Infection of South American coatis (Nasua nasua) with highly pathogenic avian influenza H5N1 virus displaying mammalian adaptive mutations.

Deadly outbreaks among poultry, wild birds, and carnivorous mammals by the highly pathogenic H5N1 virus of the clade 2.3.4.4b have been reported in South America. The increasing virus incidence in various mammal species poses a severe zoonotic and pandemic threat. In Uruguay, the clade 2.3.4.4b viruses were first detected in February 2023, affecting wild birds and backyard poultry. Three months after the first reported case in Uruguay, the disease affected a population of 23 coatis (Nasua) in an ecological park. Most animals became infected, likely directly or indirectly from wild birds in the park, and experienced sudden death. Five animals from the colony survived, and four of them developed antibodies. The genomes of the H5N1 strains infecting coatis belonged to the B3.2 genotype of the clade 2.3.4.4b. Genomes from coatis were closely associated with those infecting backyard poultry, but transmission likely occurred through wild birds. Notable, two genomes have a 627K substitution in the RNA polymerase PB2 subunit, a hallmark amino acid linked to mammalian adaptation. Our findings support the ability of the avian influenza virus of the 2.3.4.4b clade to infect and transmit among terrestrial mammals with high pathogenicity and undergo rapid adaptive changes. It also highlights the coatis' ability to develop immunity and naturally clear the infection.

Mortality in sea lions is associated with the introduction of the H5N1 clade 2.3.4.4b virus in Brazil October 2023: whole genome sequencing and phylogenetic analysis.

Clade 2.3.4.4b highly pathogenic avian influenza (HPAI) H5N1 virus was detected in the South American sea lions found dead in Santa Catarina, Brazil, in October 2023. Whole genome sequencing and comparative phylogenetic analysis were conducted to investigate the origin, genetic diversity, and zoonotic potentials of the H5N1 viruses. The H5N1 viruses belonged to the genotype B3.2 of clade 2.3.4.4b H5N1 virus, which was identified in North America and disseminated to South America. They have acquired new amino acid substitutions related to mammalian host affinity. Our study provides insights into the genetic landscape of HPAI H5N1 viruses in Brazil, highlighting the continuous evolutionary processes contributing to their possible adaptation to mammalian hosts.

Phylodynamics of avian influenza A(H5N1) viruses from outbreaks in Brazil.

Our study identified strains of the A/H5N1 virus in analyzed samples of subsistence poultry, wild birds, and mammals, belonging to clade 2.3.4.4b, genotype B3.2, with very high genetic similarity to strains from Chile, Uruguay, and Argentina. This suggests a migratory route for wild birds across the Pacific, explaining the phylogenetic relatedness. The Brazilian samples displayed similarity to strains that had already been previously detected in South America. Phylogeographic analysis suggests transmission of US viruses from Europe and Asia, co-circulating with other lineages in the American continent. As mutations can influence virulence and host specificity, genomic surveillance is essential to detect those changes, especially in critical regions, such as hot spots in the HA, NA, and PB2 sequences. Mutations in the PB2 gene (D701N and Q591K) associated with adaptation and transmission in mammals were detected suggesting a potential zoonotic risk. Nonetheless, resistance to neuraminidase inhibitors (NAIs) was not identified, however, continued surveillance is crucial to detect potential resistance. Our study also mapped the spread of the virus in the Southern hemisphere, identifying possible entry routes and highlighting the importance of surveillance to prevent outbreaks and protect both human and animal populations.

Highly pathogenic avian influenza A(H5N1) virus of clade 2.3.4.4b isolated from a human case in Chile causes fatal disease and transmits between co-housed ferrets.

Clade 2.3.4.4b highly pathogenic avian influenza A(H5N1) viruses have caused large outbreaks within avian populations on five continents, with concurrent spillover into a variety of mammalian species. Mutations associated with mammalian adaptation have been sporadically identified in avian isolates, and more frequently among mammalian isolates following infection. Reports of human infection with A(H5N1) viruses following contact with infected wildlife have been reported on multiple continents, highlighting the need for pandemic risk assessment of these viruses. In this study, the pathogenicity and transmissibility of A/Chile/25945/2023 HPAI A(H5N1) virus, a novel reassortant with four gene segments (PB1, PB2, NP, MP) from North American lineage, isolated from a severe human case in Chile, was evaluated in vitro and using the ferret model. This virus possessed a high capacity to cause fatal disease, characterized by high morbidity and extrapulmonary spread in virus-inoculated ferrets. The virus was capable of transmission to naïve contacts in a direct contact setting, with contact animals similarly exhibiting severe disease, but did not exhibit productive transmission in respiratory droplet or fomite transmission models. Our results indicate that the virus would need to acquire an airborne transmissible phenotype in mammals to potentially cause a pandemic. Nonetheless, this work warrants continuous monitoring of mammalian adaptations in avian viruses, especially in strains isolated from humans, to aid pandemic preparedness efforts.

Highly Pathogenic Avian Influenza A(H5N1) from Wild Birds, Poultry, and Mammals, Peru.

We identified highly pathogenic avian influenza A(H5N1) virus clade 2.3.4.4b in wild birds, poultry, and a lion in Peru during November 2022-February 2023 and markers associated with transmission adaptation and antiviral drug resistance. Continuous genomic surveillance is needed to inform public health measures and avoid mass animal deaths.

Highly pathogenic avian influenza A (H5N1) in marine mammals and seabirds in Peru.

Highly pathogenic avian influenza (HPAI) A/H5N1 viruses (lineage 2.3.4.4b) are rapidly invading the Americas, threatening wildlife, poultry, and potentially evolving into the next global pandemic. In November 2022 HPAI arrived in Peru, triggering massive pelican and sea lion die-offs. We report genomic characterization of HPAI/H5N1 in five species of marine mammals and seabirds (dolphins, sea lions, sanderlings, pelicans and cormorants). Peruvian viruses belong to lineage 2.3.4.4b, but they are 4:4 reassortants where 4 genomic segments (PA, HA, NA and MP) position within the Eurasian lineage that initially entered North America from Eurasia, while the other 4 genomic segments (PB2, PB1, NP and NS) position within the American lineage (clade C) that circulated in North America. These viruses are rapidly accruing mutations, including mutations of concern, that warrant further examination and highlight an urgent need for active local surveillance to manage outbreaks and limit spillover into other species, including humans.

Spreading of the High-Pathogenicity Avian Influenza (H5N1) Virus of Clade 2.3.4.4b into Uruguay.

BACKGROUND: Avian influenza viruses (genus Alphainfluenzavirus, family Orthomyxoviridae) infect avian and mammal hosts. In 2022, the high pathogenicity avian influenza virus (H5N1) spread to South America, resulting in the loss of thousands of wild birds, including endangered species, and severely impacting the global poultry industry. OBJECTIVES: We analyzed the complete genomes of influenza viruses obtained from wild birds and backyard poultry in Uruguay between February and May 2023. METHODS: Twelve complete genomes were obtained in 2023 from cloacal swabs using Illumina sequencing. Genomes were phylogenetically analyzed with regional and global strains. FINDINGS: The identified strains have multiple basic amino acids at the hemagglutinin cleavage sites, which is typical for highly pathogenic strains. The Uruguayan viruses belonged to hemagglutinin clade 2.3.4.4b of the H5N1 subtype. A reassortment in North America has resulted in some segments of South American strains being of Eurasian or North American origins. The Uruguayan viruses shared a common ancestor with South American strains from Argentina and Chile. The influenza viruses displayed a spatiotemporal divergence pattern rather than being host-specific. MAIN CONCLUSIONS: The arrival of the 2.3.4.4b clade in Uruguay may have been mediated by birds that acquired the virus from Argentine and Chilean waterfowl migrating in the Pacific Flyway.

Detection and phylogenetic analysis of highly pathogenic A/H5N1 avian influenza clade 2.3.4.4b virus in Chile, 2022.

Highly pathogenic avian influenza (HPAI) A/H5N1 viruses continue to pose a significant threat to animal and human health worldwide. In late 2022, the first confirmed case of HPAI A/H5N1 infection in wild birds in Chile near the Chilean-Peruvian border was reported. Active surveillance by our group in the adyacent Lluta river estuary revealed an increase in A/H5N1 prevalence coinciding with the arrival of migratory birds from the Northern Hemisphere. Genomic analysis of A/H5N1-positive samples demonstrated a close genetic relationship to strains detected in Peru during the same period, which originated from A/H5N1 viruses causing outbreaks in North America. Notably, we identified genetic mutations that did not correlate with known enhanced transmission or binding traits to mammalian receptors. In summary, this study provides valuable genomic insights into the A/H5N1 Clade 2.3.4.4b viruses in wild birds in Chile, emphasizing the need for enhanced surveillance and response strategies to mitigate the threat posed by these highly pathogenic avian influenza viruses in South America.

Phylogenetic analysis reveals that the H5N1 avian influenza A outbreak in poultry in Ecuador in November 2022 is associated with the highly pathogenic clade 2.3.4.4b.

The ongoing H5N1 outbreak in the Americas caused by clade 2.3.4.4 is causing unprecedented impact in poultry and wild birds. In November 2022, a highly pathogenic avian influenza A outbreak was declared in poultry in Ecuador, affecting more than 1.1 million heads of poultry in two farms by February 2023. Phylogenetic analysis shows that the virus clade is 2.3.4.4b, and to the best of our knowledge, this is the first scientific publication reporting this clade in South America.

Impact of tetramerization on the ligand recognition of N1 influenza neuraminidase via MMGBSA approach.

Influenza virus neuraminidase (NA) is a homotetrameric surface protein that, in contrast to other non-influenza NAs, requires a quaternary assembly to exhibit enzymatic activity, suggesting that the oligomeric state significantly impacts the active site of influenza NA. Nevertheless, most structure-based drug design studies have been reported by employing the monomeric state in the closed or open-loop due to the computational cost of employing the tetrameric NA. In this work, we present MD simulations coupled to the MMGBSA approach of avian N1 type NA in its monomeric and tetrameric closed and open-loop state both with and without the inhibitor oseltamivir and its natural substrate, sialic acid. Structural and energetic analyses revealed that the tetrameric state impacts flexibility as well as the map of interactions participating in stabilizing the protein-ligand complexes with respect to the monomeric state. It was observed that the tetrameric state exerts dissimilar effects in binding affinity, characteristic of positive and negative cooperativity for oseltamivir and sialic acid, respectively. Based on our results, to perform a confident structure-based drug design, as well as to evaluate the impact of key mutations through MD simulations, it is important to consider the tetrameric state closed-loop state.

In Silico Identification of Highly Conserved Epitopes of Influenza A H1N1, H2N2, H3N2, and H5N1 with Diagnostic and Vaccination Potential.

The unpredictable, evolutionary nature of the influenza A virus (IAV) is the primary problem when generating a vaccine and when designing diagnostic strategies; thus, it is necessary to determine the constant regions in viral proteins. In this study, we completed an in silico analysis of the reported epitopes of the 4 IAV proteins that are antigenically most significant (HA, NA, NP, and M2) in the 3 strains with the greatest world circulation in the last century (H1N1, H2N2, and H3N2) and in one of the main aviary subtypes responsible for zoonosis (H5N1). For this purpose, the HMMER program was used to align 3,016 epitopes reported in the Immune Epitope Database and Analysis Resource (IEDB) and distributed in 34,294 stored sequences in the Pfam database. Eighteen epitopes were identified: 8 in HA, 5 in NA, 3 in NP, and 2 in M2. These epitopes have remained constant since they were first identified (~91 years) and are present in strains that have circulated on 5 continents. These sites could be targets for vaccination design strategies based on epitopes and/or as markers in the implementation of diagnostic techniques.

Avian-derived NS gene segments alter pathogenicity of the A/Puerto Rico/8/34 virus.

While the effect of the influenza A virus non-structural protein (NS) on cytokine production during viral infection is well known, inconsistent results have been observed with some other influenza A virus backbone studied. In this study, in order to focus on the impact of the avian NS gene segments on viral virulence, the NS genes encoded by different strains of avian influenza A viruses were incorporated into an identical [A/Puerto Rico/8/1934(H1N1), PR8] virus background to generate various NS recombinant viruses. Thus, PR8NS, PR8×[A/Hong Kong/483/97(H5N1) 483NS, PR8×[A/Ck/Korea/150/03(H9N2) 150NS, and PR8×[A/EM/Korea/W149/06(H5N1) W149NS were constructed utilizing reverse genetics. Here, we show the effects of each of these recombinant viruses upon viral pathogenesis and cytokine production during viral replication in vivo. In this regard, we found that infection of mice with the PR8×150NS recombinant virus resulted in the lowest pathogenicity (6.0×10(4)MLD50), yet elicited the highest levels of TNF-α production in bronchoalveolar lavage (BAL) fluid compared to infection with the other recombinant influenza viruses. In contrast, infection with the PR8 virus showed the highest pathogenicity (1.0×10(2)MLD50) as well as relatively high cytokine levels (IL-1α, IL-1ß, IL-17, and eotaxin) in mouse BAL fluid. In addition, the PR8 and PR8×483NS viruses induced severe and extensive inflammation in infected lungs compared with that of PR8×150 NS recombinant virus-infected mice. These results clearly demonstrate that the NS genes of diverse influenza A strains can variable impact pathogenicity, histopathology, and cytokine production in mice even when expressed in an identical genetic background.

Improvement of influenza vaccine strain A/Vietnam/1194/2004 (H5N1) growth with the neuraminidase packaging sequence from A/Puerto Rico/8/34.

H5N1 influenza candidate vaccine viruses were developed using the "6+2" approach. The hemagglutinin (HA) and neuraminidase (NA) genes were derived from the popular H5N1 virus and the remaining six internal segments were derived from the A/Puerto Rico/8/34 strain (H1N1, PR8). However, some of these candidate strains have been reported to produce relatively low yields in vaccine manufacture. In this study, we found that the NA vRNA of the A/Vietnam/1194/2004 strain (H5N1, VN1194) was poorly packaged into recombinant viruses with a backbone of PR8 genes, which resulted in the formation of defective virions that did not include the NA vRNA in the genome. Using recombinant DNA techniques, we constructed a chimeric NA gene with the coding region of VN1194 NA flanked by the packaging signal sequence of the PR8 NA gene (41 bp form the 3' end of the vRNA and 67 bp from the 5' end). The packaging of the NA vRNA was restored to normal levels in the recombinant viruses containing the chimeric NA gene. Recombinant viruses containing the chimeric NA replicated much better in chicken embryonated eggs than viruses with the wild-type NA from VN1194. These findings suggest a novel strategy to improve in ovo growth of vaccine strains and to increase the number of vaccine doses available to save people if a pandemic were to occur.

Improving global influenza surveillance: trends of A(H5N1) virus in Africa and Asia.

BACKGROUND: Highly pathogenic avian influenza A(H5N1) viruses are an important health problem in many Asian and African countries. The current increase in human cases demonstrates that influenza A(H5N1) is still a significant global pandemic threat. Many health organizations have recognized the need for new strategies to improve influenza global surveillance. Specifically, the World Health Organization through the global technical consultation for influenza surveillance have called for a detailed picture of the current limitations, especially at the nation level, to evaluate, standardize and strength reporting systems. The main goal of our study is to demonstrate the value of genetic surveillance as part of a strategic surveillance plan. As a proof of concept, we evaluated the current situation of influenza A(H5N1) in Asian and Africa. RESULTS: Our analysis revealed a power-law distribution in the number of sequences of A(H5N1) viruses analyzed and/or reported to influenza surveillance networks. The majority of the Asian and African countries at great risk of A(H5N1) infections have very few (approximately three orders of magnitude) sequenced A(H5N1) viruses (e.g. hemagglutinin genes). This suggests that countries under pandemic alert for avian influenza A(H5N1) have very limited participation (e.g. data generation, genetic analysis and data share) in avian influenza A(H5N1) surveillance. More important, this study demonstrates the usefulness of influenza genetic surveillance to detect emerging pandemic threat viruses. CONCLUSIONS: Our study reveals that some countries suffering from human cases of avian influenza have limited participation (e.g. genetic surveillance or data share) with global surveillance networks. Also, we demonstrate that the implementation of genetic surveillance programs could increase and strengthen worldwide epidemic and pandemic preparedness. We hope that this work promotes new discussions between policy makers and health surveillance organizations to improve current methodologies and regulations.

Experimental adaptation of an influenza H5HA strain confers respiratory droplet transmission to reassortant H5H/H1N1 virus strain in ferrets.

An evaluation of the role - if any - of censorship in scientific papers, based on the publishing of the article that shows it is possible to modify the genetics of the H5N1 aviary influenza virus, enhancing its transmission among mammals, which was followed by much ado and ethical discussion.

Effects of magnesium ions on recombinant human furin: selective activation of hydrolytic activity upon substrates derived from virus envelope glycoprotein.

Here we report a detailed analysis of magnesium (Mg²+) ion effects on furin hydrolysis of fluorescent resonance energy transfer decapeptide substrates derived from canonical R-X-K/R-R furin cleavage motifs within certain viral envelope glycoproteins and eukaryotic proproteins. Using virus-derived sequences a selective activation of furin by Mg²+) ions was observed as a result of cooperativity between furin subsites. Furin hydrolysis of the peptides Abz-SRRHKR↓FAGV-Q-EDDnp (from measles virus fusion protein F0 and Abz-RERRRKKR↓GLFG-Q-EDDnp (from Asian avian influenza A, H5N1) was activated between 60- and 80-fold by MgCl2. It appears that virus envelope glycoprotein mutations have been selected to increase their susceptibility to furin within cells, a location where Mg²+ is present in adequate concentrations for activation. Both the pH profile of furin and its intrinsic fluorescence were modified by Mg²+ ions, which bind to furin with a K(d) value of 1.1 mM.

Influenza in the tropics.

PURPOSE OF REVIEW: Data on influenza in tropical countries remain scarce compared with that in temperate countries. However, the emergence of avian influenza virus (H5N1) and the 2009 pandemic represented a major stimulus for advances in knowledge about influenza in many of these countries. This review summarizes recent data on viral and epidemiologic aspects of the condition in tropical countries. RECENT FINDINGS: Avian influenza and the influenza pandemic of 2009 have contributed to pioneer production of data on influenza in the tropical settings. Preliminary data on epidemiology, genetic diversity, and antiviral resistance of influenza viruses (seasonal and pandemic) in tropical countries have been published recently. SUMMARY: The seasonality of influenza is very diverse in tropical countries. Recent studies on the subject confirm the year-round activity of these viruses in many countries and the regular occurrence of epidemic outbreaks in others. The use of molecular diagnostic methods has led to prevalence rates comparable to those seen in temperate countries. Phylogenetic analysis of hemagglutinin of influenza isolates allows understanding of the evolution and divergence of avian (H5N1) and human influenza viruses (A/H1N1, A/H3N2, and B) in tropical settings. Influenza viruses resistant to oseltamivir and zanamivir have been detected in tropical countries despite the rare use of these drugs in the treatment of influenza in people living in these areas.

Evaluation of the protection induced by avian influenza vaccines containing a 1994 Mexican H5N2 LPAI seed strain against a 2008 Egyptian H5N1 HPAI virus belonging to clade 2.2.1 by means of serological and in vivo tests.

Since 2006 Egypt has been facing an extensive epidemic of H5N1 highly pathogenic avian influenza (HPAI) with a huge number of outbreaks both in rural and intensively reared poultry areas. The use of efficacious vaccines in this country has been, and still remains, essential for the control and possible eradication of HPAI. The present study was performed to establish whether the administration of inactivated vaccines containing an H5 virus belonging to a different lineage to the Eurasian H5N1 HPAI viruses guarantees protection from clinical signs, provides significant immune response and is able to achieve a reduction of viral shedding in the face of a challenge with a contemporary H5N1 virus isolated in Egypt. Despite the genetic and antigenic differences between the vaccine strain (H5N2/Mexico) and the challenge strain (H5N1/Egypt), confirmed by molecular and serological (haemagglutination inhibition) tests, it was established that the immune response induced by these conventional vaccines is sufficient to prevent infection in the majority of birds challenged with a contemporary H5N1 Egyptian strain. The data reported in this study also indicate that there may be a low degree of correlation between haemagglutination inhibition titres, clinical protection and reduction of shedding.