Highly pathogenic avian influenza A (H5N1) virus outbreak in Peru in 2022-2023.

Background: An epizootic of highly pathogenic avian influenza A (H5N1) has spread worldwide since 2022. Even though this virus has been extensively studied for many decades, little is known about its evolution in South America. Methods: Here, we describe the sequencing and characterization of 13 H5N1 genomes collected from wild birds, poultry, and wild mammals in Peru during the genomic surveillance of this outbreak. Results: The samples belonged to the highly pathogenic avian influenza (H5N1) 2.3.4.4b clade. Chilean and Peruvian samples clustered in the same group and therefore share a common ancestor. An analysis of the hemagglutinin and neuraminidase genes detected new mutations, some dependent upon the host type. Conclusions: The genomic surveillance of highly pathogenic avian influenza is necessary to promote the One Health policy and to overcome the new problems entailed by climate change, which may alter the habitats of resident and migratory birds.

Strong and consistent effects of waterbird composition on HPAI H5 occurrences across Europe.

Since 2014, highly pathogenic avian influenza (HPAI) H5 viruses of clade 2.3.4.4 have been dominating the outbreaks across Europe, causing massive deaths among poultry and wild birds. However, the factors shaping these broad-scale outbreak patterns, especially those related to waterbird community composition, remain unclear. In particular, we do not know whether these risk factors differ from those of other H5 clades. Addressing this knowledge gap is important for predicting and preventing future HPAI outbreaks. Using extensive waterbird survey datasets from about 6883 sites, we here explored the effect of waterbird community composition on HPAI H5Nx (clade 2.3.4.4) spatial patterns in the 2016/2017 and 2020/2021 epidemics in Europe, and compared it with the 2005/2006 HPAI H5N1 (clade 2.2) epidemic. We showed that HPAI H5 occurrences in wild birds in the three epidemics were strongly associated with very similar waterbird community attributes, which suggested that, in nature, similar interspecific transmission processes operate between the HPAI H5 subtypes or clades. Importantly, community phylogenetic diversity consistently showed a negative association with H5 occurrence in all three epidemics, suggesting a dilution effect of phylogenetic diversity. In contrast, waterbird community variables showed much weaker associations with HPAI H5Nx occurrence in poultry. Our results demonstrate that models based on previous epidemics can predict future HPAI H5 patterns in wild birds, implying that it is important to include waterbird community factors in future HPAI studies to predict outbreaks and improve surveillance activities.

Genomic characterization of highly pathogenic avian influenza A H5N1 virus newly emerged in dairy cattle.

In March 2024, the emergence of highly pathogenic avian influenza (HPAI) A (H5N1) infections in dairy cattle was detected in the United Sates for the first time. We genetically characterize HPAI viruses from dairy cattle showing an abrupt drop in milk production, as well as from two cats, six wild birds, and one skunk. They share nearly identical genome sequences, forming a new genotype B3.13 within the 2.3.4.4b clade. B3.13 viruses underwent two reassortment events since 2023 and exhibit critical mutations in HA, M1, and NS genes but lack critical mutations in PB2 and PB1 genes, which enhance virulence or adaptation to mammals. The PB2 E627 K mutation in a human case associated with cattle underscores the potential for rapid evolution post infection, highlighting the need for continued surveillance to monitor public health threats.

Design, synthesis and biological evaluation of sulfamethazine derivatives as potent neuraminidase inhibitors.

Aim: The purpose of this study is to design and synthesize a new series of sulfamethazine derivatives as potent neuraminidase inhibitors. Materials & methods: A sulfamethazine lead compound, ZINC670537, was first identified by structure-based virtual screening technique, then some novel inhibitors X1-X10 based on ZINC670537 were designed and synthesized. Results: Compound X3 exerts the most good potency in inhibiting the wild-type H5N1 NA (IC50 = 6.74 µM) and the H274Y mutant NA (IC50 = 21.09 µM). 150-cavity occupation is very important in determining activities of these inhibitors. The sulfamethazine moiety also plays an important role. Conclusion: Compound X3 maybe regard as a good anti-influenza candidate to preform further study.

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Novel Genotypes of Highly Pathogenic Avian Influenza H5N1 Clade 2.3.4.4b Viruses, Germany, November 2023.

Several subtypes and many different genotypes of highly pathogenic avian influenza viruses of subtype H5 clade 2.3.4.4b have repeatedly caused outbreaks in Germany. Four new highly pathogenic avian influenza genotypes emerged in November 2023 after reassortment with low pathogenicity precursors, replacing genotype BB, which had dominated in Europe since 2022.

Construction of a monoclonal molecular imprinted sensor with high affinity for specific recognition of influenza a virus subtype.

Although molecular imprinting technology has been widely used in the construction of virus sensors, it is still a great challenge to identify subtypes viruses specifically because of their high similarity in morphology, size and structure. Here, a monoclonal molecular imprinted polymers (MIPs) sensor for recognition of H5N1 is constructed to permit the accurate distinguishing of H5N1 from other influenza A virus (IAV) subtypes. Firstly, H5N1 are immobilized on magnetic microspheres to produce H5N1-MagNPs, then the high affinity nanogel H5N1-MIPs is prepared by solid phase imprinting technique. When H5N1-MIPs is combined with MagNP-H5N1, different concentrations of H5N1 are added for competitive substitution. The quantitative detection of H5N1 is realized by the change of fluorescence intensity of supernatant. As expected, the constructed sensor shows satisfactory selectivity, and can identify the target virus from highly similar IAV subtypes, such as H1N1, H7N9 and H9N2. The sensor was highly sensitive, with a detection limit of 0.58 fM, and a selectivity factor that is comparable to that of other small MIPs sensors is achieved. In addition, the proposed sensor is cheap, with a cost of only RMB 0.08 yuan. The proposed monoclonal sensor provides a new method for the specific recognition of designated virus subtype, which is expected to be used for large-scale screening and accurate treatment of infected people.

Preventive, safety and control measures against Avian Influenza A(H5N1) in occupationally exposed groups: A scoping review.

Introduction: During the outbreak of avian influenza, A (H5N1) (IA) in wild and domestic birds recorded in January 2023, the epidemiological alert has been extended due to its potential contagion to humans, particularly in those exposed occupational groups. Objective: to identify the primary occupational risk groups, as well as the preventive, safety, and control measures against IA intended or implemented in these positions. Material and methods: A systematic search was conducted in Pubmed, Scopus, Web of science, Scielo and literature databases. Scientific articles, normative documents, and technical reports identifying vulnerable occupational groups and preventive measures against IA were included. Two authors conducted a full-text review, extracting information independently, and findings were summarized narratively. Results: A total of 5518 documents were identified, and 30 reports were included. 20% of the reports were published in 2023, 13/30 were affiliated to a university institution. Occupationally exposed groups were identified both directly and indirectly. 63.3% of reports identified breeders, poultry farmers and sellers as the most concerning occupational group, while 60% identified biosecurity practices (use of PPE, handwashing) as the primary measure against IA, followed by strategies such as education (training and capacity-building). Conclusion: Occupational groups of interest were identified, primarily those involved in sales, commerce, and the handling of bird waste with potential exposure to IA. Furthermore, the maintenance of biosecurity measures, cleaning-disinfection practices, and educational strategies in workplace settings are recommended.

Harnessing high-throughput OMICS in emerging zoonotic virus preparedness and response activities.

Emerging and re-emerging viruses pose unpredictable and significant challenges to global health. Emerging zoonotic infectious diseases, which are transmitted between humans and non-human animals, have been estimated to be responsible for nearly two-thirds of emerging infectious disease events and emergence events attributed to these pathogens have been increasing in frequency with the potential for high global health and economic burdens. In this review we will focus on the application of highthroughput OMICS approaches to emerging zoonotic virus investigtations. We highlight the key contributions of transcriptome and proteome investigations to emerging zoonotic virus preparedness and response activities with a focus on SARS-CoV-2, avian influenza virus subtype H5N1, and Orthoebolavirus investigations.

Detection and characterization of H5N1 HPAIV in environmental samples from a dairy farm.

The recent expansion of HPAIV H5N1 infections in terrestrial mammals in the Americas, most recently including the outbreak in dairy cattle, emphasizes the critical need for better epidemiological monitoring of zoonotic diseases. In this work, we detected, isolated, and characterized the HPAIV H5N1 from environmental swab samples collected from a dairy farm in the state of Kansas, USA. Genomic sequencing of these samples uncovered two distinctive substitutions in the PB2 (E249G) and NS1 (R21Q) genes which are rare and absent in recent 2024 isolates of H5N1 circulating in the mammalian and avian species. Additionally, approximately 1.7% of the sequence reads indicated a PB2 (E627K) substitution, commonly associated with virus adaptation to mammalian hosts. Phylogenetic analyses of the PB2 and NS genes demonstrated more genetic identity between this environmental isolate and the 2024 human isolate (A/Texas/37/2024) of H5N1. Conversely, HA and NA gene analyses revealed a closer relationship between our isolate and those found in other dairy cattle with almost 100% identity, sharing a common phylogenetic subtree. These findings underscore the rapid evolutionary progression of HPAIV H5N1 among dairy cattle and reinforces the need for more epidemiological monitoring which can be done using environmental sampling.

Avian influenza viruses in wild birds in Canada following incursions of highly pathogenic H5N1 virus from Eurasia in 2021-2022.

Following the detection of novel highly pathogenic avian influenza virus (HPAIV) H5N1 clade 2.3.4.4b in Newfoundland, Canada, in late 2021, avian influenza virus (AIV) surveillance in wild birds was scaled up across Canada. Herein, we present the results of Canada's Interagency Surveillance Program for Avian Influenza in Wild Birds during the first year (November 2021-November 2022) following the incursions of HPAIV from Eurasia. The key objectives of the surveillance program were to (i) identify the presence, distribution, and spread of HPAIV and other AIVs; (ii) identify wild bird morbidity and mortality associated with HPAIV; (iii) identify the range of wild bird species infected by HPAIV; and (iv) genetically characterize detected AIV. A total of 6,246 sick and dead wild birds were tested, of which 27.4% were HPAIV positive across 12 taxonomic orders and 80 species. Geographically, HPAIV detections occurred in all Canadian provinces and territories, with the highest numbers in the Atlantic and Central Flyways. Temporally, peak detections differed across flyways, though the national peak occurred in April 2022. In an additional 11,295 asymptomatic harvested or live-captured wild birds, 5.2% were HPAIV positive across 3 taxonomic orders and 19 species. Whole-genome sequencing identified HPAIV of Eurasian origin as most prevalent in the Atlantic Flyway, along with multiple reassortants of mixed Eurasian and North American origins distributed across Canada, with moderate structuring at the flyway scale. Wild birds were victims and reservoirs of HPAIV H5N1 2.3.4.4b, underscoring the importance of surveillance encompassing samples from sick and dead, as well as live and harvested birds, to provide insights into the dynamics and potential impacts of the HPAIV H5N1 outbreak. This dramatic shift in the presence and distribution of HPAIV in wild birds in Canada highlights a need for sustained investment in wild bird surveillance and collaboration across interagency partners. IMPORTANCE: We present the results of Canada's Interagency Surveillance Program for Avian Influenza in Wild Birds in the year following the first detection of highly pathogenic avian influenza virus (HPAIV) H5N1 on the continent. The surveillance program tested over 17,000 wild birds, both sick and apparently healthy, which revealed spatiotemporal and taxonomic patterns in HPAIV prevalence and mortality across Canada. The significant shift in the presence and distribution of HPAIV in Canada's wild birds underscores the need for sustained investment in wild bird surveillance and collaboration across One Health partners.

Farm biosecurity practices affecting avian influenza virus circulation in commercial chicken farms in Bangladesh.

Avian influenza virus (AIV) is of major concern to livestock, wildlife, and human health. In many countries in the world, including Bangladesh, AIV is endemic in poultry, requiring improving biosecurity. In Bangladesh, we investigated how variation in biosecurity practices in commercial chicken farms affected their AIV infection status to help guide AIV mitigation strategies. We collected pooled fecal swabs from 225 farms and tested the samples for the AIV matrix gene followed by H5, H7, and H9 subtyping using rRT-PCR. We found that 39.6% of chicken farms were AIV positive, with 13% and 14% being positive for subtypes H5 and H9, respectively. Using a generalized linear mixed effects model, we identified as many as 12 significant AIV risk factors. Two major factors promoting AIV risk that cannot be easily addressed in the short term were farm size and the proximity of the farm to a live bird market. However, the other ten significant determinants of AIV risk can be more readily addressed, of which the most important ones were limiting access by visitors (reducing predicted AIV risk from 42 to 6%), isolation and treatment of sick birds (42 to 7%), prohibiting access of vehicles to poultry sheds (38 to 8%), improving hand hygiene (from 42 to 9%), not sharing farm workers across farms (37 to 8%), and limiting access by wild birds to poultry sheds (37 to 8%). Our findings can be applied to developing practical and cost-effective measures that significantly decrease the prevalence of AIV in chicken farms. Notably, in settings with limited resources, such as Bangladesh, these measures can help governments strengthen biosecurity practices in their poultry industry to limit and possibly prevent the spread of AIV.

Outbreaks of H5N1 High Pathogenicity Avian Influenza in South Africa in 2023 Were Caused by Two Distinct Sub-Genotypes of Clade 2.3.4.4b Viruses.

In 2023, South Africa continued to experience sporadic cases of clade 2.3.4.4b H5N1 high-pathogenicity avian influenza (HPAI) in coastal seabirds and poultry. Active environmental surveillance determined that H5Nx, H7Nx, H9Nx, H11Nx, H6N2, and H12N2, amongst other unidentified subtypes, circulated in wild birds and ostriches in 2023, but that H5Nx was predominant. Genome sequencing and phylogenetic analysis of confirmed H5N1 HPAI cases determined that only two of the fifteen sub-genotypes that circulated in South Africa in 2021-2022 still persisted in 2023. Sub-genotype SA13 remained restricted to coastal seabirds, with accelerated mutations observed in the neuraminidase protein. SA15 caused the chicken outbreaks, but outbreaks in the Paardeberg and George areas, in the Western Cape province, and the Camperdown region of the KwaZulu-Natal province were unrelated to each other, implicating wild birds as the source. All SA15 viruses contained a truncation in the PB1-F2 gene, but in the Western Cape SA15 chicken viruses, PA-X was putatively expressed as a novel isoform with eight additional amino acids. South African clade 2.3.4.4b H5N1 viruses had comparatively fewer markers of virulence and pathogenicity compared to European strains, a possible reason why no spillover to mammals has occurred here yet.

Natural Infection with Highly Pathogenic Avian Influenza A/H5N1 Virus in Pet Ferrets.

The study involved five ferrets from one household in Poland, comprising three sick 9-week-old juveniles, their healthy mother, and another clinically normal adult, admitted to the veterinary clinic in June 2023. The juvenile ferrets displayed significant lethargy and a pronounced unwillingness to move with accompanying pulmonary distress. Prompted by concurrent outbreaks of A/H5N1 influenza virus infections in Polish cats, point-of-care tests were conducted that revealed type A influenza antigens in the throat swabs of all five ferrets. Despite treatment, one juvenile ferret exhibited dyspnea and neurological symptoms and eventually died. The two remaining ferrets recovered fully, including one severely affected showing persistent dyspnea and incoordination without fever that recovered after 11 days of treatment. In the RT-qPCR, the throat swabs collected from all surviving ferrets as well as the samples of lungs, trachea, heart, brain, pancreas, liver, and intestine of the succumbed ferret were found positive for A/H5N1 virus RNA. To our best knowledge, this is the first documented natural A/H5N1 avian influenza in domestic ferrets kept as pets. In addition, this outbreak suggests the possibility of asymptomatic A/H5N1 virus shedding by ferrets, highlighting their zoonotic potential and the advisability of excluding fresh or frozen poultry from their diet to reduce the A/H5N1 virus transmission risks.

Fatal Infection in Ferrets after Ocular Inoculation with Highly Pathogenic Avian Influenza A(H5N1) Virus.

Ocular inoculation of a clade 2.3.4.4b highly pathogenic avian influenza A(H5N1) virus caused severe and fatal infection in ferrets. Virus was transmitted to ferrets in direct contact. The results highlight the potential capacity of these viruses to cause human disease after either respiratory or ocular exposure.

Dual Gene Detection of H5N1 Avian Influenza Virus Based on Dual RT-RPA.

The H5N1 avian influenza virus seriously affects the health of poultry and humans. Once infected, the mortality rate is very high. Therefore, accurate and timely detection of the H5N1 avian influenza virus is beneficial for controlling its spread. This article establishes a dual gene detection method based on dual RPA for simultaneously detecting the HA and M2 genes of H5N1 avian influenza virus, for the detection of H5N1 avian influenza virus. Design specific primers for the conserved regions of the HA and M2 genes. The sensitivity of the dual RT-RPA detection method for HA and M2 genes is 1 × 10-7 ng/µL. The optimal primer ratio is 1:1, the optimal reaction temperature is 40 °C, and the optimal reaction time is 20 min. Dual RT-RPA was used to detect 72 samples, and compared with RT-qPCR detection, the Kappa value was 1 (p value < 0.05), and the clinical sample detection sensitivity and specificity were both 100%. The dual RT-RPA method is used for the first time to simultaneously detect two genes of the H5N1 avian influenza virus. As an accurate and convenient diagnostic tool, it can be used to diagnose the H5N1 avian influenza virus.

Highly pathogenic avian influenza A(H5N1) virus infections on fur farms connected to mass mortalities of black-headed gulls, Finland, July to October 2023.

Highly pathogenic avian influenza (HPAI) has caused widespread mortality in both wild and domestic birds in Europe 2020-2023. In July 2023, HPAI A(H5N1) was detected on 27 fur farms in Finland. In total, infections in silver and blue foxes, American minks and raccoon dogs were confirmed by RT-PCR. The pathological findings in the animals include widespread inflammatory lesions in the lungs, brain and liver, indicating efficient systemic dissemination of the virus. Phylogenetic analysis of Finnish A(H5N1) strains from fur animals and wild birds has identified three clusters (Finland I-III), and molecular analyses revealed emergence of mutations known to facilitate viral adaptation to mammals in the PB2 and NA proteins. Findings of avian influenza in fur animals were spatially and temporally connected with mass mortalities in wild birds. The mechanisms of virus transmission within and between farms have not been conclusively identified, but several different routes relating to limited biosecurity on the farms are implicated. The outbreak was managed in close collaboration between animal and human health authorities to mitigate and monitor the impact for both animal and human health.

Does pasteurization inactivate bird flu virus in milk?

Recently, an outbreak of highly pathogenic avian influenza A (H5N1), which carries the clade 2.3.4.4b hemagglutinin (HA) gene and has been prevalent among North American bird populations since the winter of 2021, was reported in dairy cows in the United States. As of 24 May 2024, the virus has affected 63 dairy herds across nine states and has resulted in two human infections. The virus causes unusual symptoms in dairy cows, including an unexpected drop in milk production, and thick colostrum-like milk. Notably, The US Food and Drug Administration reported that around 20% of tested retail milk samples contained H5N1 viruses, with a higher percentage of positive results from regions with infected cattle herds. Data are scant regarding how effectively pasteurization inactivates the H5N1 virus in milk. Therefore, in this study, we evaluated the thermal stability of the H5 clade 2.3.4.4b viruses, along with one human H3N2 virus and other influenza subtype viruses, including H1, H3, H7, H9, and H10 subtype viruses. We also assessed the effectiveness of pasteurization in inactivating these viruses. We found that the avian H3 virus exhibits the highest thermal stability, whereas the H5N1 viruses that belong to clade 2.3.4.4b display moderate thermal stability. Importantly, our data provide direct evidence that the standard pasteurization methods used by dairy companies are effective in inactivating all tested subtypes of influenza viruses in raw milk. Our findings indicate that thermally pasteurized milk products do not pose a safety risk to consumers.