Low Pathogenic Avian Influenza Subtype Research Articles

First Report of Low Pathogenic Avian Influenza Subtype H9N2 in African Houbara Bustards (Chlamydotis undulata undulata) and Gamebirds in Morocco: Clinico-Pathological Findings, Molecular Characterization, and Associated Coinfections.

We report in this paper the first detection of low pathogenic avian influenza (LPAI) subtype H9N2 in houbara bustards and in gamebirds in Morocco. Starting in 2019, an increase in mortality rates related to respiratory distress was recorded in these species. Necropsy of the specimens revealed fibrinous sinusitis and tracheitis with intra-bronchial fibrin casts, which are consistent with H9N2 infection in chickens; therefore, implication of the virus in these outbreaks was strongly suspected. Consequently, between January 2020 and June 2023, birds with respiratory signs were necropsied for pathological lesions, tissue samples were examined by histopathology, and samples of trachea, lungs, and cecal tonsils were analyzed using quantitative real-time PCR for the detection of H9N2 virus. In addition, the sequencing of isolates was performed and lastly differential diagnosis with other respiratory pathogens was carried out. During the study period, 93 samples were collected from suspected H9N2 outbreaks, of which 30 tested positive for H9N2 virus: 23 Houbara bustards, 4 partridges, 2 quails, and 1 pheasant. Moreover, sequencing of the HA gene of the virus showed 97.33% nucleotide identity with strains reported previously in broilers in Morocco in 2017 and in 2022. Phylogenetic analysis grouped the Moroccan partridge isolates in the same cluster as viruses isolated in Morocco between 2016 and 2022, Algeria (2017), Burkina Faso (2017), Nigeria (2019), and Togo (2020). Additionally, 10 house sparrows from the premises of these birds were examined for the presence of H9N2 virus, revealing a 30% positivity rate. In conclusion, LPAIV H9N2 is circulating in houbara bustards and gamebirds in Morocco, and house sparrows might be a possible source of the infection. To our knowledge, this is the first report of LPAI H9N2 in the African species of houbara bustards worldwide and in gamebirds in Morocco.

Emerging threats and vaccination strategies of H9N2 viruses in poultry in Indonesia: A review

Avian influenza virus subtype H9N2 was first documented in Indonesia in 2017. It has become prevalent in chickens in many provinces of Indonesia as a result of reassortment in live bird markets. Low pathogenic avian influenza subtype H9N2 virus-infected poultry provides a new direction for the influenza virus. According to the latest research, the Indonesian H9N2 viruses may have developed through antigenic drift into a new genotype, posing a significant hazard to poultry and public health. The latest proof of interspecies transmission proposes that the next human pandemic variant will be the avian influenza virus subtype H9N2. Manipulation and elimination of H9N2 viruses in Indonesia, constant surveillance of viral mutation, and vaccine updates are required to achieve effectiveness. The current review examines should be investigates/assesses/report on the development and evolution of newly identified H9N2 viruses in Indonesia and their vaccination strategy.

Emerging threat and vaccination strategies of H9N2 viruses in poultry in Indonesia: A review.

Avian influenza virus subtype H9N2 was first documented in Indonesia in 2017. It has become prevalent in chickens in many provinces of Indonesia as a result of reassortment in live bird markets. Low pathogenic avian influenza subtype H9N2 virus-infected poultry provides a new direction for influenza virus. According to the latest research, the Indonesian H9N2 viruses may have developed through antigenic drift into new genotype, posing a significant hazard to poultry and public health. The latest proof of interspecies transmission proposes that, the next human pandemic variant will be avian influenza virus subtype H9N2. Manipulation and elimination of H9N2 viruses in Indonesia, constant surveillance of viral mutation, and vaccines updates are required to achieve effectiveness. The current review examines should be investigates/assesses/report on the development and evolution of newly identified H9N2 viruses in Indonesia and their vaccination strategy.

Vaccination with inactivated virus against low pathogenic avian influenza subtype H9N2 does not prevent virus transmission in chickens

H9N2 subtype avian influenza has spread dramatically in China ever since first reported in the 1990s. A national vaccination program for poultry was initiated in 1998. Field isolation data show that the widely used inactivated H9N2 vaccine does not provide effective control of the transmission of this low pathogenic avian influenza (LPAI) virus in poultry. Current research has focused on two reasons: (i) insufficient immune response triggered by the vaccination with the inactivated virus, (ii) the occurrence of escape mutants selected by vaccine-induced immune pressure. However, the lack of effectivity of the inactivated virus vaccine to sufficiently reduce transmission has been noticed. We mimicked the natural infection and transmission process of the H9N2 virus in vaccinated and non-vaccinated chickens. A statistical model was used to estimate the transmission rate parameters among vaccinated chickens, varying in serum hemagglutinin inhibition titers (HIT) and non-vaccinated chickens. We demonstrate, for the first time, that the transmission is not sufficiently reduced by the H9N2 vaccine, even when vaccinated chickens have an IgG serum titer (HIT>23), which is considered protective for vaccination against homologous highly pathogenic avian influenza (HPAI) virus. Our study does, on the other hand, cast new light on virus transmission and immune escape of LPAI H9N2 virus in vaccinated chickens populations, and shows that new mitigation strategies against LPAI viruses in poultry are needed.

Influenza A Virus in Backyard Pigs and Poultry in Rural Cambodia.

Surveillance of influenza virus in humans and livestock is critical, given the worldwide public health threats and livestock production losses. Livestock farming involving close proximity between humans, pigs and poultry is often practised by smallholders in low-income countries and is considered an important driver of influenza virus evolution. This study determined the prevalence and genetic characteristics of influenza A virus (IAV) in backyard pigs and poultry in Cambodia. A total of 751 animals were tested by matrix gene-based rRT-PCR, and influenza virus was detected in 1.5% of sampled pigs, 1.4% of chickens and 1.0% of ducks, but not in pigeons. Full-length genome sequencing confirmed triple reassortant H3N2 in all IAV-positive pigs and various low pathogenic avian influenza subtypes in poultry. Phylogenetic analysis of the swine influenza viruses revealed that these had haemagglutinin and neuraminidase genes originating from human H3N2 viruses previously isolated in South-East Asia. Phylogenetic analysis also revealed that several of the avian influenza subtypes detected were closely related to internal viral genes from highly pathogenic H5N1 and H9N2 formerly sequenced in the region. High sequence homology was likewise found with influenza A viruses circulating in pigs, poultry and wild birds in China and Vietnam, suggesting transboundary introduction and cocirculation of the various influenza subtypes. In conclusion, highly pathogenic subtypes of influenza virus seem rare in backyard poultry, but virus reassortment, involving potentially zoonotic and pandemic subtypes, appears to occur frequently in smallholder pigs and poultry. Increased targeted surveillance and monitoring of influenza circulation on smallholdings would further improve understanding of the transmission dynamics and evolution of influenza viruses in humans, pigs and poultry in the Mekong subregion and could contribute to limit the influenza burden.

Circulation of the low pathogenic avian influenza subtype H5N2 virus in ducks at a live bird market in Ibadan, Nigeria.

BackgroundTo monitor carrier hosts of avian influenza in Nigeria, we randomly collected cloaca swab specimens from 155 ducks at a live bird market (LBM) in Ibadan, southwest Nigeria, between July 2011 and July 2012.MethodsThe samples were analyzed by real-time reverse transcription-polymerase chain reaction (RT-PCR) and virus isolation was carried out in embryonated chicken eggs. Partial sequencing of the antigenic cleavage site of the haemagglutinin (HA) gene was performed, multiple sequence alignment was carried out using ClustalW, and a phylogenetic tree was constructed using the neighbor joining method.ResultsTwenty (13%) of the 155 samples were positive for avian influenza subtype H5N2 by real-time RT-PCR and three isolates were obtained from embryonated chicken eggs. Partial sequencing of the amino acid cleavage site of the HA genes of two isolates corresponded to a PQRETGL*F sequence that is common in low pathogenic avian influenza (LPAI). Phylogenetically, the HA genes of the two influenza viruses are monophyletic and clustered with H5N2 viruses detected in wild ducks from Africa.ConclusionThe occurrence of LPAI in domestic ducks in Nigeria underscores the importance of continuous surveillance and monitoring of the virus (in a country that is considered to be free of avian influenza) in order to prevent the emergence of virulent strains that may spread to commercial poultry and humans.Electronic supplementary materialThe online version of this article (doi:10.1186/2049-9957-3-38) contains supplementary material, which is available to authorized users.

Low pathogenic avian influenza H7N7 in domestic poultry in Germany in 2011

Between May and July 2011, a total of 23 outbreaks of low pathogenic avian influenza (LPAI) subtype H7N7 occurred in poultry farms in Germany. The first cases were detected in...

An outbreak of avian influenza subtype H9N8 among chickens in South Korea

Low pathogenic avian influenza subtype H9N8 was diagnosed on a Korean native chicken farm in Gyeonggi province, South Korea, in late April 2004. Clinical signs included moderate respiratory distress, depression, mild diarrhoea, loss of appetite and a slightly elevated mortality (1.4% in 5 days). Pathologically, mucopurulent tracheitis and air sacculitis were prominently found with urate renal deposition. The isolated A/chicken/Kr/164/04 (H9N8) had an Ala-Ser-Gly-Arg (A/S/G/R) motif at the cleavage site of haemagglutinin, which has been commonly found in H9N2 isolated from Korean poultry. Phylogenetic analysis of the haemagglutinin and neuraminidase genes of the H9N8 avian influenza virus (AIV) isolate showed that reassortment had occurred. Its haemagglutinin gene was similar to that of Korean H9N2 AIVs, but its neuraminidase gene was closely related to that of A/WBF/Kr/KCA16/03 (H3N8) isolated from the faeces of wild birds in Korea. The pathogenicity of the isolate was tested on 6-week-old specific pathogen free chickens. The inoculated virus (H9N8) was recovered from most tested organs, including the trachea, lung, kidney, spleen, and caecal tonsil. This is the first report of an outbreak of low pathogenic avian influenza in chickens caused by AIV subtype H9N8.

Discrimination between Highly Pathogenic and Low Pathogenic H5 Avian Influenza A Viruses

To the Editor: To thoroughly investigate avian influenza outbreaks, identifying highly pathogenic avian influenza (HPAI) and low pathogenic avian influenza (LPAI) is essential. Currently, determination of inserted basic amino acids within the hemagglutinin cleavage site of HPAI relies on nucleotide sequencing (1–3). Direct sequencing is relatively time-consuming and laborious and thus is not suitable for local and regional diagnostic laboratories that receive large numbers of samples that may contain HPAI or LPAI subtype H5N1. Therefore, a rapid diagnostic assay was developed to discriminate between HPAI and LPAI subtype H5 viruses by 1-step real-time reverse transcription–polymerase chain reaction (RT-PCR) with melting curve analysis. H5 primers flanking the cleavage site were designed from conserved regions among HPAI and LPAI strains by using nucleotide sequences obtained from GenBank and our previous studies (4–6). The primers consisted of a forward primer H5F3+ (nucleotides 1001–1021: 5´-AACAGATTAGTCCTTGCGACTG-3´) and a reverse primer H5R2+ (nucleotides 1124–1103: 5´-CATCTACCATTCCCTGCCATCC-3´), which yielded products of ≈124 bp and ≈112 bp, corresponding to HPAI and LPAI, respectively. Consequently, the sizes of the amplicons and percentage guanine-cytosine content were different, allowing discrimination between HPAI and LPAI by melting curve analysis (7). One-step, real-time RT-PCR with melting curve analysis was performed in an ABI 7500 system (Applied Biosystems, Foster City, CA, USA). In each reaction, 3.0 μL of RNA sample was combined with a reaction mixture containing 10 μL 2× SYBR Green PCR Master Mix (Applied Biosystems), 0.5 μL 40× MultiScribe (Applied Biosystems) and RNase inhibitor, each primer (at final concentration of 0.5 μmol/L), 1.5 mmol/L MgCl2, and RNase-free water in a final volume of 20 μL. The thermal profile began with incubation at 48°C for 45 min (reverse transcription), then incubation at 95°C for 10 min (predenaturation), followed by 40 cycles of amplification alternating between 94°C for 15 s (denaturation) and 68°C for 40 s (annealing/extension). The SYBR Green I fluorescent signal was obtained once per cycle at the end of the extension step. After amplification, melting curve analysis was performed by heating the sample to 95°C for 15 s, then cooling it to 70°C for 1 min, followed by a linear temperature increase to 95°C at a rate of 0.5°C/s, while continuously monitoring the fluorescent signal. Data were analyzed by the 7500 System SDS Software version 1.2 (Applied Biosystems). To develop the assay, samples previously identified as influenza A/chicken/Nakorn-Patom/Thailand/CU-K2/04 (H5N1) served as a control for HPAI, and A/duck/Hong Kong/308/78 (H5N3) served as a control for LPAI. The H5 genes (nucleotides 914–1728) of each strain were inserted into pGEM-T Easy Vector and then transcribed in vitro by using RiboMAX Large Scale RNA Production System-T7 (Promega, Madison, WI, USA). Serial 10-fold dilutions of the standard H5 RNA were subjected to a sensitivity test (8). The fluorescent signal can be detected at RNA dilutions as low as 102 copies/μL. To assess the specificity, viral RNA extracted from other subtypes of influenza A viruses (H1–H4 and H6–H15) was tested. The assay was specific for the H5 subtype, since no amplification was detected from other subtypes. Three preliminary melting curve analyses showed that this assay was effective in discriminating between the melting peaks of HPAI and LPAI (Figure). The variations of melting temperature (Tm) between runs were experimentally determined. The mean (standard deviation) of Tm values for HPAI and LPAI were 77.43°C (0.21°C) and 79.57°C (0.23°C), respectively. This assay provided high reliability and reproducibility, since the coefficients of variation were <0.30. Figure Discrimination between highly pathogenic avian influenza (HPAI) and low pathogenic avian influenza (LPAI) by melting curve analysis based on real-time reverse transcription–polymerase chain reaction of the H5 HA gene with SYBR Green I fluorescent ... Seventy-eight specimens of influenza A virus were used to validate the assay. The 75 HPAI samples were isolated during the 2004 outbreak in Thailand; 3 LPAI samples, including A/avian/NY/01 (H5N2), A/Chicken/Mexico/31381-3/94 (H5N2), and A/shoveler/Egypt/03 (H5N2), were provided by the Centers for Disease Control and Prevention. The viruses were isolated in embryonated chicken eggs as described previously (9). RNA was extracted from 140 μL of allantoic fluid, and RT-PCR was performed as described above. Melting curve analysis showed that all H5N1 isolates from the 2004 outbreak in Thailand were interpreted as HPAI, whereas the 3 samples of H5N2 subtype were classified as LPAI. The Tm varied from 77.2°C to 78.1°C for HPAI and 78.75°C to 79.5°C for LPAI. The melting curve analysis results were completely concordant with the results of direct sequence analysis of the H5 gene for all samples tested. The melting curve analysis described here provides a rapid, accurate, and high-throughput approach to discriminate between HPAI and LPAI. This assay is particularly attractive for large-scale screening of suspected subtype H5 influenza A virus during outbreaks to identify candidate LPAI that could be used as vaccine strains.

Detection of highly pathogenic and low pathogenic avian influenza subtype H5 (Eurasian lineage) using NASBA

Nucleic acid sequence-based amplification (NASBA) is a technique that allows the rapid amplification of specific regions of nucleic acid obtained from a diverse range of sources. It is especially suitable for amplifying RNA sequences. A NASBA technique has been developed that allows the detection of avian influenza A subtype H5 from allantoic fluid harvested from inoculated chick embryos. The amplified viral RNA is detected by electrochemiluminescence. The NASBA technique described below is rapid and specific for the identification of influenza A subtype H5 viruses of the Eurasian lineage. More importantly, it can be used to distinguish highly pathogenic and low pathogenic strains of the H5 subtype.