Field and laboratory investigation of highly pathogenic avian influenza H5N6 and H5N8 in Quang Ninh province, Vietnam, 2020 to 2021.

BACKGROUND: Avian influenza (AI) is a contagious disease that causes illness and death in poultry and humans. High pathogenicity AI (HPAI) H5N6 outbreaks commonly occur in Quang Ninh province bordering China. In June 2021, the first HPAI H5N8 outbreak occurred at a Quang Ninh chicken farm. OBJECTIVES: This study examined the risk factors associated with HPAI H5N6 and H5N8 outbreaks in Quang Ninh. METHODS: A retrospective case-control study was conducted in Quang Ninh from Nov 2021 to Jan 2022. The cases were households with susceptible poultry with two or more clinical signs and tested positive by real-time reverse transcription polymerase chain reaction. The controls were households in the same village as the cases but did not show clinical symptoms of the disease. Logistic regression models were constructed to assess the risk factors associated with HPAI outbreaks at the household level. RESULTS: There were 38 cases with H5N6 clade 2.3.4.4h viruses (n = 35) and H5N8 clade 2.3.4.4b viruses (n = 3). Compared to the 112 controls, raising poultry in uncovered or partially covered ponds (odds ratio [OR], 7.52; 95% confidence interval [CI], 1.44-39.27), poultry traders visiting the farm (OR, 8.66; 95% CI, 2.7-27.69), farms with 50-2,000 birds (OR, 3.00; 95% CI, 1.06-8-51), and farms with ≥ 2,000 birds (OR, 11.35; 95% CI, 3.07-41.94) were significantly associated with HPAI outbreaks. CONCLUSIONS: Combining biosecurity measures, such as restricting visitor entry and vaccination in farms with more than 50 birds, can enhance the control and prevention of HPAI in Quang Ninh and its spread across borders.

Isolation and genetic characteristics of Novel H4N1 Avian Influenza viruses in ChongQing, China.

BACKGROUND: Avian influenza viruses (AIVs) constitute significant zoonotic pathogens encompassing a broad spectrum of subtypes. Notably, the H4 subtype of AIVs has a pronounced ability to shift hosts. The escalating prevalence of the H4 subtype heightens the concern for its zoonotic potential, signaling an urgent need for vigilance. METHODS: During the period from December 2021 to November 2023, we collected AIV-related environmental samples and assessed them using a comprehensive protocol that included nucleic acid testing, gene sequencing, isolation culture, and resequencing. RESULTS: In this study, a total of 934 environmental samples were assessed, revealing a remarkably high detection rate (43.66%, 289/662) of AIV in the live poultry market. Notably, the H4N1 subtype AIV (cs2301) was isolated from the live poultry market and its complete genome sequence was successfully determined. Subsequent analysis revealed that cs2301, resulting from a reassortment event between wild and domesticated waterfowl, exhibits multiple mutations and demonstrates potential for host transfer. CONCLUSIONS: Our research once again demonstrates the significant role of wild and domesticated waterfowl in the reassortment process of avian influenza virus, enriching the research on the H4 subtype of AIV, and emphasizing the importance of proactive monitoring the environment related to avian influenza virus.

Molecular characterization of the whole genome of H9N2 avian influenza virus isolated from Egyptian poultry farms.

H9N2 avian influenza viruses (AIVs) affect both poultry and humans on a global level, and they are especially prevalent in Egypt. In this study, we sequenced the entire genome of AIV H9N2 isolated from chickens in Egypt in 2021, using next-generation sequencing (NGS) technology. Phylogenetic analysis of the resulting sequences showed that the studied strain was generally monophyletic and grouped within the G1 sublineage of the Eurasian lineage. Four segments (polymerase basic 2 [PB2], polymerase basic 1 [PB1], polymerase acidic [PA], and non-structural [NS]) were related to Egyptian genotype II, while the nucleoprotein (NP), neuraminidase (NA), matrix (M), and haemagglutinin (HA) segments were related to Egyptian genotype I. Molecular analysis revealed that HA protein contained amino acid residues (191H and 234L) that suggested a predilection for attaching to human-like receptors. The antigenic sites of HA had two nonsynonymous mutations: V194I at antigenic site A and M40K at antigenic site B. Furthermore, the R403W and S372A mutations, which have been observed in H3N2 and H2N2 strains that caused human pandemics, were found in the NA protein of the detected strain. The internal proteins contained virulence markers: 504V in the PB2 protein, 622G, 436Y, 207K, and 677T in the PB1 protein, 127V, 550L, and 672L in PA protein, and 64F and 69P in the M protein. These results show that the detected strain had undergone intrasubtype reassortment. Furthermore, it contains changes in the viral proteins that make it more likely to be virulent, raising a question about the tendency of AIV H9N2 to become highly pathogenic in the future for both poultry and humans.

Automated quantification of avian influenza virus antigen in different organs.

As immunohistochemistry is valuable for determining tissue and cell tropism of avian influenza viruses (AIV), but time-consuming, an artificial intelligence-based workflow was developed to automate the AIV antigen quantification. Organ samples from experimental AIV infections including brain, heart, lung and spleen on one slide, and liver and kidney on another slide were stained for influenza A-matrixprotein and analyzed with QuPath: Random trees algorithms were trained to identify the organs on each slide, followed by threshold-based quantification of the immunoreactive area. The algorithms were trained and tested on two different slide sets, then retrained on both and validated on a third set. Except for the kidney, the best algorithms for organ selection correctly identified the largest proportion of the organ area. For most organs, the immunoreactive area assessed following organ selection was significantly and positively correlated to a manually assessed semiquantitative score. In the validation set, intravenously infected chickens showed a generally higher percentage of immunoreactive area than chickens infected oculonasally. Variability between the slide sets and a similar tissue texture of some organs limited the ability of the algorithms to select certain organs. Generally, suitable correlations of the immunoreactivity data results were achieved, facilitating high-throughput analysis of AIV tissue tropism.

Mammalian adaptation risk in HPAI H5N8: a comprehensive model bridging experimental data with mathematical insights.

Understanding the mammalian pathogenesis and interspecies transmission of HPAI H5N8 virus hinges on mapping its adaptive markers. We used deep sequencing to track these markers over five passages in murine lung tissue. Subsequently, we evaluated the growth, selection, and RNA load of eight recombinant viruses with mammalian adaptive markers. By leveraging an integrated non-linear regression model, we quantitatively determined the influence of these markers on growth, adaptation, and RNA expression in mammalian hosts. Furthermore, our findings revealed that the interplay of these markers can lead to synergistic, additive, or antagonistic effects when combined. The elucidation distance method then transformed these results into distinct values, facilitating the derivation of a risk score for each marker. In vivo tests affirmed the accuracy of scores. As more mutations were incorporated, the overall risk score of virus heightened, and the optimal interplay between markers became essential for risk augmentation. Our study provides a robust model to assess risk from adaptive markers of HPAI H5N8, guiding strategies against future influenza threats.

Evolution of H6N6 viruses in China between 2014 and 2019 involves multiple reassortment events.

H6N6 avian influenza viruses (AIVs) have been widely detected in wild birds, poultry, and even mammals. Recently, H6N6 viruses were reported to be involved in the generation of H5 and H7 subtype viruses. To investigate the emergence, evolutionary pattern, and potential for an epidemic of H6N6 viruses, the complete genomes of 198 H6N6 viruses were analyzed, including 168 H6N6 viruses deposited in the NCBI and GISAID databases from inception to January 2019 and 30 isolates collected from China between November 2014 and January 2019. Using phylogenetic analysis, the 198 strains of H6N6 viruses were identified as 98 genotypes. Molecular clock analysis indicated that the evolution of H6N6 viruses in China was constant and not interrupted by selective pressure. Notably, the laboratory isolates reassorted with six subtype viruses: H6N2, H5N6, H7N9, H5N2, H4N2, and H6N8, resulting in nine novel H6N6 reassortment events. These results suggested that H6N6 viruses can act as an intermediary in the evolution of H5N6, H6N6, and H7N9 viruses. Animal experiments demonstrated that the 10 representative H6N6 viruses showed low pathogenicity in chickens and were capable of infecting mice without prior adaptation. Our findings suggest that H6N6 viruses play an important role in the evolution of AIVs, and it is necessary to continuously monitor and evaluate the potential epidemic of the H6N6 subtype viruses.

Genetic and virological characteristics of a reassortant avian influenza A H6N1 virus isolated from wild birds at a live-bird market in Egypt.

The first detection of a human infection with avian influenza A/H6N1 virus in Taiwan in 2013 has raised concerns about this virus. During our routine surveillance of avian influenza viruses (AIVs) in live-bird markets in Egypt, an H6N1 virus was isolated from a garganey duck and was characterized. Phylogenetic analysis indicated that the Egyptian H6N1 strain A/Garganey/Egypt/20869C/2022(H6N1) has a unique genomic constellation, with gene segments inherited from different subtypes (H5N1, H3N8, H7N3, H6N1, and H10N1) that have been detected previously in AIVs from Egypt and some Eurasian countries. We examined the replication of kinetics of this virus in different mammalian cell lines (A549, MDCK, and Vero cells) and compared its pathogenicity to that of the ancestral H6N1 virus A/Quail/HK/421/2002(H6N1). The Egyptian H6N1 virus replicated efficiently in C57BL/6 mice without prior adaptation and grew faster and reached higher titers than in A549 cells than the ancestral strain. These results show that reassortant H6 AIVs might pose a potential threat to human health and highlight the need to continue surveillance of H6 AIVs circulating in nature.

Worries about bird flu in U.S. cattle intensify.

Farm worker becomes infected as H5N1 appears to spread between dairy cows in five states.

Highly pathogenic avian influenza A(H5N1) virus in a common bottlenose dolphin (Tursiops truncatus) in Florida.

Since late 2021, highly pathogenic avian influenza (HPAI) viruses of A/goose/Guangdong/1/1996 (H5N1) lineage have caused widespread mortality in wild birds and poultry in the United States. Concomitant with the spread of HPAI viruses in birds are increasing numbers of mammalian infections, including wild and captive mesocarnivores and carnivores with central nervous system involvement. Here we report HPAI, A(H5N1) of clade 2.3.4.4b, in a common bottlenose dolphin (Tursiops truncatus) from Florida, United States. Pathological findings include neuronal necrosis and inflammation of the brain and meninges, and quantitative real time RT-PCR reveal the brain carried the highest viral load. Virus isolated from the brain contains a S246N neuraminidase substitution which leads to reduced inhibition by neuraminidase inhibitor oseltamivir. The increased prevalence of A(H5N1) viruses in atypical avian hosts and its cross-species transmission into mammalian species highlights the public health importance of continued disease surveillance and biosecurity protocols.

Importancia de los Centros Nacionales de Gripe en la vigilancia de virus aviares de alta patogenicidad. El momento para One-Healthes ahora / Importance of National Influenza Centers in the surveillance of highly pathogenic avian viruses. The time for One-Health is now

Desde el año 1996 el subtipo de gripe aviar de alta patogenicidad A(H5N1) ha estado casi de forma ininterrumpida causando brotes en aves salvajes y domésticas, además de casos en seres humanos con una mortalidad cercana al 50%. Sin embargo, los años de mayor circulación han sido precisamente los años posteriores a la pandemia de COVID-19, en los que se han registrado diversos casos en humanos en lugares donde nunca antes habían aparecido, además de múltiples casos en mamíferos salvajes, domésticos y peri domésticos, que entrañan cierta preocupación por el riesgo que puede suponer para el salto del virus al ser humano través de cadenas de transmisión de mayor o menor extensión. El brote actual de A(H5N1) nos muestra que el concepto One-Health debe estar más vivo que nunca para aunar esfuerzos entre profesionales de diferentes sectores de la sanidad humana, animal y medio ambiental para evitar o minimizar estos riesgos, de tal forma que los laboratorios de referencia como los Centros Nacionales de Gripe dispongan de los medios humanos y materiales para ofrecerinformación rápida y relevante en el menor tiempo posible antes emergencias de este tipo. Las herramientas de diagnóstico y seguimiento que se deben utilizar en estos casos deben estar disponibles para cualquier eventualidad, y llegar más allá de los datos básicos debe ser una premisa indispensable para poder hacer un seguimiento pormenorizado que sirva para acotar brotes, limitar la difusión de la enfermedad, y ayudar al diseño de futuras vacunas pandémicas frente a virus aviares. (AU)

Since 1996, the highly pathogenic avian influenza subtype A(H5N1) has been causing almost uninterrupted outbreaks in wild and domestic birds, as well as cases in humans with a mortality rate close to 50%. However, the years of greatest circulation have been precisely the years following the COVID-19 pandemic, in which several cases have been recorded in humans in places where they had never appeared before, in addition to multiple cases in wild, domestic and peri-domestic mammals, which raise some concern about the risk that the virus may jump to humans through chains of transmission of greater or lesser extent. The current outbreak of A(H5N1) shows us that the One-Health concept should be more alive than ever to join efforts between professionals from different sectors of human, animal and environmental health to avoid or minimize these risks, so that reference laboratories such as the National Influenza Centers have the human and material resources to provide rapid and relevant information in the shortest possible time before emergencies of this type. The diagnostic and monitoring tools to be used in these cases must be available for any eventuality, and going beyond the basic data must be an indispensable premise to be able to carry out a detailed monitoring that serves to limit outbreaks, limit the spread of the disease, and help in the design of future pandemic vaccines against avian viruses. (AU)

Cross-Species Transmission Potential of H4 Avian Influenza Viruses in China: Epidemiological and Evolutionary Study.

H4 avian influenza viruses (AIVs) have been widely detected in live poultry markets in China. However, the potential public health impact of H4 AIVs remains largely uncertain. Here, we fully analyzed the distribution and phylogenetic relationship of H4 AIVs in China. We obtained 31 isolates of H4 viruses in China during 2009-2022 through surveillance in poultry-associated environments, such as live poultry markets and poultry farms. Genomic sequence analysis together with publicly available data revealed that frequent reassortment and introduction of H4 AIV from wild birds to poultry may have occurred. We identified 62 genotypes among 127 whole genome sequences of H4 viruses in China, indicating that H4 AIVs had great genetic diversity in China. We also investigated molecular markers and found that drug resistance mutations frequently occurred in the M2 protein and a few mutations related to receptor binding and the host signature in H4 AIVs. Our study demonstrates the cross-species transmission potential of H4 AIVs in China and provides some reference significance for its risk assessment.

Genetics of H5N1 and H5N8 High-Pathogenicity Avian Influenza Viruses Isolated in Japan in Winter 2021-2022.

In winter 2021-2022, H5N1 and H5N8 high-pathogenicity avian influenza (HPAI) viruses (HPAIVs) caused serious outbreaks in Japan: 25 outbreaks of HPAI at poultry farms and 107 cases in wild birds or in the environment. Phylogenetic analyses divided H5 HPAIVs isolated in Japan in the winter of 2021-2022 into three groups-G2a, G2b, and G2d-which were disseminated at different locations and times. Full-genome sequencing analyses of these HPAIVs revealed a strong relationship of multiple genes between Japan and Siberia, suggesting that they arose from reassortment events with avian influenza viruses (AIVs) in Siberia. The results emphasize the complex of dissemination and reassortment events with the movement of migratory birds, and the importance of continual monitoring of AIVs in Japan and Siberia for early alerts to the intrusion of HPAIVs.

Insights from Avian Influenza: A Review of Its Multifaceted Nature and Future Pandemic Preparedness.

Avian influenza viruses (AIVs) have posed a significant pandemic threat since their discovery. This review mainly focuses on the epidemiology, virology, pathogenesis, and treatments of avian influenza viruses. We delve into the global spread, past pandemics, clinical symptoms, severity, and immune response related to AIVs. The review also discusses various control measures, including antiviral drugs, vaccines, and potential future directions in influenza treatment and prevention. Lastly, by summarizing the insights from previous pandemic control, this review aims to direct effective strategies for managing future influenza pandemics.

Avian H6 Influenza Viruses in Vietnamese Live Bird Markets during 2018-2021.

Avian influenza viruses of the H6 subtype are prevalent in wild ducks and likely play an important role in the ecology of influenza viruses through reassortment with other avian influenza viruses. Yet, only 152 Vietnamese H6 virus sequences were available in GISAID (Global Initiative on Sharing All Influenza Data) prior to this study with the most recent sequences being from 2018. Through surveillance in Vietnamese live bird markets from 2018 to 2021, we identified 287 samples containing one or several H6 viruses and other influenza A virus subtypes, demonstrating a high rate of co-infections among birds in Vietnamese live bird markets. For the 132 H6 samples with unique influenza virus sequences, we conducted phylogenetic and genetic analyses. Most of the H6 viruses were similar to each other and closely related to other H6 viruses; however, signs of reassortment with other avian influenza viruses were evident. At the genetic level, the Vietnamese H6 viruses characterized in our study encode a single basic amino acid at the HA cleavage site, consistent with low pathogenicity in poultry. The Vietnamese H6 viruses analyzed here possess an amino acid motif in HA that confers binding to both avian- and human-type receptors on host cells, consistent with their ability to infect mammals. The frequent detection of H6 viruses in Vietnamese live bird markets, the high rate of co-infections of birds with different influenza viruses, and the dual receptor-binding specificity of these viruses warrant their close monitoring for potential infection and spread among mammals.

Seroprevalence of Avian Influenza A(H5N6) Virus Infection, Guangdong Province, China, 2022.

In 2022, we assessed avian influenza A virus subtype H5N6 seroprevalence among the general population in Guangdong Province, China, amid rising numbers of human infections. Among the tested samples, we found 1 to be seropositive, suggesting that the virus poses a low but present risk to the general population.