Highly Pathogenic Avian Influenza A(H5N1) Viruses from Multispecies Outbreak, Argentina, August 2023.

We report full-genome characterization of highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b virus from an outbreak among sea lions (August 2023) in Argentina and possible spillover to fur seals and terns. Mammalian adaptation mutations in virus isolated from marine mammals and a human in Chile were detected in mammalian and avian hosts.

Dual-Action Heteromultivalent Glycopolymers Stringently Block and Arrest Influenza A Virus Infection In Vitro and Ex Vivo.

Here, we demonstrate the concerted inhibition of different influenza A virus (IAV) strains using a low-molecular-weight dual-action linear polymer. The 6'-sialyllactose and zanamivir conjugates of linear polyglycerol are optimized for simultaneous targeting of hemagglutinin and neuraminidase on the IAV surface. Independent of IAV subtypes, hemagglutination inhibition data suggest better adsorption of the heteromultivalent polymer than homomultivalent analogs onto the virus surface. Cryo-TEM images imply heteromultivalent compound-mediated virus aggregation. The optimized polymeric nanomaterial inhibits >99.9% propagation of various IAV strains 24 h postinfection in vitro at low nM concentrations and is up to 10000× more effective than the commercial zanamivir drug. In a human lung ex vivo multicyclic infection setup, the heteromultivalent polymer outperforms the commercial drug zanamivir and homomultivalent analogs or their physical mixtures. This study authenticates the translational potential of the dual-action targeting approach using small polymers for broad and high antiviral efficacy.

Comparative histopathological and immunological study of two field strains of chicken anemia virus.

Infection of poultry with chicken anemia virus (CAV) is implicated in several field problems in broiler flocks due to the immunosuppression generated and, consequently, the increased susceptibility to secondary infections. Recently, we have reported an increased occurrence of clinical cases caused by CAV strains distantly related to those commonly used for vaccination. In order to understand the behavior of two Argentinean CAV strains (CAV-10 and CAV-18) in two-week-old chickens, an immune and histopathological study was performed. Neither mortality nor clinical signs were observed in the infected or control groups. Thymus lobes from chickens infected with both CAV viruses were smaller compared to the negative control group. At 14 days post-infection (dpi), only chickens inoculated with CAV-10 show a severe depletion of lymphocytes in the thymus cortex and in follicles from the bursa of Fabricius. Also thymopoiesis disorders, such as reduction in the percentage of total DP (CD4 + CD8α+) thymocytes and alteration in the percentages of DP subpopulations, were more important in animals inoculated with the CAV-10 than the CAV-18 strain. In addition, only animals infected with CAV-10 show a decrease in CD8αß splenocytes. Altogether our results show that, although both Argentinean CAV strains produce subclinical infections in chickens causing immunosuppression at 14 dpi, they might differ in their in vivo pathogenicity.

Identification and characterization of novel alphacoronaviruses in Tadarida brasiliensis (Chiroptera, Molossidae) from Argentina: insights into recombination as a mechanism favoring bat coronavirus cross-species transmission.

Bats are reservoirs of various coronaviruses that can jump between bat species or other mammalian hosts, including humans. This article explores coronavirus infection in three bat species (Tadarida brasiliensis, Eumops bonariensis, and Molossus molossus) of the family Molossidae from Argentina using whole viral metagenome analysis. Fecal samples of 47 bats from three semiurban or highly urbanized areas of the province of Santa Fe were investigated. After viral particle enrichment, total RNA was sequenced using the Illumina NextSeq 550 instrument; the reads were assembled into contigs and taxonomically and phylogenetically analyzed. Three novel complete Alphacoronavirus (AlphaCoV) genomes (Tb1-3) and two partial sequences were identified in T. brasiliensis (Tb4-5), and an additional four partial sequences were identified in M. molossus (Mm1-4). Phylogenomic analysis showed that the novel AlphaCoV clustered in two different lineages distinct from the 15 officially recognized AlphaCoV subgenera. Tb2 and Tb3 isolates appeared to be variants of the same virus, probably involved in a persistent infectious cycle within the T. brasiliensis colony. Using recombination analysis, we detected a statistically significant event in Spike gene, which was reinforced by phylogenetic tree incongruence analysis, involving novel Tb1 and AlphaCoVs identified in Eptesicus fuscus (family Vespertilionidae) from the U.S. The putative recombinant region is in the S1 subdomain of the Spike gene, encompassing the potential receptor-binding domain of AlphaCoVs. This study reports the first AlphaCoV genomes in molossids from the Americas and provides new insights into recombination as an important mode of evolution of coronaviruses involved in cross-species transmission. IMPORTANCE This study generated three novel complete AlphaCoV genomes (Tb1, Tb2, and Tb3 isolates) identified in individuals of Tadarida brasiliensis from Argentina, which showed two different evolutionary patterns and are the first to be reported in the family Molossidae in the Americas. The novel Tb1 isolate was found to be involved in a putative recombination event with alphacoronaviruses identified in bats of the genus Eptesicus from the U.S., whereas isolates Tb2 and Tb3 were found in different collection seasons and might be involved in persistent viral infections in the bat colony. These findings contribute to our knowledge of the global diversity of bat coronaviruses in poorly studied species and highlight the different evolutionary aspects of AlphaCoVs circulating in bat populations in Argentina.

Phylogenetic analysis of H6 influenza viruses isolated from rosy-billed pochards (Netta peposaca) in Argentina reveals the presence of different HA gene clusters.

Until recently, influenza A viruses from wild waterfowl in South America were rarely isolated and/or characterized. To explore the ecology of influenza A viruses in this region, a long-term surveillance program was established in 2006 for resident and migratory water birds in Argentina. We report the characterization of 5 avian influenza viruses of the H6 hemagglutinin (HA) subtype isolated from rosy-billed pochards (Netta peposaca). Three of these viruses were paired to an N2 NA subtype, while the other two were of the N8 subtype. Genetic and phylogenetic analyses of the internal gene segments revealed a close relationship with influenza viruses from South America, forming a unique clade and supporting the notion of independent evolution from influenza A viruses in other latitudes. The presence of NS alleles A and B was also identified. The HA and NA genes formed unique clades separate from North American and Eurasian viruses, with the exception of the HA gene of one isolate, which was more closely related to the North American lineage, suggesting possible interactions between viruses of North American and South American lineages. Animal studies suggested that these Argentine H6 viruses could replicate and transmit inefficiently in chickens, indicating limited adaptation to poultry. Our results highlight the importance of continued influenza virus surveillance in wild birds of South America, especially considering the unique evolution of these viruses.

Few Amino Acid Mutations in H6 Influenza A Virus From South American Lineage Increase Viral Replication Efficiency in Poultry.

In chickens, infections due to influenza A virus (IAV) can be mild to severe and lethal. The study of IAV infections in poultry has been mostly limited to strains from the North American and Eurasian lineages, whereas limited information exists on similar studies with strains from the South American lineage (SAm). To better evaluate the risk of introduction of a prototypical SAm IAV strain into poultry, chickens were infected with a wild-type SAm origin strain (WT557/H6N2). The resulting virus progeny was serially passaged in chickens 20 times, and the immunopathological effects of the last passage virus, 20Ch557/H6N2, in chickens were compared to those of the parental strain. A comparison of complete viral genome sequences indicated that the 20Ch557/H6N2 strain contained 13 amino acid differences compared to the wild-type strain. Five of these mutations are in functionally relevant regions of the viral surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). However, despite higher and more prolonged virus shedding in chickens inoculated with the 20Ch557/H6N2 strain compared to those that received the WT557/H6N2 strain, transmission to naïve chickens was not observed for either group. Analyses by flow cytometry of mononuclear cells and lymphocyte subpopulations from the lamina propria and intraepithelial lymphocytic cells (IELs) from the ileum revealed a significant increase in the percentages of CD3+TCRγδ+ IELs in chickens inoculated with the 20Ch557/H6N2 strain compared to those inoculated with the WT557/H6N2 strain.

Outbreak of swine influenza in Argentina reveals a non-contemporary human H3N2 virus highly transmissible among pigs.

Sporadic outbreaks of human H3N2 influenza A virus (IAV) infections in swine populations have been reported in Asia, Europe and North America since 1970. In South America, serological surveys in pigs indicate that IAVs of the H3 and H1 subtypes are currently in circulation; however, neither virus isolation nor characterization has been reported. In November 2008, an outbreak of respiratory disease in pigs consistent with swine influenza virus (SIV) infection was detected in Argentina. The current study describes the clinical epidemiology, pathology, and molecular and biological characteristics of the virus. Phylogenetic analysis revealed that the virus isolate shared nucleotide identities of 96-98 % with H3N2 IAVs that circulated in humans from 2000 to 2003. Antigenically, sera from experimentally inoculated animals cross-reacted mainly with non-contemporary human-origin H3N2 influenza viruses. In an experimental infection in a commercial swine breed, the virus was of low virulence but was transmitted efficiently to contact pigs and caused severe disease when an infected animal acquired a secondary bacterial infection. This is the first report of a wholly human H3N2 IAV associated with clinical disease in pigs in South America. These studies highlight the importance of two-way transmission of IAVs and SIVs between pigs and humans, and call for enhanced influenza surveillance in the pig population worldwide.

Pandemic (H1N1) 2009 outbreak on pig farm, Argentina.

In June-July 2009, an outbreak of pandemic (H1N1) 2009 infection occurred on a pig farm in Argentina. Molecular analysis indicated that the virus was genetically related to the pandemic (H1N1) 2009 influenza virus strain. The outbreak presumably resulted from direct human-to-pig transmission.

Molecular characterization of avian infectious bronchitis virus strains from outbreaks in Argentina (2001-2008).

Twenty infectious bronchitis virus isolates were recovered from broilers and layers in different outbreaks amongst commercial poultry flocks in different geographic regions of Argentina from 2001 to 2008. The viruses were isolated from the tracheas, lungs, and caecal tonsils of birds that were showing respiratory signs. Further analysis based on their nucleotide and amino acid sequences in hypervariable region (HVR) 1 and the intervening sequence including HVRs 1 and 2 (HVR1/2) of the S1 gene was done to determine the genetic relationships among them and reference strains. Five isolates were highly related to the Massachusetts or Connecticut serotypes, indicating the probability of the detection and isolation of vaccine strains. The other Argentinean isolates formed three separate clusters (A, B and C), distant from the vaccine serotypes, with no correlation between the generated clusters and a geographic pattern. These observations could explain the failure of the Massachusetts serotype vaccination programmes to control IBV in these flocks. In addition, the utilization of HVR1/2 and HVR1 sequences resulted in trees with similar topology but the phylogenetic relationships using HVR1/2 nucleotide sequences were better supported by higher bootstrap values. Therefore, the sequences of the HVR1/2 region are recommended for phylogenetic studies.

Evidence of a fixed internal gene constellation in influenza A viruses isolated from wild birds in Argentina (2006-2016).

Wild aquatic birds are the major reservoir of influenza A virus. Cloacal swabs and feces samples (n = 6595) were collected from 62 bird species in Argentina from 2006 to 2016 and screened for influenza A virus. Full genome sequencing of 15 influenza isolates from 6 waterfowl species revealed subtypes combinations that were previously described in South America (H1N1, H4N2, H4N6 (n = 3), H5N3, H6N2 (n = 4), and H10N7 (n = 2)), and new ones not previously identified in the region (H4N8, H7N7 and H7N9). Notably, the internal gene segments of all 15 Argentine isolates belonged to the South American lineage, showing a divergent evolution of these viruses in the Southern Hemisphere. Time-scaled phylogenies indicated that South American gene segments diverged between ~ 30 and ~ 140 years ago from the most closely related influenza lineages, which include the avian North American (PB1, HA, NA, MP, and NS-B) and Eurasian lineage (PB2), and the equine H3N8 lineage (PA, NP, and NS-A). Phylogenetic analyses of the hemagglutinin and neuraminidase gene segments of the H4, H6, and N8 subtypes revealed recent introductions and reassortment between viruses from the Northern and Southern Hemispheres in the Americas. Remarkably and despite evidence of recent hemagglutinin and neuraminidase subtype introductions, the phylogenetic composition of internal gene constellation of these influenza A viruses has remained unchanged. Considering the extended time and the number of sampled species of the current study, and the paucity of previously available data, our results contribute to a better understanding of the ecology and evolution of influenza virus in South America.

Epidemiological and virological findings during multiple outbreaks of equine influenza in South America in 2012.

BACKGROUND: In 2012, equine influenza (EI) virus was confirmed as the cause of outbreaks of respiratory disease in horses throughout South America. In Uruguay and Argentina, hundreds of vaccinated thoroughbred horses in training and racing facilities were clinically affected. OBJECTIVE: To characterise the EI viruses detected during the outbreak in Uruguay and Argentina. METHODS: Virus was detected in nasopharyngeal swabs by a pan-reactive influenza type A real-time RT-PCR. The nucleotide sequence of the HA1 gene was determined and analysed phylogenetically using mega 5 software. Amino acid sequences alignments were constructed and virus was antigenically characterised with specific ferret antisera. Paired serum samples were tested by haemagglutination inhibition and single radial haemolysis. RESULTS: The diagnosis of EIV was confirmed by real-time RT-PCR, virus isolation and serological testing. The phylogenetic analysis of HA1 gene sequences of 18 EI viruses indicated that all of them belong to clade 1 of the Florida sublineage of the American lineage and are closely related to viruses isolated in the United States in 2012. The HA1 of viruses identified in horses in racing facilities in Maroñas, Uruguay, and in Palermo, Argentina, displayed 100% amino acid sequence identity and were identical to that of a virus isolated in Dubai in 2012, from vaccinated endurance horses recently imported from Uruguay. CONCLUSIONS: The surveillance data reported illustrate the international spread of EI viruses and support the recommendations of the OIE expert surveillance panel to include viruses of the Florida sublineage in vaccines.

Isolation and characterization of an H9N2 influenza virus isolated in Argentina.

As part of our ongoing efforts on animal influenza surveillance in Argentina, an H9N2 virus was isolated from a wild aquatic bird (Netta peposaca), A/rosy-billed pochard/Argentina/CIP051-559/2007 (H9N2) - herein referred to as 559/H9N2. Due to the important role that H9N2 viruses play in the ecology of influenza in nature, the 559/H9N2 isolate was characterized molecularly and biologically. Phylogenetic analysis of the HA gene revealed that the 559/H9N2 virus maintained an independent evolutionary pathway and shared a sister-group relationship with North American viruses, suggesting a common ancestor. The rest of the genome segments clustered with viruses from South America. Experimental inoculation of the 559/H9N2 in chickens and quail revealed efficient replication and transmission only in quail. Our results add to the notion of the unique evolutionary trend of avian influenza viruses in South America. Our study increases our understanding of H9N2 viruses in nature and emphasizes the importance of expanding animal influenza surveillance efforts to better define the ecology of influenza viruses at a global scale.

Evidence of reassortment of pandemic H1N1 influenza virus in swine in Argentina: are we facing the expansion of potential epicenters of influenza emergence?

In this report, we describe the occurrence of two novel swine influenza viruses (SIVs) in pigs in Argentina. These viruses are the result of two independent reassortment events between the H1N1 pandemic influenza virus (H1N1pdm) and human-like SIVs, showing the constant evolution of influenza viruses at the human-swine interface and the potential health risk of H1N1pdm as it appears to be maintained in the swine population. It must be noted that because of the lack of information regarding the circulation of SIVs in South America, we cannot discard the possibility that ancestors of the H1N1pdm or other SIVs have been present in this part of the world. More importantly, these findings suggest an ever-expanding geographic range of potential epicenters of influenza emergence with public health risks.

Genetic characterization of porcine circovirus type 2 from pigs with porcine circovirus associated diseases in Argentina.

Porcine circovirus type 2 (PCV-2) has been associated with syndromes grouped by the term porcine circovirus associated diseases (PCVAD). The PCV-2 isolates have been grouped into two major groups or genotypes according to their nucleotide sequence of whole genomes and/or ORF-2: PCV-2b, which have, in turn, been subdivided into three clusters (1A-1C), and PCV-2a, which has been subdivided into five clusters (2A-2E). In the present study, we obtained 16 sequences of PCV-2 from different farms from 2003 to 2008, from animals with confirmatory diagnosis of PCVAD. Since results showed an identity of 99.8% among them, they were grouped within a common cluster 1A-B. This preliminary study suggests a stable circulation of PCV-2b among the Argentinean pig population.

Avian influenza virus isolated in wild waterfowl in Argentina: evidence of a potentially unique phylogenetic lineage in South America.

Avian influenza (AI) viruses have been sporadically isolated in South America. The most recent reports are from an outbreak in commercial poultry in Chile in 2002 and its putative ancestor from a wild bird in Bolivia in 2001. Extensive surveillance in wild birds was carried out in Argentina during 2006-2007. Using RRT-PCR, 12 AI positive detections were made from cloacal swabs. One of those positive samples yielded an AI virus isolated from a wild kelp gull (Larus dominicanus) captured in the South Atlantic coastline of Argentina. Further characterization by nucleotide sequencing reveals that it belongs to the H13N9 subtype. Phylogenetic analysis of the 8 viral genes suggests that the 6 internal genes are related to the isolates from Chile and Bolivia. The analysis also indicates that a cluster of phylogenetically related AI viruses from South America may have evolved independently, with minimal gene exchange, from influenza viruses in other latitudes. The data produced from our investigations are valuable contributions to the study of AI viruses in South America.