical samples collected from live-poultry markets or poultry farms. The detection limit of this assay was as low as 0.1 EID50 of H7N9 viruses, which is similar to the detection limit of the real-time RT-PCR assay released by the Word Health Organization. The coefficients of variation (CVs) of both inter-assay and intra-assay reproducibility were less than 1.55 %, showing good reproducibility. No cross-reactivity was observed with RNA of other subtypes of influenza virus or other avian respiratory viruses. The assay can effectively detect H7N9 influenza virus RNA from multiple sources, including chickens, pigeons, ducks, humans, and the environment. Furthermore, the RRT-PCR assay was evaluated with more than 700 clinical samples collected from live-poultry markets and 120 experimentally infected chicken samples. Together, these results indicate that the duplex RRT-PCR assay is a specific, sensitive, and efficient diagnostic method for the epidemiological surveillance and diagnosis of H7N9 virus from different sources, particularly poultry samples.

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.)

Detection of avian influenza viruses (AIVs) is important for diagnosis, surveillance and control of avian influenza which is of great economic and public health significance. Proper transport and storage of samples is critical for the detection when the samples cannot be detected immediately. As recommended by some international or national authoritative entities and some publications, phosphate buffered saline (PBS), PBS-glycerol and brain heart infusion broth (BHIB) are frequently used for transport and storage of the samples collected for detection of AIVs worldwide. In this study, we compared these three media for transport and storage of simulated and authentic swab and feces samples collected for detection of AIVs using virus isolation and reverse transcription-PCR. The results suggest that PBS-glycerol is superior to PBS and BHIB as the sample transport and storage media. The results also suggest that the samples collected for detection of AIVs should be detected as soon as possible because the virus concentration of the samples may decline rapidly during storage within days at 4 or -20°C., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Real-time and label-free detection of antibodies from avian influenza (anti-AI) in an aqueous solution is demonstrated with the use of a nanowire field effect transistor. A real-time measurement system is constructed without leakage paths through the solution medium. The current through the nanowire changes significantly after an injection of an anti-AI solution onto the device, which was previously functionalized by the antigen of AI as a probe of anti-AI. In contrast, no significant response arises when an anti-AI solution is injected onto a non-functionalized device. Therefore, the real-time detection of specific antibody-antigen interaction of the AI is successfully implemented for a chip-based biosensor.

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., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

Since its invention in 1985 the polymerase chain reaction (PCR) has become a well-established method for amplification and detection of segments of double-stranded DNA. Incorporation of fluorogenic probe or DNA intercalating dyes (such as SYBR Green) into the PCR mixture allowed real-time reaction monitoring and extraction of quantitative information (qPCR). Probes with different excitation spectra enable multiplex qPCR of several DNA segments using multi-channel optical detection systems. Here we show multiplex qPCR using an economical EvaGreen-based system with single optical channel detection. Previously reported non quantitative multiplex real-time PCR techniques based on intercalating dyes were conducted once the PCR is completed by performing melting curve analysis (MCA). The technique presented in this paper is both qualitative and quantitative as it provides information about the presence of multiple DNA strands as well as the number of starting copies in the tested sample. Besides important internal control, multiplex qPCR also allows detecting concentrations of more than one DNA strand within the same sample. Detection of the avian influenza virus H7N9 by PCR is a well established method. Multiplex qPCR greatly enhances its specificity as it is capable of distinguishing both haemagglutinin (HA) and neuraminidase (NA) genes as well as their ratio.

Bifunctional magnetic nanobeads (bi-MBs) were fabricated by co-immobilizing target recognition molecules and signal molecules on a magnetic nanobead surface, which were used as both separation and enrichment carriers and signal carriers. The bi-MBs could capture and separate avian influenza A (H7N9) virus (H7N9 AIV) from complex samples efficiently based on the specific reaction between antigen-antibody and their good magnetic response, which simplified sample pretreatment and saved the detection time. Taking advantages of their high surface to volume ratio and rich surface functional groups, multiple alkaline phosphatase (ALP) signal molecules were tethered on the surface of bi-MBs which greatly amplified the detection signal. As an efficient signal amplification strategy, enzyme-induced metallization had been integrated with bi-MBs and anodic stripping voltammetry to construct an ultrasensitive electrochemical immunosensor for H7N9 AIV detection. Under the optimal conditions, the introduction of bi-MBs could amplify the detection signal in about four times compared with the same immunoassay without MBs, and the method showed a wide linear range of 0.01-20 ng/mL with a detection limit of 6.8 pg/mL. The electrochemical immunosensor provides a simple and reliable platform with high sensitivity and selectivity which shows great potential in early diagnosis of diseases., (Copyright © 2015 Elsevier B.V. All rights reserved.)

An integrated reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) microdevice which consists of microbead-assisted RNA purification and RT-LAMP with real-time monitoring by a miniaturized optical detector was demonstrated. The integrated RT-LAMP microdevice includes four reservoirs for a viral RNA sample (purified influenza A viral RNA or lysates), a washing solution (70% ethanol), an elution solution (RNase-free water), and an RT-LAMP cocktail, and two chambers (a waste chamber and an RT-LAMP reaction chamber). The separate reservoirs for a washing solution, an elution solution, and an RT-LAMP cocktail were designed with capillary valves for stable storage. Three influenza A virus strains (A/H1N1, A/H3N2, and A/H5N1) were used for RNA templates, and RT-LAMP primer sets were designed to detect hemagglutinin (HA) and conserved M gene. Sequential sample flow to the microbeads for RNA purification was achieved by centrifugal force with optimization of capillary valves and a siphon channel. Furthermore, the purified RNA solution was successfully isolated from the waste solution by changing the rotational direction, and combined with the RT-LAMP cocktail in the RT-LAMP reaction chamber for target gene amplification. Total process from the sample injection to the result was completed in 47 min. Influenza A H1N1 virus was confirmed on the integrated RT-LAMP microdevice even with 10 copies of viral RNAs, which revealed 10-fold higher sensitivity than that of a conventional RT-PCR. Subtyping and specificity test of influenza A H1N1 viral lysates were also performed and clinical samples were successfully genotyped to confirm influenza A virus on our proposed integrated microdevice., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Previous studies demonstrated that the H3 avian influenza virus (AIV) in China is isolated most frequently from wild birds and live poultry markets. However, there is no subtype-specific real-time polymerase chain reaction (RT-PCR) available for the rapid and highly sensitive identification of H3 AIV. In this study, a TaqMan minor groove binder (MGB) probe and a pair of primers were designed based on a conserved region in the hemagglutinin gene of H3 AIV. These were used to generate an H3-MGB RT-PCR assay that recognizes only H3 AIV. The detection limit of the H3-MGB RT-PCR was 10 copies of DNA per reaction when 10-fold serial dilutions of T-H3HA plasmid were used as the template. This was 1000-times more sensitive than conventional RT-PCR. In experimental samples obtained from oropharyngeal swabs or cloacal swabs, the virus was detected in all ducks using H3-MGB RT-PCR, whereas only one duck tested positive for the virus in oropharyngeal swabs tested using conventional RT-PCR. The H3-MGB RT-PCR assay developed in this study is a sensitive and rapid tool for screening H3 AIV in China., (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.

In January 2014, an outbreak of infection with highly pathogenic avian influenza (HPAI) A(H5N8) virus began on a duck farm in South Korea and spread to other poultry farms nearby. During this outbreak, many sick or dead wild birds were found around habitats frequented by migratory birds. To determine the causes of death, we examined 771 wild bird carcasses and identified HPAI A(H5N8) virus in 167. Gross and histologic lesions were observed in pancreas, lung, brain, and kidney of Baikal teals, bean geese, and whooper swans but not mallard ducks. Such lesions are consistent with lethal HPAI A(H5N8) virus infection. However, some HPAI-positive birds had died of gunshot wounds, peritonitis, or agrochemical poisoning rather than virus infection. These findings suggest that susceptibility to HPAI A(H5N8) virus varies among species of migratory birds and that asymptomatic migratory birds could be carriers of this virus.

Background: A novel reassortant H7N9 influenza A virus has crossed the species barrier from poultry to cause human infections in China in 2013 and 2014. Rapid detection of the novel H7N9 virus is important to detect this virus in poultry and reduce the risk of an epidemic in birds or humans., Findings: In this study, a multiplex real-time RT-PCR (rRT-PCR) assay for rapid detection of H7N9 and other influenza A viruses was developed. To evaluate the assay, various influenza A viruses, other avian respiratory viruses, and 1,070 samples from poultry were tested. Fluorescence signals corresponding to H7 and N9 subtypes were detected only when H7 and N9 subtypes were present, while the fluorescence signal for the influenza A M gene was detected in all specimens with influenza A strains. The fluorescent signal can be detected in dilutions as low as 56 copies per reaction for the H7, N9 and M genes. Intra- and inter-assay variability tests showed a reliable assay. In poultry samples, a comparison of rRT-PCR with virus isolation showed a high level of agreement., Conclusions: The multiplex rRT-PCR assay in this study has good specificity, sensitivity and reproducibility, and will be useful for laboratory surveillance and rapid diagnosis of H7N9 and other influenza A viruses.

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.

Avian influenza (AI) is an infectious disease caused by avian influenza viruses (AIVs) which belong to the influenza virus A group. AI causes tremendous economic losses in poultry industry and pose great threatens to human health. Active serologic surveillance is necessary to prevent and control the spread of AI. In this study, a protein microarray using nucleoprotein (NP) of H5N1 AIV expressed in insect cells was developed to detect antibodies against AIV NP protein. The protein microarray was used to test Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), AIV positive and negative sera. The results indicated that the protein microarray could hybridize specifically with antibodies against AIV with strong signals and without cross-hybridization. Moreover, 76 field serum samples were detected by microarray, enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition test (HI). The positive rate was 92.1% (70/76), 93.4% (71/76) and 89.4% (68/76) by protein microarray, ELISA and HI test, respectively. Compared with ELISA, the microarray showed 100% (20/20) agreement ratio in chicken and 98.2% (55/56) in ornamental bird. In conclusion, this method provides an alternative serological diagnosis for influenza antibody screening and will provide a basis for the development of protein microarrays that can be used to respectively detect antibodies of different AIV subtypes and other pathogens.

A 1:1 matched case-control study was conducted to identify risk factors for avian influenza subtype H9N2 infection on commercial poultry farms in 16 districts of Punjab, and 1 administrative unit of Pakistan. One hundred and thirty-three laboratory confirmed positive case farms were matched on the date of sample submission with 133 negative control farms. The association between a series of farm-level characteristics and the presence or absence of H9N2 was assessed by univariable analysis. Characteristics associated with H9N2 risk that passed the initial screening were included in a multivariable conditional logistic regression model. Manual and automated approaches were used, which produced similar models. Key risk factors from all approaches included selling of eggs/birds directly to live bird retail stalls, being near case/infected farms, a previous history of infectious bursal disease (IBD) on the farm and having cover on the water storage tanks. The findings of current study are in line with results of many other studies conducted in various countries to identify similar risk factors for AI subtype H9N2 infection. Enhancing protective measures and controlling risks identified in this study could reduce spread of AI subtype H9N2 and other AI viruses between poultry farms in Pakistan.

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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.)

A serological test for detecting N9-specific antibodies may be useful as a DIVA strategy to differentiate vaccinated from infected animals or simply for direct serological detection of infection with N9-subtype virus. The method currently recommended for the detection of antibodies against neuraminidase is neuraminidase inhibition (NI), which is a laborious method using toxic chemicals and has low sensitivity. The present study describes the development and validation of an N9-specific ELISA. Data obtained with this N9 ELISA were compared to those obtained with nucleoprotein-based ELISA, haemagglutination inhibition test using homologous antigen and NI assay. 785 sera from ducks and chickens were used, from flocks previously determined to be AI negative or from experimentally infected or immunized flocks. Sensitivity and specificity were evaluated, and a ROC curve and kappa values, which provide a comparison between methods, were calculated. The results obtained in this study indicate that the N9 based-ELISA is effective in detecting N9-specific antibodies with high specificity and with better sensitivity than the recommended NI method; using data from 177 common sera tested with N9 ELISA and NI assay both compared to NP-based ELISA, their specificity were evaluated at 93.6% and 91.5% respectively, and sensitivity at 90.8% and 39.2% respectively., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Variant high pathogenicity avian influenza (HPAI) H5 viruses have recently emerged as a result of reassortment of the H5 haemagglutinin (HA) gene with different neuraminidase (NA) genes, including NA1, NA2, NA5, NA6 and NA8. These viruses form a newly proposed HA clade 2.3.4.4 (previously provisionally referred to as clade 2.3.4.6), and have been implicated in disease outbreaks in poultry in China, South Korea, Laos, Japan and Vietnam and a human fatality in China. There is real concern that this new clade may be wide spread and not readily identified using existing diagnostic algorithms., Findings: Fluorescent probe based reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) assays were developed to facilitate the identification of novel clade 2.3.4.4 viruses of H5N6 subtype emerging in Asia. Assays were aimed at the haemagglutinin (HA) gene for clade identification and at the NA gene to identify N6. The HA assay employing a minor groove binder (MGB) probe was able to detect and differentiate A/duck/Laos/XBY004/2014(H5N6) and related influenza A(H5N6) virus isolates belonging to the proposed clade 2.3.4.4 from other H5 HPAI viruses. In addition, an Eurasian N6 assay was able to differentiate N6 from other NA subtypes., Conclusions: Laos influenza A(H5N6) virus representative of proposed clade 2.3.4.4, was detected and differentiated from viruses in other H5N1 clades using a clade-specific HA RT-qPCR assay whereas the N6-NA subtype was determined by an Eurasian N6 RT-qPCR assay. Such a clade-specific assay would be of particular value for surveillance and in diagnostic laboratories where sequencing is not readily available.

The recent H5N2 flu epidemic in US Midwest has led to deaths of millions of turkeys and farm bred poultry. While no human infections are reported to date, the rapid mutations in flu viruses can lead to more pathogenic subtypes. We have investigated such possibilities and have shown that H5N4, H5N9 and H5N6 are the most likely candidates for next round of viral reassortments, amongst which H5N9, if reassorted from Asiatic strains, could be highly pathogenic. We discuss here possibilities of anticipatory rational vaccine design based on work done earlier.

Biotechnology-based detection systems and sensors are in use for a wide range of applications in biomedicine, including the diagnostics of viral pathogens. In this review, emerging detection systems and their applicability for diagnostics of viruses, exemplified by the case of avian influenza virus, are discussed. In particular, nano-diagnostic assays presently under development or available as prototype and their potentials for sensitive and rapid virus detection are highlighted.

The goal of this work was to demonstrate the results of the development of the enzyme-linked immunosorbent tests with chemiluminescence detection and colorimetric detection of specific viral antigens and antibodies for identifying the avian influenza and the Newcastle disease viruses: high sensitivity and specificity of the immuno- chemiluminescence assay, which are 10-50 times higher than those of the ELISA colorimetric method. The high effectiveness of the results and the automation of the process of laboratory testing (using a luminometer) allow these methods to be recommended for including in primary screening tests for these infectious diseases.

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.

We have developed a simple and sensitive assay for the detection of the RNA genetic marker associated with high pathogenicity influenza (HPAI) virus. The assay constituted of an array of Raman label tagged hairpin-DNA immobilized on a surface-enhanced Raman scattering (SERS) active substrate as the molecular sentinel (MS) reporter. Upon incubation of the assay with the target RNA, the structure of the hairpin-DNA probe changed from stem-loop configuration (closed state) to DNA/RNA hybridization configuration (open state) so that the Raman label tag will be physically separated from the SERS substrate and induce a decrease of Raman scattering intensity. A metal film over nanosphere (MFON) substrate was developed with a SERS enhancement of about 1.7 × 10(5). Based on this MS-modified substrate, the SERS signal showed a linear relationship to the target RNA in the range of 0-60 attomoles and the limit of detect is 2.67 attomoles. The non-complementary RNA sequences control was also detected and no spectral response was observed. The sensing process only required a single hybridization step and post-hybridization washing could also be omitted. Given that this ultrasensitive biosensor assay is free of polymerase chain reaction (PCR) amplification, it would be a potential diagnostic tool for point-of-care HPAI virus detection., (Copyright © 2014. Published by Elsevier B.V.)