Molecular diagnostics of Avian influenza virus

The success of supervizing an infectious disease depends on the ability for speedy detection and characterization of the cause and the forming of a corresponding system for examining the success of control implemented in order to prevent a recurrence of the disease. Since influenza viruses continue to circle, causing significant morbidity and mortality both among the human population and among animals all over the world, it is essential to secure the timely identification and monitoring of the strains that are in circulation. The speedy detection and characterization of new highly-virulent varieties is one of the priorities of the World Health Organization monitoring network. The implementation of molecular methods has an increasingly significant role in diagnostics and the monitoring of the influenza virus. Among a large number of molecular methods, the one particularly in use is the reverse transcription-polimerase chain reaction (PT-PCR). Technological progress in the area of the conducting of molecular methods has enabled that we can prove, in one day, using the RT-PCR method even very small quantities of the infective agent in a sample. In an obtained PCR product, we can relatively easily establish the nucleotide sequence, a detailed analysis and molecular epidemiology of the circulating strains. The molecular diagnostics procedure (RT-PCR) is based on the correct choice or designing of primers depending on the desired knowledge. In order to obtain a specific diagnosis of influenza A, B or C, primers are used which multiply internal genes, such as the nucleoprotein (NP) or matrix gene (M), because these are genes that are highly conserved among the virus types. In the event that we are interested in the subtype of influenza A, after obtaining a positive reaction, primers for genes of surface antigens are selected, such as hemagglutinin. Following the correct detection of the H subtype, it is possible to establish the virus virulence through the direct sequencing of the PCR product. The possibility of typization using molecular methods is based on the big difference at the amino acid and nucleotide levels between different HA subtypes (from 20- 74%), while the differences between strains of the same HA subtype are relatively small (0- 9%). The basic advantage in the detection and typization of influenza viruses using the RTPCR method is that it saves time. Namely, it can be performed directly from the samples taken in the field, and the result can be obtained within the same day, contrary to conventional methods that take 7 to 10 days. The obtained PCR product can also be sequenced immediately, which can provide an answer to the possible virulent potential of the isolate and its further spreading. The establishment of changes in the HA gene sequence can provide us with the information about the direction of the development of the genetic drift. The paper will describe in detail the possibilities for the implementation of molecular methods in diagnostics and typization, in fact, in the molecular epizootiology of avian influenza.

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Characterization of H9N2 influenza A viruses isolated from chicken products imported into Japan from China

Epidemiology and Infection ◽

10.1017/s0950268806006728 ◽

2006 ◽

Vol 135 (3) ◽

pp. 386-391 ◽

Cited By ~ 13

Author(s):

M. MASE ◽

M. ETO ◽

K. IMAI ◽

K. TSUKAMOTO ◽

S. YAMAGUCHI

Keyword(s):

Influenza Virus ◽

Amino Acid ◽

Avian Influenza ◽

Avian Influenza Virus ◽

Influenza A ◽

H9n2 Virus ◽

Amino Acid Substitutions ◽

Influenza A Viruses ◽

Potential Sources

We characterized eleven H9N2 influenza A viruses isolated from chicken products imported from China. Genetically they were classified into six distinct genotypes, including five already known genotypes and one novel genotype. This suggested that such multiple genotypes of the H9N2 virus have possibly already become widespread and endemic in China. Two isolates have amino-acid substitutions that confer resistance to amantadine in the M2 region, and this supported the evidence that this mutation might be a result of the wide application of amantadine for avian influenza treatment in China. These findings emphasize the importance of surveillance for avian influenza virus in this region, and of quarantining imported chicken products as potential sources for the introduction of influenza virus.

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Avian influenza and Newcastle disease virusindead chickens in markets in Dhaka, Bangladesh in 2011-2012

Bangladesh Veterinarian ◽

10.3329/bvet.v33i1.33308 ◽

2017 ◽

Vol 33 (1) ◽

pp. 8-15

Author(s):

LR Barman ◽

RD Sarker ◽

BC Das ◽

EH Chowdhury ◽

PM Das ◽

...

Keyword(s):

Influenza Virus ◽

Avian Influenza ◽

Newcastle Disease ◽

Rapid Test ◽

Type A ◽

Antigen Test ◽

Rt Pcr ◽

Test Kit ◽

Live Bird

A virological survey for avian influenza (AI) and Newcastle disease (ND) was conducted in two selected live bird markets (LBMs), namely Kaptan Bazar and Karwan Bazar in Dhaka city, Bangladesh from August 2011 to July 2012. A total of 513 dead chickens were collected. An immune-chromatographic rapid antigen test for Type A influenza virus and both conventional and real time RT-PCR were used for the detection and characterization of AI and ND viruses. All carcasses were first screened by the rapid antigen test kit and 93 were positive for Type A influenza virus. RT-PCR on a representative number of rapid antigen test positive samples (n = 24) confirmed the presence of Type A influenza virus and mostly H5 influenza virus (22 out of 24 tested samples). Influenza rapid test negative samples (n = 420) were subjected to routine necropsy. Heat stress, suffocation and physical injury were the most common cause of mortality (163 cases), followed by ND, suspected to be the cause of 85 deaths. On molecular investigation of these 85 samples, the presence of ND virus was confirmed in 59 and AI virus in 6; 15 were negative for both ND and AI viruses and 5 were unsuitable for investigation. Among the 59 ND confirmed cases 18 also contained AI virus. In summary, out of 513 carcasses 117 (22.81%) contained AI virus and 59 (11.50%) contained ND virus. Eighteen (3.51%) carcasses contained both AI and ND viruses. The findings suggest that both AI and ND should be considered as major threats to the poultry industry.Bangl. vet. 2016. Vol. 33, No. 1, 8-15

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Equine and canine influenza: a review of current events

Animal Health Research Reviews ◽

10.1017/s1466252310000046 ◽

2010 ◽

Vol 11 (1) ◽

pp. 43-51 ◽

Cited By ~ 28

Author(s):

E. Paul J. Gibbs ◽

Tara C. Anderson

Keyword(s):

Influenza Virus ◽

Avian Influenza ◽

Influenza A ◽

One Health ◽

Control Strategies ◽

Close Association ◽

Companion Animals ◽

Emerging Diseases ◽

Influenza Viruses ◽

Canine Influenza

AbstractIn the past decade, the pandemics of highly pathogenic avian influenza H5N1 and the novel H1N1 influenza have both illustrated the potential of influenza viruses to rapidly emerge and spread widely in animals and people. Since both of these viruses are zoonotic, these pandemics have been the driving force behind a renewed commitment by the medical and veterinary professions to practice One World, One Health for the control of infectious diseases. The discovery in 2004 that an equine origin H3N8 influenza virus was the cause of an extensive epidemic of respiratory disease in dogs in the USA came as a surprise; at that time dogs were thought to be refractory to infection with influenza viruses. In 2007, a second emerging canine influenza was confirmed in Korea, but this time the causal virus was an H3N2 avian influenza virus. This review focuses on recent events associated with equine and canine influenza viruses. While these viruses do not appear to be zoonotic, the close association between humans and dogs, and to a lesser extent horses, demands that we develop better surveillance and control strategies for emerging diseases in companion animals within the context of One World, One Health.

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Methods for Molecular Surveillance of Influenza Used in Macedonia

PRILOZI ◽

10.2478/prilozi-2014-0003 ◽

2014 ◽

Vol 35 (2) ◽

pp. 25-30

Author(s):

Golubinka Bosevska ◽

Elizabeta Janceska ◽

Gordana Kuzmanovska ◽

Vladimir Mikik ◽

Nikola Panovski

Keyword(s):

Influenza Virus ◽

Predictive Value ◽

Influenza A ◽

Rna Extraction ◽

Influenza Virus Infection ◽

Laboratory Diagnosis ◽

Influenza Viruses ◽

Rt Pcr ◽

Two Samples ◽

Nose And Throat

AbstractThe aim: To present and compare different Nucleic Acid Testing assays used for laboratory diagnosis of influenza virus infection in our country.Materials and methods: Respiratory samples used were nose and throat swabs. The RNA extraction was performed with a QIAamp viral RNA kit. During the season 2009–2010 the first 25 samples were tested with: conventional gel-based RT-PCR and CDC rtRT-PCR using published specific matrix and HA gene primers and probes for influenza virus typing and subtyping.Results: Of 25 samples tested with conventional RT-PCR7(28%) were positive for influenza A, but negative for A/H1seasonal and A/H3. Retested with rtRT-PCR 9(36%) were positive for influenza A, 8(32%) were positive for A/H1pdm and 1(4%) was A/H3. Two samples positive with rtRT-PCR for influenza A were negative with RT-PCR. The sensitivity of the RT-PCR in comparison with rtRT-PCR is 100% and the specificity is 88.89%. Positive predictive value for RT-PCR is 77.78%, and negative predictive value is 100%. RT-PCR is a four-step and rtRT-PCR a one-step procedure. The turn-around time of RT-PCR is 6 hours and for rtRT-PCR it is 2 hours.Discussion and conclusion: For surveillance purposes nose and throat swabs are the more easy and practical to collect. It was proved that RT-PCR is too laborious, multi-step and time-consuming. The sensitivity of both assays is equal. The specificity of rtRT-PCR is higher. NAT assays for detection of influenza viruses have become an integral component of the surveillance programme in our country. They provide a fast, accurate and sensitive detection of influenza.

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Development of a duplex TaqMan real-time RT-PCR assay for simultaneous detection of newly emerged H5N6 influenza viruses

Virology Journal ◽

10.1186/s12985-019-1229-2 ◽

2019 ◽

Vol 16 (1) ◽

Cited By ~ 1

Author(s):

Lin Liu ◽

Ying Zhang ◽

Pengfei Cui ◽

Congcong Wang ◽

Xianying Zeng ◽

...

Keyword(s):

Influenza Virus ◽

Avian Influenza ◽

Simultaneous Detection ◽

Reaction System ◽

Taxonomic Status ◽

Human Infection ◽

Influenza Viruses ◽

Rt Pcr ◽

Avian Influenza Viruses ◽

Pcr Assay

Abstract Background In 2017–2018, a new highly pathogenic H5N6 avian influenza virus (AIV) variant appeared in poultry and wild birds in Asian and European countries and caused multiple outbreaks. These variant strains are different from the H5N6 virus associated with human infection in previous years, and their genetic taxonomic status and antigenicity have changed. Therefore, revision of the primers and probes of fluorescent RT-PCR is important to detect the new H5N6 subtype AIV in poultry and reduce the risk of an epidemic in birds or humans. Methods In this study, the primers and probes including three groups of HA and four groups of NA for H5N6 influenza virus were evaluated. Then a set of ideal primer and probes were selected to further optimize the reaction system and established a method of double rRT-PCR assay. The specificity of this method was determined by using H1~H16 subtype AIV. Results The results showed that fluorescence signals were obtained for H5 virus in FAM channel and N6 virus in VIC channel, and no fluorescent signal was observed in other subtypes of avian influenza viruses. The detection limit of this assay was 69 copies for H5 and 83 copies for N6 gene. And, the variability tests of intra- and inter-assay showed excellent reproducibility. Moreover, this assay showed 100% agreement with virus isolation method in detecting samples from poultry. Conclusion The duplex rRT-PCR assay presented here has high specificity, sensitivity and reproducibility, and can be used for laboratory surveillance and rapid diagnosis of newly emerged H5N6 subtype avian influenza viruses.

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International Laboratory Comparison of Influenza Microneutralization Assays for A(H1N1)pdm09, A(H3N2), and A(H5N1) Influenza Viruses by CONSISE

Clinical and Vaccine Immunology ◽

10.1128/cvi.00278-15 ◽

2015 ◽

Vol 22 (8) ◽

pp. 957-964 ◽

Cited By ~ 28

Author(s):

Karen L. Laurie ◽

Othmar G. Engelhardt ◽

John Wood ◽

Alan Heath ◽

Jacqueline M. Katz ◽

...

Keyword(s):

Influenza Virus ◽

Incubation Time ◽

Influenza A ◽

Influenza Viruses ◽

Enzyme Linked Immunosorbent Assay ◽

Influenza A Viruses ◽

H5n1 Influenza ◽

The World ◽

International Laboratory ◽

The Impact

ABSTRACTThe microneutralization assay is commonly used to detect antibodies to influenza virus, and multiple protocols are used worldwide. These protocols differ in the incubation time of the assay as well as in the order of specific steps, and even within protocols there are often further adjustments in individual laboratories. The impact these protocol variations have on influenza serology data is unclear. Thus, a laboratory comparison of the 2-day enzyme-linked immunosorbent assay (ELISA) and 3-day hemagglutination (HA) microneutralization (MN) protocols, using A(H1N1)pdm09, A(H3N2), and A(H5N1) viruses, was performed by the CONSISE Laboratory Working Group. Individual laboratories performed both assay protocols, on multiple occasions, using different serum panels. Thirteen laboratories from around the world participated. Within each laboratory, serum sample titers for the different assay protocols were compared between assays to determine the sensitivity of each assay and were compared between replicates to assess the reproducibility of each protocol for each laboratory. There was good correlation of the results obtained using the two assay protocols in most laboratories, indicating that these assays may be interchangeable for detecting antibodies to the influenza A viruses included in this study. Importantly, participating laboratories have aligned their methodologies to the CONSISE consensus 2-day ELISA and 3-day HA MN assay protocols to enable better correlation of these assays in the future.

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Detection of avian influenza virus and newcastle disease virus by duplex one step RT PCR

Open Life Sciences ◽

10.2478/s11535-013-0164-7 ◽

2013 ◽

Vol 8 (6) ◽

pp. 520-526 ◽

Cited By ~ 1

Author(s):

Dawid Nidzworski ◽

Krzysztof Smietanka ◽

Zenon Minta ◽

Bogusław Szewczyk

Keyword(s):

Influenza Virus ◽

Avian Influenza ◽

Newcastle Disease Virus ◽

Disease Virus ◽

Newcastle Disease ◽

Egg Production ◽

Influenza Viruses ◽

Detection Methods ◽

Rt Pcr ◽

One Step

AbstractNewcastle disease Virus (NDV), a member of the Paramyxoviridae family, and Influenza virus, from the Orthomyxoviridae family, are two main avian pathogens that cause serious economic problems in poultry farming. NDV strains are classified into three major pathotypes: velogenic, mesogenic, and lentogenic. Avian influenza viruses (AIV) are also divided into: low pathogenic (LPAI) and highly pathogenic (HPAI) strains. Both viruses are enveloped, single stranded, negative-sense RNA viruses which give similar symptoms ranging from sub-clinical infections to severe disease, including loss in egg production, acute respiratory syndrome, and high mortality, depending on their level of pathogenicity. This similarity hinders diagnosis when based solely on clinical and post mortem examination. Most of the currently available molecular detection methods are also pathogenspecific, so that more than one RT-PCR is then required to confirm or exclude the presence of both pathogens. To overcome this disadvantage, we have applied a One Step Duplex RT-PCR method to distinguish between those two pathogens. The main objective of the project was to develop a universal, fast, and inexpensive method which could be used in any veterinary laboratory.

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Resurrected ancestral influenza A viruses recapitulate the avian-to-mammalian adaptation of European avian-like swine influenza virus

10.21203/rs.3.rs-436907/v1 ◽

2021 ◽

Author(s):

Wen Su ◽

Rhodri Harfoot ◽

Yvonne Su ◽

Jennifer DeBeauchamp ◽

Udayan Joseph ◽

...

Keyword(s):

Influenza Virus ◽

Avian Influenza ◽

Influenza A ◽

Swine Influenza Virus ◽

Swine Influenza ◽

Polymerase Activity ◽

Influenza Viruses ◽

Mammalian Host ◽

Influenza A Viruses ◽

Sequence Reconstruction

Abstract The emergence of a pandemic influenza virus may be better anticipated if we better understand the evolutionary steps taken by avian influenza viruses as they adapt to mammals. We used ancestral sequence reconstruction to resurrect viruses representing initial adaptive stages of the European avian-like H1N1 virus as it transitioned from avian to swine hosts. We demonstrate that efficient transmissibility in pigs was gained through stepwise adaptation after 1983. These time-dependent adaptations resulted in changes in hemagglutinin receptor binding specificity and increased viral polymerase activity. An NP-R351K mutation under strong positive selection increased the transmissibility of a reconstructed virus. The stepwise-adaptation of a wholly avian influenza virus to a mammalian host suggests a window where targeted intervention may have highest impact. Successful intervention will, however, require strategic coordination of surveillance and risk assessment activities to identify these adapting viruses and guide pandemic preparedness resources.

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Detecting influenza and emerging avian influenza virus by influenza and pneumonia surveillance systems in a large city in China, 2005 to 2016

10.21203/rs.2.1751/v1 ◽

2019 ◽

Author(s):

Xiaorong Guo ◽

Dong Yang ◽

Ruchun Liu ◽

Yaman Li ◽

Qingqing Hu ◽

...

Keyword(s):

Influenza Virus ◽

Avian Influenza ◽

Avian Influenza Virus ◽

Influenza A ◽

Influenza Viruses ◽

Surveillance Systems ◽

Avian Influenza Viruses ◽

Influenza A H1n1 ◽

Death Cases ◽

Positive Results

Abstract Background: Detecting avian influenza virus has become an important public health strategy for controlling the emerging infectious disease. This study aimed to analyze the efficiency of two surveillance systems in detecting the emerging avian influenza viruses. Methods: A modified influenza surveillance system (ISS) and a new built pneumonia surveillance system (PSS) have been used to monitor the viruses in Changsha City, China. The ISS is based on monitoring outpatients in two sentinel hospitals to detect mild influenza and avian influenza cases, and PSS is based on monitoring inpatients in all 49 hospitals to detect severe and death influenza cases. Results: During the study period, 3551917 outpatients were monitored by the ISS system, among which 126076 were influenza-like illness (ILI) cases, with the ILI% of 3.55%. Totally, 14913 throat swabs were collected by the ISS system, among which 2016 were tested positive of influenza or avian influenza virus. Among the positive results, 621 were H3N2, 135 were seasonal H1N1, 610 were influenza A/H1N1 (pandemic in 2009), 106 were untyped influenza A, 540 were B, 1 was H5N6, 1 was H7N9, and 2 were H9N2 virus. 5491560 inpatient people were monitored by the PSS system, among which 6.61% (362743/5491560) were pneumonia cases. 10.55% (38260/362743) of reported pneumonia was severe or death cases. 3401 throat swab or lower respiratory tract samples were collected, among which 2094 were tested positive of influenza or avian influenza virus. Among the positive results, 78 were H3N2, 17 were seasonal H1N1, 1871 were influenza A/H1N1, 103 were untyped influenza A, 16 were B, 1 was H5N6, and 8 were H7N9 virus. Of 15 avian influenza cases reported from January, 2005 to September, 2016, 26.7% (4/15) were mild cases detected by the ISS system, while 60.0% (9/15) were severe or death cases detected by the PSS system. Two H5N1 severe cases were missed by the ISS system in January, 2009 when the PSS system was not available. Conclusion: The two systems seem to be of high efficiency in detecting the emerging avian influenza viruses but need to be verified in other cities or countries.

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A Novel Neuraminidase-Dependent Hemagglutinin Cleavage Mechanism Enables the Systemic Spread of an H7N6 Avian Influenza Virus

mBio ◽

10.1128/mbio.02369-19 ◽

2019 ◽

Vol 10 (6) ◽

Cited By ~ 2

Author(s):

Hyeok-il Kwon ◽

Young-Il Kim ◽

Su-Jin Park ◽

Eun-Ha Kim ◽

Semi Kim ◽

...

Keyword(s):

Influenza Virus ◽

Avian Influenza ◽

Influenza A ◽

Systemic Infection ◽

Influenza Viruses ◽

Mallard Duck ◽

Virulence Markers ◽

Systemic Spread ◽

Cleavage Motif

ABSTRACT In this study, we demonstrate a novel mechanism for hemagglutinin (HA) activation in a naturally occurring H7N6 avian influenza A virus strain, A/mallard duck/Korea/6L/2007 (A/Mdk/6L/07). This novel mechanism allows for systemic infection of chickens, ducks, and mice, and A/Mdk/6L/07 can replicate in vitro without exogenous trypsin and exhibits broad tissue tropism in animals despite the presence of a monobasic HA cleavage motif (PEIPKGR/G). The trypsin-independent growth phenotype requires the N6 neuraminidase and the specific recognition of glycine at the P2 position of the HA cleavage motif by a thrombin-like protease. Correspondingly, viral growth is significantly attenuated by the addition of a thrombin-like protease inhibitor (argatroban). These data provide evidence for a previously unrecognized virus replication mechanism and support the hypothesis that thrombin-mediated HA cleavage is an important virulence marker and potential therapeutic target for H7 influenza viruses. IMPORTANCE The identification of virulence markers in influenza viruses underpins risk assessment programs and the development of novel therapeutics. The cleavage of the influenza virus HA is a required step in the viral life cycle, and phenotypic differences in viruses can be caused by changes in this process. Here, we describe a novel mechanism for HA cleavage in an H7N6 influenza virus isolated from a mallard duck. The mechanism requires the N6 protein and full activity of thrombin-like proteases and allows the virus to cause systemic infection in chickens, ducks, and mice. The thrombin-mediated cleavage of HA is thus a novel virulence determinant of avian influenza viruses.

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