Evolution Of Equine Influenza Viruses Research Articles

Full-Length Genome of the Equine Influenza A Virus Subtype H3N8 from 2019 Outbreak in Saudi Arabia.

Simple SummaryEquine influenza is a highly contagious respiratory viral disease. The current study is the first to provide a description of the full-length genome sequence and surveillance of recent exposure to the equine influenza virus (EIV) during the 2019 epidemic in Saudi Arabia. This epidemic was benign, since it resulted in low case fatality (0.45%, 1/224). The viruses detected in the current study were found to be related to subtype H73N8 clade 1 of the Florida sublineage. Full-length genome sequencing revealed no evidence of major genetic changes or of reassortment among the eight segments of the viral genome. However, the Saudi strains showed a considerable number of amino acid substitutions in the signal peptide (2 amino acid substitutions), HA1 (10 amino acid substitutions) and HA2 (4 amino acid substitutions) in the haemagglutinin glycoprotein in comparison to clade 1 Florida sublineage vaccinal strains. These findings should be considered during selection of the equine influenza vaccine strains approved for use in Saudi Arabia.Equine influenza is a major cause of respiratory infections in horses and can spread rapidly despite the availability of commercial vaccines. This study aimed to screen the incidence of equine influenza virus (EIV) and molecularly characterize the haemagglutinin and neuraminidase from positive EIV field samples collected from Saudi Arabia. Six-hundred twenty-one horses from 57 horse barns were screened for the presence of the clinical signs, suggestive for equine influenza, from different parts of Saudi Arabia. Nasopharyngeal swabs were collected from each horse showing respiratory distress. Samples from the same horse barn were pooled together and screened for the presence of the influenza A virus using quantitative real time reverse transcriptase polymerase chain reaction (qRT-PCR). Selective positive samples were subjected to full-length genome sequencing using MiSeq Illumina. Out of the total 57 pools, 39 were found positive to EIV using qRT-PCR. Full-length gene sequences were compared with representative EIV strains selected from the GenBank database. Phylogenetic analysis of the HA and NA genes revealed that the identified virus strains belong to H3N8 clade 1 of the Florida sublineage and were very similar to viruses identified in USA in 2019, with no current evidence for reassortment. This is one of the first reports providing detailed description and characterization of EIVs in Saudi Arabia. Detailed surveillance and genetic information sharing could allow genetic evolution of equine influenza viruses to be monitored more effectively on a global basis and aid in refinement of vaccine strain selection for EIV.

Equine Influenza Virus in Asia: Phylogeographic Pattern and Molecular Features Reveal Circulation of an Autochthonous Lineage.

Equine influenza virus (EIV) causes severe acute respiratory disease in horses. Currently, the strains belonging to the H3N8 subtype are divided into two clades, Florida clade 1 (FC1) and Florida clade 2 (FC2), which emerged in 2002. Both FC1 and FC2 clades were reported in Asian and Middle East countries in the last decade. In this study, we described the evolution, epidemiology, and molecular characteristic of the EIV lineages, with focus on those detected in Asia from 2007 to 2017. The full genome phylogeny showed that FC1 and FC2 constituted separate and divergent lineages, without evidence of reassortment between the clades. While FC1 evolved as a single lineage, FC2 showed a divergent event around 2004 giving rise to two well-supported and coexisting sublineages, European and Asian. Furthermore, two different spread patterns of EIV in Asian countries were identified. The FC1 outbreaks were caused by independent introductions of EIV from the Americas, with the Asian isolates genetically similar to the contemporary American lineages. On the other hand, the FC2 strains detected in Asian mainland countries conformed to an autochthonous monophyletic group with a common ancestor dated in 2006 and showed evidence of an endemic circulation in a local host. Characteristic aminoacidic signature patterns were detected in all viral proteins in both Asian-FC1 and FC2 populations. Several changes were located at the top of the HA1 protein, inside or near antigenic sites. Further studies are needed to assess the potential impact of these antigenic changes in vaccination programs.IMPORTANCE The complex and continuous antigenic evolution of equine influenza viruses (EIVs) remains a major hurdle for vaccine development and the design of effective immunization programs. The present study provides a comprehensive analysis showing the EIV evolutionary dynamics, including the spread and circulation within the Asian continent and its relationship to global EIV populations over a 10-year period. Moreover, we provide a better understanding of EIV molecular evolution in Asian countries and its consequences on the antigenicity. The study underscores the association between the global horse movement and the circulation of EIV in this region. Understanding EIV evolution is imperative in order to mitigate the risk of outbreaks affecting the horse industry and to help with the selection of the viral strains to be included in the formulation of future vaccines.

Mammalian Adaptation of an Avian Influenza A Virus Involves Stepwise Changes in NS1.

ABSTRACTInfluenza A viruses (IAVs) are common pathogens of birds that occasionally establish endemic infections in mammals. The processes and mechanisms that result in IAV mammalian adaptation are poorly understood. The viral nonstructural 1 (NS1) protein counteracts the interferon (IFN) response, a central component of the host species barrier. We characterized the NS1 proteins of equine influenza virus (EIV), a mammalian IAV lineage of avian origin. We showed that evolutionarily distinct NS1 proteins counteract the IFN response using different and mutually exclusive mechanisms: while the NS1 proteins of early EIVs block general gene expression by binding to cellular polyadenylation-specific factor 30 (CPSF30), NS1 proteins from more evolved EIVs specifically block the induction of IFN-stimulated genes by interfering with the JAK/STAT pathway. These contrasting anti-IFN strategies are associated with two mutations that appeared sequentially and were rapidly selected for during EIV evolution, highlighting the importance of evolutionary processes in immune evasion mechanisms during IAV adaptation.IMPORTANCE Influenza A viruses (IAVs) infect certain avian reservoir species and occasionally transfer to and cause epidemics of infections in some mammalian hosts. However, the processes by which IAVs gain the ability to efficiently infect and transmit in mammals remain unclear. H3N8 equine influenza virus (EIV) is an avian-origin virus that successfully established a new lineage in horses in the early 1960s and is currently circulating worldwide in the equine population. Here, we analyzed the molecular evolution of the virulence factor nonstructural protein 1 (NS1) and show that NS1 proteins from different time periods after EIV emergence counteract the host innate immune response using contrasting strategies, which are associated with two mutations that appeared sequentially during EIV evolution. The results shown here indicate that the interplay between virus evolution and immune evasion plays a key role in IAV mammalian adaptation.

Evolution of equine influenza viruses (H3N8) during a Brazilian outbreak, 2015.

Equine influenza is one of the major respiratory infectious diseases in horses. An equine influenza virus outbreak was identified in vaccinated and unvaccinated horses in a veterinary school hospital in São Paulo, SP, Brazil, in September 2015. The twelve equine influenza viruses isolated belonged to Florida Clade 1. The hemagglutinin and neuraminidase amino acid sequences were compared with the recent isolates from North and South America and the World Organisation for Animal Health recommended Florida Clade 1 vaccine strain. The hemagglutinin amino acid sequences had nine substitutions, compared with the vaccine strain. Two of them were in antigenic site A (A138S and G142R), one in antigenic site E (R62K) and another not in antigenic site (K304E). The four substitutions changed the hydrophobicity of hemagglutinin. Three distinct genetic variants were identified during the outbreak. Eleven variants were found in four quasispecies, which suggests the equine influenza virus evolved during the outbreak. The use of an out of date vaccine strain or updated vaccines without the production of protective antibody titers might be the major contributing factors on virus dissemination during this outbreak.

Analysis of codon usage bias and evolution of equine influenza viruses

Analysis of codon usage bias and evolution of equine influenza viruses

Phylogenetically distinct equine influenza viruses show different tropism for the swine respiratory tract.

Influenza A viruses circulate in a wide range of animals. H3N8 equine influenza virus (EIV) is an avian-origin virus that has established in dogs as canine influenza virus (CIV) and has also been isolated from camels and pigs. Previous work suggests that mutations acquired during EIV evolution might have played a role in CIV emergence. Given the potential role of pigs as a source of human infections, we determined the ability of H3N8 EIVs to replicate in pig cell lines and in respiratory explants. We show that phylogenetically distinct EIVs display different infection phenotypes along the pig respiratory tract, but not in cell lines. Our results suggest that EIV displays a dynamic host range along its evolutionary history, supporting the view that evolutionary processes play important roles in host range and tropism and also underscoring the utility of using explant cultures to study influenza pathogenesis.

An efficient genome sequencing method for equine influenza [H3N8] virus reveals a new polymorphism in the PA-X protein.

BackgroundH3N8 equine influenza virus (EIV) has caused disease outbreaks in horses across the world since its first isolation in 1963. However, unlike human, swine and avian influenza, there is relatively little sequence data available for this virus. The majority of published sequences are for the segment encoding haemagglutinin (HA), one of the two surface glycoproteins, making it difficult to study the evolution of the other gene segments and determine the level of reassortment occurring between sub-lineages.MethodsTo facilitate the generation of full genome sequences for EIV, we developed a simple, cost-effective and efficient method. M13-tagged primers were used to amplify short, overlapping RT-PCR products, which were then sequenced using Sanger dideoxynucleotide sequencing technology. We also modified a previously published method, developed for human H3N2 and avian H5N1 influenza viruses, which was based on the ligation of viral RNA and subsequent amplification by RT-PCR, to sequence the non-coding termini (NCRs). This necessitated the design of novel primers for an N8 neuraminidase segment.ResultsTwo field isolates were sequenced successfully, A/equine/Lincolnshire/1/07 and A/equine/Richmond/1/07, representative of the Florida sublineage clades 1 and 2 respectively. A total of 26 PCR products varying in length from 400–600 nucleotides allowed full coverage of the coding sequences of the eight segments, with sufficient overlap to allow sequence assembly with no primer-derived sequences. Sequences were also determined for the non-coding regions and revealed cytosine at nucleotide 4 in the polymerase segments. Analysis of EIV genomes sequenced using these methods revealed a novel polymorphism in the PA-X protein in some isolates.ConclusionsThese methods can be used to determine the genome sequences of EIV, including the NCRs, from both clade 1 and clade 2 of the Florida sublineage. Full genomes were covered efficiently using fewer PCR products than previously reported methods for influenza A viruses, the techniques used are affordable and the equipment required is available in most research laboratories. The adoption of these methods will hopefully allow for an increase in the number of full genomes available for EIV, leading to improved surveillance and a better understanding of EIV evolution.Electronic supplementary materialThe online version of this article (doi:10.1186/1743-422X-11-159) contains supplementary material, which is available to authorized users.

Development and Efficacy of the Recombinant Canarypox‐Based Equine Influenza (EI) Vaccine Updated According to the Last OIE Expert Surveillance Panel Recommendations

Reasons for performing studySince 2010, the OIE expert surveillance panel (ESP) recommends that EI vaccines contain representative equine influenza virus (EIV) strains from the Florida clade 1 and 2 sub‐lineages for optimal protection against EIV currently circulating worldwide. To date, no EI vaccine commercially available in the EU meets this recommendation.ObjectivesThis report summarises the development process of a fully updated recombinant canarypox‐based EI vaccine, including clinical efficacy results against EIV strain A/eq/Richmond/1/07.Study designThe EI vaccine ProteqFlu containing 2 recombinant canarypox viruses expressing the haemagglutinin of EIV strains was updated by replacing the A/eq/Newmarket/2/93 strain with the A/eq/Richmond/1/07 isolate (Florida clade 2 sub‐lineage) and keeping A/eq/Ohio/03 (Florida clade 1 sub‐lineage), to meet the last OIE recommendations. The updated EI vaccine was tested for efficacy in the Welsh mountain pony model.MethodsThe mode of action, production steps and efficacy of the updated EI vaccine will be presented. Efficacy was tested in a group of 7 ponies vaccinated twice, 5 weeks apart. Protective antibody response were measured and challenged by experimental infection with the A/eq/Richmond/1/07 EIV strain. Clinical signs of disease and virus shedding were compared with control unvaccinated ponies (n = 7).ResultsSignificant protection was measured in vaccinated ponies, which supports the vaccine registration.ConclusionsThe recombinant canarypox‐based EI vaccine was already the first EI vaccine to meet the 2004 OIE ESP recommendations and was successfully used in Australia during the 2007 EI outbreak. This new version will be the first fully updated EI vaccine available in the EU, which will help to minimise the increasing risk of vaccine breakdown due to constant EIV evolution through antigenic drift. EI vaccination remains one of the most effective tools to prevent or limit the impact of EI, as clearly illustrated by the limited frequency and scale of EI outbreaks.Ethical animal research: All animal work received ethical approval. Sources of funding: The study was funded by Merial SAS. Competing interests: R.P. reports no conflict of interest. L.L., A.D., J‐L.T. and J.M. are employed by the study sponsor.

Antigenic and Genetic Evolution of Equine Influenza A (H3N8) Virus from 1968 to 2007

Equine influenza virus is a major respiratory pathogen in horses, and outbreaks of disease often lead to substantial disruption to and economic losses for equestrian industries. The hemagglutinin (HA) protein is of key importance in the control of equine influenza because HA is the primary target of the protective immune response and the main component of currently licensed influenza vaccines. However, the influenza virus HA protein changes over time, a process called antigenic drift, and vaccine strains must be updated to remain effective. Antigenic drift is assessed primarily by the hemagglutination inhibition (HI) assay. We have generated HI assay data for equine influenza A (H3N8) viruses isolated between 1968 and 2007 and have used antigenic cartography to quantify antigenic differences among the isolates. The antigenic evolution of equine influenza viruses during this period was clustered: from 1968 to 1988, all isolates formed a single antigenic cluster, which then split into two cocirculating clusters in 1989, and then a third cocirculating cluster appeared in 2003. Viruses from all three clusters were isolated in 2007. In one of the three clusters, we show evidence of antigenic drift away from the vaccine strain over time. We determined that a single amino acid substitution was likely responsible for the antigenic differences among clusters.

Genetic evolution of equine influenza viruses isolated in China

China experienced an outbreak of equine influenza during 2007-2008. Meanwhile, its neighbor countries, such as Mongolia, India and Japan, have also been affected by various influenza virus strains in each country. Phylogenetic analysis showed that the newly emerging Chinese strains belong to Florida sublineage clade 2, as well as the Indian strain Jammu-Katra/6/08 and the Mongolian strain Mongolia/1/08. All of these strains were derived from European strains of this clade, such as the Newmarket/1/07 and Cheshire/1/07 strains, but these were not related to Japanese strains isolated around the same time (Florida sublineage clade 1) or to Chinese strains isolated in the 1990s (European lineage). Some unique amino acid changes were found in the antigenic sites in Asian strains of Florida sublineage clade 2. Moreover, the loss of a glycosylation site was found in the Liaoning/9/08 strain. From these studies, we have determined that equine influenza viruses in China have evolved with some new characteristics during recent years, and this emphasizes the importance of continued equine influenza virus surveillance in China.