Sequence-based epitope mapping of high pathogenicity avian influenza H5 clade 2.3.4.4b in Latin America.

High Pathogenicity Avian Influenza (HPAI) poses a significant threat to public and animal health. Clade 2.3.4.4b recently emerged from the Eastern hemisphere and disseminated globally, reaching the Latin American (LATAM) region in late 2022 for the first time. HPAI in LATAM has resulted in massive mortalities and culling of poultry and wild birds, causing infection in mammals and humans. Despite its meaningful impact in the region, only occasional evidence about the genetic and epitope characteristics of the introduced HPAI is reported. Hence, this study seeks to phylogenetically characterize the molecular features and the source of HPAI in LATAM by evaluating potential antigenic variations. For such a purpose, we analyzed 302 whole genome sequences. All Latin American viruses are descendants of the 2.3.4.4b clade of the European H5N1 subtype. According to genomic constellations deriving from European and American reassortments, the identification of three major subtypes and eight sub-genotypes was achievable. Based on the variation of antigenic motifs at the HA protein in LATAM, we detected three potential antigenic variants, indicating the HA-C group as the dominant variant. This study decidedly contributes to unraveling the origin of the 2.3.4.4b clade in LATAM, its geographic dissemination, and evolutionary dynamics within Latin American countries. These findings offer useful information for public health interventions and surveillance initiatives planned to prevent and manage the transmission of avian influenza viruses.

Zooming in on the molecular characteristics of swine influenza virus circulating in Colombia before and after the H1N1pdm09 virus.

Influenza is one of the most critical viral agents involved in the respiratory disease complex affecting swine production systems worldwide. Despite the absence of vaccination against swine influenza virus in Colombia, the serologic reactivity to classic H1N1 and H3N2 subtypes reported since 1971 indicates the virus has been circulating in the country's swine population for several decades. However, successful isolation and sequencing of field virus from pigs was nonexistent until 2008, when H1N1 classical influenza virus was identified. One year later, due to the emergence of the influenza A (H1N1) pdm09 virus, responsible for the first global flu pandemic of the 21st century, it was introduced in the country. Therefore, to understand the impact of the introduction of the H1N1pdm09 virus in Colombia on the complexity and dynamics of influenza viruses previously present in the swine population, we carried out a study aiming to characterize circulating viruses genetically and establish possible reassortment events that might have happened between endemic influenza viruses before and after the introduction of the pandemic virus. A phylogenetic analysis of ten swine influenza virus isolates from porcine samples obtained between 2008 and 2015 was conducted. As a result, a displacement of the classical swine influenza virus with the pdmH1N1 virus in the swine population was confirmed. Once established, the pandemic subtype exhibited phylogenetic segregation based on a geographic pattern in all the evaluated segments. The evidence presents reassortment events with classic viruses in one of the first H1N1pdm09 isolates. Thus, this study demonstrates complex competition dynamics and variations in Colombian swine viruses through Drift and Shift.

Molecular Evidence of Orthomyxovirus Presence in Colombian Neotropical Bats.

The Orthomyxoviridae family includes the genera Influenzavirus, Isavirus, Quaranjavirus, and Thogotovirus. In turn, Influenzavirus can be classified into four types: α, ß, γ, and δ (Formerly A, B, C, and D), from which Alphainfluenzavirus (AIV) has the broadest host range, including birds, mammals, reptiles, and amphibians. Additionally, AIV has shown global epidemiological relevance owing to its pandemic potential. The epidemiological relevance of Chiropteran due to its multiple functional characteristics makes them ideal reservoirs for many viral agents. Recently, new influenza-like subtypes have been reported in Neotropical bats, but little is known about the relevance of bats as natural reservoirs of influenza viruses. Therefore, the current study aimed to determine the presence of AIV and new influenza-like subtypes in South American bats. For a better understanding of the drivers and interactions between AIV and bats, we used molecular assays with different gene targets (i.e., M, NP, and PB1) to identify AIV in New World bats. A housekeeping gene (CytB) PCR was used to check for nucleic acid preservation and to demonstrate the bat-origin of the samples. A total of 87 free-living bats belonging to 25 different species of the families Phyllostomidae and Vespertilionidae were collected in Casanare, Colombia. As a result, this study found seven AIV-positive bat species, three of them reported for the first time as AIV prone hosts. Neither of the AIV-like analyzed samples were positive for H17N10/H18/N11 subtypes. Although additional information is needed, the presence of a completely new or divergent AIV subtype in neotropical bats cannot be discarded. Collectively, the results presented here expand the epidemiological knowledge and distribution of AIV in neotropical free-ranging bats and emphasize the need to continue studying these viruses to establish the role they could play as a threat to animal and public health.

Molecular characterization of Marek's disease virus in a poultry layer farm from Colombia.

Marek's disease (MD) is a lymphoproliferative disease caused by an Alphaherpesvirus, genus Mardivirus, serotype 1 (Gallid Herpesvirus 2, GaHV-2) that includes all known pathogenic strains. In addition to Marek's disease virus (MDV) serotype 1, the genus includes 2 distinct nonpathogenic serotypes: serotype 2 (GaHV-3) and serotype 3 (Meleagridis Herpesvirus 1, MeHV-1) which are used in commercially available vaccines against MD. As a result of vaccination, clinical signs are not commonly observed, and new cases are usually associated with emerging variant strains against which the vaccines are less effective. In this study, a commercial layer farm showing clinical signs compatible with MDV infection was evaluated. Histological lesions and positive immunohistochemistry in the sciatic nerve and thymus were compatible with cytolytic phase of MD. GaHV-2, GaHV-3 and MeHV-1 were identified by PCR and qPCR in blood samples from 17 birds with suspected MD. Analysis of the Meq gene of the Colombian GaHV-2 isolate revealed a 99% sequence identity with Asian strains, and in the phylogenetic analysis clustered with vv+ MDV. The analysis of amino acid alignments demonstrated an interruption of the proline rich region in P176A, P217A and P233L positions, which are generally associated with vv+ strains. Some of these changes, such as P233L and L258S positions have not been reported previously. In addition, primary cell cultures inoculated with lymphocytes isolated from the spleen showed typical cytopathic effect of GaHV-2 at 5 d post infection. Based on the molecular analysis, the results from this study indicate the presence of vv+ MDV infection in commercial birds for the first time in Colombia. It is recommended to perform further assays in order to demonstrate the pathotype characteristics in vivo.

Multivalent HA DNA vaccination protects against highly pathogenic H5N1 avian influenza infection in chickens and mice.

BACKGROUND: Sustained outbreaks of highly pathogenic avian influenza (HPAI) H5N1 in avian species increase the risk of reassortment and adaptation to humans. The ability to contain its spread in chickens would reduce this threat and help maintain the capacity for egg-based vaccine production. While vaccines offer the potential to control avian disease, a major concern of current vaccines is their potency and inability to protect against evolving avian influenza viruses. METHODOLOGY / PRINCIPAL FINDINGS: The ability of DNA vaccines encoding hemagglutinin (HA) proteins from different HPAI H5N1 serotypes was evaluated for its ability to elicit neutralizing antibodies and to protect against homologous and heterologous HPAI H5N1 strain challenge in mice and chickens after DNA immunization by needle and syringe or with a pressure injection device. These vaccines elicited antibodies that neutralized multiple strains of HPAI H5N1 when given in combinations containing up to 10 HAs. The response was dose-dependent, and breadth was determined by the choice of the influenza virus HA in the vaccine. Monovalent and trivalent HA vaccines were tested first in mice and conferred protection against lethal H5N1 A/Vietnam/1203/2004 challenge 68 weeks after vaccination. In chickens, protection was observed against heterologous strains of HPAI H5N1 after vaccination with a trivalent H5 serotype DNA vaccine with doses as low as 5 microg DNA given twice either by intramuscular needle injection or with a needle-free device. CONCLUSIONS/SIGNIFICANCE: DNA vaccines offer a generic approach to influenza virus immunization applicable to multiple animal species. In addition, the ability to substitute plasmids encoding different strains enables rapid adaptation of the vaccine to newly evolving field isolates.