Transboundary spread of equine influenza viruses (H3N8) in West and Central Africa: Molecular characterization of identified viruses during outbreaks in Niger and Senegal, in 2019.

Since November 2018, several countries in West and Central Africa have reported mortalities in donkeys and horses. Specifically, more than 66,000 horses and donkeys have succumbed to disease in Burkina Faso, Chad, Cameroon, The Gambia, Ghana, Mali, Niger, Nigeria, and Senegal. Strangles caused by Streptococcus equi subsp equi, African Horse Sickness (AHS) virus, and Equine influenza virus (EIV) were all suspected as potential causative agents. This study reports the identification of EIV in field samples collected in Niger and Senegal. Phylogenetic analysis of the hemagglutinin and neuraminidase genes revealed that the identified viruses belonged to clade 1 of the Florida sublineage and were very similar to viruses identified in Nigeria in 2019. Interestingly, they were also more similar to EIVs from recent outbreaks in South America than to those in Europe and the USA. This is one of the first reports providing detailed description and characterization of EIVs in West and Central Africa region.

Characterizing Emerging Canine H3 Influenza Viruses.

The continual emergence of novel influenza A strains from non-human hosts requires constant vigilance and the need for ongoing research to identify strains that may pose a human public health risk. Since 1999, canine H3 influenza A viruses (CIVs) have caused many thousands or millions of respiratory infections in dogs in the United States. While no human infections with CIVs have been reported to date, these viruses could pose a zoonotic risk. In these studies, the National Institutes of Allergy and Infectious Diseases (NIAID) Centers of Excellence for Influenza Research and Surveillance (CEIRS) network collaboratively demonstrated that CIVs replicated in some primary human cells and transmitted effectively in mammalian models. While people born after 1970 had little or no pre-existing humoral immunity against CIVs, the viruses were sensitive to existing antivirals and we identified a panel of H3 cross-reactive human monoclonal antibodies (hmAbs) that could have prophylactic and/or therapeutic value. Our data predict these CIVs posed a low risk to humans. Importantly, we showed that the CEIRS network could work together to provide basic research information important for characterizing emerging influenza viruses, although there were valuable lessons learned.

Prevalence of Influenza A Viruses in Ducks Sampled in Northwestern Minnesota and Evidence for Predominance of H3N8 and H4N6 Subtypes in Mallards, 2007-2016.

Long-term comprehensive studies of avian influenza virus subtypes in ducks not only contribute to understanding variations and patterns of subtype diversity, but also can be important in defining seasonal and temporal risks associated with transmission of potentially highly pathogenic H5 and H7 subtypes to domestic poultry. We analyzed influenza A virus (IAV) surveillance data from dabbling ducks collected at an important migratory stopover site in northwestern Minnesota from 2007-2016 and identified prevalence and subtype diversity throughout this period. In total, 13,228 cloacal and oropharyngeal swabs from waterfowl were tested over the 10-year period; the majority of these waterfowl were mallards sampled from late August through late September (n = 9133). From these, 1768 IAVs were isolated (19.4% mean annual prevalence, ranging from 11.0% in 2007 to 32.8% in 2011), and both hemagglutinin (HA) and neuraminidase were identified for 1588. Although subtype diversity and prevalence varied by year, H3 and H4 HA subtypes predominated in all years, accounting for 65.7% of the observed HA subtype diversity. The mechanisms driving this consistent pattern of subtype diversity and predominance are not understood but may include factors at the host, population, and virus level.

Prevalencia de virus de influenza A en patos muestreados en el noroeste de Minnesota y evidencia de predominio de los subtipos H3N8 y H4N6 en patos de collar entre los años 2007 al 2016. Los estudios exhaustivos a largo plazo de subtipos de virus de la influenza aviar en patos no solo contribuyen a comprender las variaciones y patrones de diversidad de subtipos, sino que también pueden ser importantes para definir los riesgos estacionales y temporales asociados con la transmisión de subtipos H5 y H7 potencialmente altamente patógenos para la avicultura comercial. Analizamos los datos de vigilancia del virus de la influenza A de patos chapoteadores recolectados en un sitio de descanso migratorio importante en el noroeste de Minnesota desde el año 2007 al 2016 y se identificó la prevalencia y la diversidad de subtipos a lo largo de este período. En total, 13,228 hisopos cloacales y orofaríngeos de aves acuáticas se analizaron durante el período de diez años; la mayoría de estas aves acuáticas eran patos silvestres muestreados desde finales de agosto hasta finales de septiembre (n = 9133). De estas muestras, 1768 virus de la influenza aviar fueron aislados (prevalencia anual media de 19.4%, y con un rango de 11.0% en el 2007 a 32.8% en 2011), y tanto la hemaglutinina (HA) como la neuraminidasa fueron identificadas para 1588 virus. Aunque la diversidad de subtipos y la prevalencia variaron por año, los subtipos de hemaglutinina H3 y H4 predominaron en todos los años, representando el 65.7% de la diversidad de subtipos observada para la hemaglutinina. Los mecanismos que impulsan este patrón consistente de diversidad de subtipos y predominio no se comprenden, pero pueden incluir factores a nivel del hospedador, de la población y del virus. Abbreviations: CL = cloacal; HA = hemagglutinin; IAV = influenza A virus; NA = neuraminidase; NWR = National Wildlife Refuge; RRT-PCR = real-time reverse transcriptase PCR; WMA = Wildlife Management Area.

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.

Isolation of H8N4 avian influenza virus from wild birds in Shanghai, China.

The H8 subtype viruses are rarely isolated from wild ducks. Shanghai is one of the important wintering or stopover sites on the East Asia-Australia Migration Flyway. An influenza virus, subtype H8N4, was firstly isolated from a common teal (Anas crecca) in Shanghai during 2017-2018 in this study. To clarify the genetic characteristics of the H8N4 virus, the whole genome sequences were analyzed. Phylogenetic analysis of the hemagglutinin and neuraminidase genes showed that they shared highest nucleotide identity (99.19%-99.64%) with the Japan duck-origin H8N4 virus collected in 2016 (A/duck/Aichi/231003/2016) and belonged to the Eurasian-like avian lineage. Six other genes of the H8N4 isolated virus were all highly similar to the corresponding genes of a wide range of AIV subtypes including H9N2, H5N7, H3N8, H1N2, H4N6 and H1N1. The results indicated that the H8N4 virus was a multiple reassortant virus. The study emphasized that the continuous surveillance of influenza virus in wild birds should be strengthened. Keywords: avian influenza virus; H8N4; phylogenetic analysis; Shanghai.

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

Abstract 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.

Isolation and characterization of an avian-origin H3N8 canine influenza virus from a dog in eastern China.

Previous studies have shown that dogs are susceptible to influenza A viruses, and the close contact between dogs and humans poses a threat to public health. In 2015, a novel H3N8 influenza virus was isolated from a dog in eastern China. This strain was characterized by whole-genome sequencing with subsequent phylogenetic analysis and genetic comparison and found to be most closely related to avian influenza viruses co-circulating in China. It was able to replicate in mice without prior adaptation. The continued circulation of this novel H3N8 influenza virus in dogs could endanger other mammalian species.

Emergence of H3N8 equine influenza virus in donkeys in China in 2017.

Equine influenza virus is a major respiratory pathogen in horses. Although both horses and donkeys belong to the genus Equus, donkey infection with influenza viruses is rare. In March 2017, an influenza outbreak occurred in donkeys in Shandong province, China. The causative virus, A/donkey/Shandong/1/2017(H3N8), was isolated from a dead donkey. Genetic analysis indicated that the virus originated from influenza A (H3N8) clade 2 of the Florida sub-lineage that has been circulating in Asian equine populations. Comparison of the deduced amino acid sequence of the HA gene of this causative virus with that of the A/equine/Richmond/1/2007 vaccine strain showed that substitutions had occurred in the antigenic regions A, B, and C. This study provides insight into the currently circulating and newly emerging H3N8 strains in donkeys in China.

Phylogenetic Analysis and Characterization of a Sporadic Isolate of Equine Influenza A H3N8 from an Unvaccinated Horse in 2015.

Equine influenza, caused by the H3N8 subtype, is a highly contagious respiratory disease affecting equid populations worldwide and has led to serious epidemics and transboundary pandemics. This study describes the phylogenetic characterization and replication kinetics of recently-isolated H3N8 virus from a nasal swab obtained from a sporadic case of natural infection in an unvaccinated horse from Montana, USA. The nasal swab tested positive for equine influenza by Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR). Further, the whole genome sequencing of the virus confirmed that it was the H3N8 subtype and was designated as A/equine/Montana/9564-1/2015 (H3N8). A BLASTn search revealed that the polymerase basic protein 1 (PB1), polymerase acidic (PA), hemagglutinin (HA), nucleoprotein (NP), and matrix (M) segments of this H3N8 isolate shared the highest percentage identity to A/equine/Tennessee/29A/2014 (H3N8) and the polymerase basic protein 2 (PB2), neuraminidase (NA), and non-structural protein (NS) segments to A/equine/Malaysia/M201/2015 (H3N8). Phylogenetic characterization of individual gene segments, using currently available H3N8 viral genomes, of both equine and canine origin, further established that A/equine/Montana/9564-1/2015 belonged to the Florida Clade 1 viruses. Interestingly, replication kinetics of this H3N8 virus, using airway derived primary cells from multiple species, such as equine, swine, bovine, and human lung epithelial cells, demonstrated appreciable titers, when compared to Madin-Darby canine kidney epithelial cells. These findings indicate the broad host spectrum of this virus isolate and suggest the potential for cross-species transmissibility.

Neutralization antibody response to booster/priming immunization with new equine influenza vaccine in Japan.

Equine influenza (EI) vaccine has been widely used. However, the causative EI virus (H3N8) undergoes continuous antigenic drift, and the vaccine strains must be periodically reviewed and if necessary, updated to maintain vaccine efficacy against circulating viruses. In 2016, the Japanese vaccine was updated by replacing the old viruses with the Florida sub-lineage Clade (Fc) 2 virus, A/equine/Yokohama/aq13/2010 (Y10). We investigated the virus neutralization (VN) antibody response to Fc2 viruses currently circulating in Europe, after booster or primary immunization with the new vaccine. These European viruses have the amino acid substitution A144V or I179V of the hemagglutinin. In horses that had previously received a primary course and bi-annual boosters with the old vaccine booster, immunization with the updated vaccine increased the VN antibody levels against the European Fc2 viruses as well as Y10. There were no significant differences in the VN titers against Y10 and the Fc2 viruses with A144V or I179V substitution in horses that had received a primary course of the updated vaccine. However, a mixed primary course where the first dose was the old vaccine and the second dose was the updated vaccine, reduced VN titers against the European viruses compared to that against Y10. In summary, the new vaccine affords horses protective level of VN titers against the Fc2 viruses carrying A144V or I179V substitution, but our results suggest that the combination of the old and new vaccines for primary immunization would not be optimum.

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.

Genetic and phylogenetic characterizations of a novel genotype of highly pathogenic avian influenza (HPAI) H5N8 viruses in 2016/2017 in South Korea.

During the outbreaks of highly pathogenic avian influenza (HPAI) H5N6 viruses in 2016 in South Korea, novel H5N8 viruses were also isolated from migratory birds. Phylogenetic analysis revealed that the HA gene of these H5N8 viruses belonged to clade 2.3.4.4, similarly to recent H5Nx viruses, and originated from A/Brk/Korea/Gochang1/14(H5N8), a minor lineage of H5N8 that appeared in 2014 and then disappeared. At least four reassortment events occurred with different subtypes (H5N8, H7N7, H3N8 and H10N7) and a chicken challenge study revealed that they were classified as HPAI viruses according to OIE criteria.

Phylogenetic analysis and pathogenicity of H3 subtype avian influenza viruses isolated from live poultry markets in China.

H3 subtype influenza A virus is one of the main subtypes that threats both public and animal health. However, the evolution and pathogenicity of H3 avian influenza virus (AIV) circulating in domestic birds in China remain largely unclear. In this study, seven H3 AIVs (four H3N2 and three H3N8) were isolated from poultry in live poultry market (LPM) in China. Phylogenetic analyses of full genomes showed that all viruses were clustered into Eurasian lineage, except N8 genes of two H3N8 isolates fell into North American lineage. Intriguingly, the N8 gene of one H3N8 and PB2, PB1, NP and NS of two H3N2 isolates have close relationship with those of the highly pathogenic H5N8 viruses circulating in Korea and United States, suggesting that the H3-like AIV may contribute internal genes to the highly pathogenic H5N8 viruses. Phylogenetic tree of HA gene and antigenic cross-reactivity results indicated that two antigenically different H3 viruses are circulating in LPM in China. Most of the H3 viruses replicated in mice lung and nasal turbinate without prior adaptation, and the representative H3 viruses infected chickens without causing clinical signs. The reassortment of H3 subtype influenza viruses warrants continuous surveillance in LPM in China.

Characterisation of the epidemic strain of H3N8 equine influenza virus responsible for outbreaks in South America in 2012.

BACKGROUND: An extensive outbreak of equine influenza occurred across multiple countries in South America during 2012. The epidemic was first reported in Chile then spread to Brazil, Uruguay and Argentina, where both vaccinated and unvaccinated animals were affected. In Brazil, infections were widespread within 3months of the first reported cases. Affected horses included animals vaccinated with outdated vaccine antigens, but also with the OIE-recommended Florida clade 1 strain South Africa/4/03. METHODS: Equine influenza virus strains from infected horses were isolated in eggs, then a representative strain was subjected to full genome sequencing using segment-specific primers with M13 tags. Phylogenetic analyses of nucleotide sequences were completed using PhyML. Amino acid sequences of haemagglutinin and neuraminidase were compared against those of vaccine strains and recent isolates from America and Uruguay, substitutions were mapped onto 3D protein structures using PyMol. Antigenic analyses were completed by haemagglutination-inhibition assay using post-infection ferret sera. RESULTS: Nucleotide sequences of the haemaglutinin (HA) and neuraminidase (NA) genes of Brazilian isolate A/equine/Rio Grande do Sul/2012 were very similar to those of viruses belonging to Florida clade 1 and clustered with contemporary isolates from the USA. Comparison of their amino acid sequences against the OIE-recommended Florida clade 1 vaccine strain A/equine/South Africa/4/03 revealed five amino acid substitutions in HA and seven in NA. Changes in HA included one within antigenic site A and one within the 220-loop of the sialic acid receptor binding site. However, antigenic analysis by haemagglutination inhibition (HI) assay with ferret antisera raised against representatives of European, Kentucky and Florida sublineages failed to indicate any obvious differences in antigenicity. CONCLUSIONS: An extensive outbreak of equine influenza in South America during 2012 was caused by a virus belonging to Florida clade 1, closely related to strains circulating in the USA in 2011. Despite reports of vaccine breakdown with products containing the recommended strain South Africa/03, no evidence was found of significant antigenic drift. Other factors may have contributed to the rapid spread of this virus, including poor control of horse movement.

The first reported Florida clade 1 virus in the Nordic countries, isolated from a Swedish outbreak of equine influenza in 2011.

Equine Influenza Virus (EIV) is a major cause of respiratory disease in horses and the virus constantly undergoes antigenic drift. Here we characterize and describe the HA1 and the NA genes of H3N8 within samples obtained from outbreaks in Sweden during November-December 2011. Both clade 1 and clade 2 viruses of the Florida sublineage were identified. The index case of clade 2 was transported to Sweden from Spain through the Netherlands, whereas the clade 1 had its origin from a Swedish stud farm. The clade 1 virus was efficiently spread between training yards by unvaccinated young horses, but vaccinated horses were also presented with clinical signs of respiratory disease. No virus of the Eurasian lineage was isolated during this outbreak. Clade 1 has previously been described in outbreaks in numerous of other countries, but this is the first time it has been detected in Sweden. The results from this study shows the importance of including both clade 1 and clade 2 of the Florida sublineage in equine influenza vaccines, supporting the ESP and OIE recommendations.

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.

Multiple alignment comparison of the non-structural genes of three strains of equine influenza viruses (H3N8) isolated in Morocco.

BACKGROUND: Three equine influenza viruses, A/equine/Nador/1/1997(H3N8), A/equine/Essaouira/2/2004(H3N8), and A/equine/Essaouira/3/2004(H3N8), were isolated from different Equidae during local respiratory disease outbreaks in Morocco in 1997 and 2004. Their non-structural (NS) genes were amplified and sequenced. RESULTS: The results show high homology of NS nucleotide sequences of A/equine/Nador/1/1997 with European strains (i.e., A/equine/newmarket/2/93 and A/equine/Grobois/1/1998) and clustered into the European lineage. However, NS gene of A/equine/Essaouira/2/2004(H3N8) and A/equine/Essaouira/3/2004(H3N8) strains indicated high homology with equine influenza strains that had circulated before 1990 (A/equine/Fontainbleu/1/1979(H3N8), which belonged to a pre-divergent phase Amino acid sequence comparison of the NS1 protein with reference strain A/equine/Miami/1963(H3N8) shows that the A/equine/Nador/1/1997(H3N8) strain has 12 substitutions at the residues D/24/N, R/44/K, S/48/I, R/67/Q, A/86/V, E/139/K, A/112/T, E/186/K, L/185/F, A/223/E, S/213/T and S/228/P. In both A/equine/Essaouira/2/2004(H3N8) and A/equine/Essaouira/3/2004(H3N8) strains, the NS1 sequences present one common mutation at the residue: S/228/P. CONCLUSION: It seems that all of these substitutions are not produced at the key residues of the RNA-binding domain (RBD) and the effector domain (ED). Consequently, we can suppose that they will not affect the potency of inhibition of cellular defences, and the virulence of the Moroccan equine strains will be maintained.

Equine influenza A(H3N8) virus isolated from Bactrian camel, Mongolia.

Because little is known about the ecology of influenza viruses in camels, 460 nasal swab specimens were collected from healthy (no overt illness) Bactrian camels in Mongolia during 2012. One specimen was positive for influenza A virus (A/camel/Mongolia/335/2012[H3N8]), which is phylogenetically related to equine influenza A(H3N8) viruses and probably represents natural horse-to-camel transmission.

Respiratory transmission of an avian H3N8 influenza virus isolated from a harbour seal.

The ongoing human H7N9 influenza infections highlight the threat of emerging avian influenza viruses. In 2011, an avian H3N8 influenza virus isolated from moribund New England harbour seals was shown to have naturally acquired mutations known to increase the transmissibility of highly pathogenic H5N1 influenza viruses. To elucidate the potential human health threat, here we evaluate a panel of avian H3N8 viruses and find that the harbour seal virus displays increased affinity for mammalian receptors, transmits via respiratory droplets in ferrets and replicates in human lung cells. Analysis of a panel of human sera for H3N8 neutralizing antibodies suggests that there is no population-wide immunity to these viruses. The prevalence of H3N8 viruses in birds and multiple mammalian species including recent isolations from pigs and evidence that it was a past human pandemic virus make the need for surveillance and risk analysis of these viruses of public health importance.

Diversity of interferon inducible Mx gene in horses and association of variations with susceptibility vis-à-vis resistance against equine influenza infection.

Equine influenza (EI) is primarily an infection of the upper respiratory tract and is one of the major infectious respiratory diseases of economic importance in equines. Re-emergence of the disease, species jumping by H3N8 virus in canines and possible threat of human pandemic due to the unpredictable nature of the virus have necessitated research on devising strategies for preventing the disease. The myxovirus resistance protein (Mx) has been reported to confer resistance to Orthomyxo virus infection by modifying cellular functions needed along the viral replication pathway. Polymorphisms and differential antiviral activities of Mx gene have been reported in pigs and chicken. Here we report the diversity of Mx gene, its expression in response to stimulation with interferon (IFN) α/ß and their association with EI resistance and susceptibility in Marwari horses. Blood samples were collected from horses declared positive for equine influenza and in contact animals with a history of no clinical signs. Mx gene was amplified by reverse transcription from total RNA isolated from peripheral blood mononuclear cells (PBMCs) stimulated with IFN α/ß using gene specific primers. The amplified gene products from representative samples were cloned and sequenced. Nucleotide sequences and deduced amino acid sequences were analyzed. Out of a total 24 amino acids substitutions sorting intolerant from tolerant (SIFT) analysis predicted 13 substitutions with functional consequences. Five substitutions (V67A, W123L, E346Y, N347Y, S689N) were observed only in resistant animals. Evolutionary distances based on nucleotide sequences with in equines ranged between 0.3-2.0% and 20-24% with other species. On phylogenetic analysis all equine sequences clustered together while other species formed separate clades.