Equine Influenza Virus Research Articles

Epidemiological and virological investigations of equine influenza outbreaks in Ireland (2010–2012)

Outbreaks of equine influenza (EI) in endemic populations cause disruption and economic loss. To identify (i) factors involved in the spread of EI (ii) virus strains responsible for outbreaks (iii) single radial haemolysis (SRH) antibody levels correlating with protection against current virus strains (iv) evidence of vaccination breakdown. RT-PCR, virus isolation and SRH were carried out on nasopharyngeal swabs and blood samples collected from horses, ponies and donkeys on affected premises. Data relating to 629 samples from 135 equidae were analysed. Outbreaks were sporadic, self limiting and associated with the movement of horses. Vaccination status and age influenced clinical signs of disease while housing and fomites contributed to virus spread. Subclinical infection as defined as a horse which tested positive by one or more of the following; RT-PCR, virus isolation and seroconversion in the absence of clinical signs, was identified in 9% of animals. Of the horses with up to date vaccination records 32% developed clinical signs. Vaccine breakdown occurred among horses vaccinated with all four commercially available vaccines. Analysis of HA1 sequence data generated for 26 viruses indicated that they all belonged to clade 2 of the Florida sublineage. Higher SRH antibody levels were required for both clinical and virological protection than reported in studies where vaccine strains were antigenically and genetically similar to those circulating in the field. The results of this study therefore support the OIE recommendations that vaccines be updated to include representatives of both clades of the Florida sublineage.

The genetics of virus particle shape in equine influenza A virus

BackgroundMany human strains of influenza A virus produce highly pleomorphic virus particles that at the extremes can be approximated as either spheres of around 100 nm diameter or filaments of similar cross‐section but elongated to lengths of many microns. The role filamentous virions play in the virus life cycle remains enigmatic.Objectives/MethodsHere, we set out to define the morphology and genetics of virus particle shape in equine influenza A virus, using reverse genetics and microscopy of infected cells.Results and ConclusionsThe majority of H3N8 strains tested were found to produce filamentous virions, as did the prototype H7N7 A/eq/Prague/56 strain. The exception was the prototype H3N8 isolate, A/eq/Miami/63. Reassortment of equine influenza virus M genes from filamentous and non‐filamentous strains into the non‐filamentous human virus A/PR/8/34 confirmed that segment 7 is a major determinant of particle shape. Sequence analysis identified three M1 amino acid polymorphisms plausibly associated with determining virion morphology, and the introduction of these changes into viruses confirmed the importance of two: S85N and N231D. However, while either change alone affected filament production, the greatest effect was seen when the polymorphisms were introduced in conjunction. Thus, influenza A viruses from equine hosts also produce filamentous virions, and the major genetic determinants are set by the M1 protein. However, the precise sequence determinants are different to those previously identified in human or porcine viruses.

Identification of equine influenza virus infection in Asian wild horses (Equus przewalskii)

An outbreak of equine influenza was observed in the Asian wild horse population in Xinjiang Province, China, in 2007. Nasal swabs were collected from wild horses and inoculated into 9-10-day SPF embryonated eggs. The complete genome of the isolate was sequenced. A comparison of the amino acid sequence revealed that the isolate was an equine influenza virus strain, which we named A/equine/Xinjiang/4/2007. Each gene of the virus was found to have greater than 99 % homology to equine influenza virus strains of the Florida-2 sublineage, which were circulating simultaneously in China, and a lesser amount of homology was found to the strain A/equine/Qinghai/1/1994 (European lineage), which was isolated during the last outbreak in China. These observations were confirmed by phylogenetic analysis. In addition, the deduced amino acid sequence of the neuraminidase of the A/equine/Xinjiang/4/2007 strain was identical to that of A/equine/California/8560/2002, an American isolate, and was found to be similar to those of Florida-2 strains found in other countries by comparing them with nine other field strains that were isolated in China from 2007 to 2008. It is suggested that the neuraminidase segment of A/equine/Xinjiang/4/2007 may have been obtained from equine influenza virus strains from other countries. We report for the first time an outbreak of equine influenza in the Asian wild horse population, and the complete genome of the virus is provided and analyzed.

Phylogenetic and Molecular Characterization of the Equine Influenza Virus A (H3N8) Causing the 1997 and 2004 Outbreaks in Morocco

Reported here are the results of antigenic and genetic characterization of equine influenza strains causing local outbreaks reported in Morocco, respectively, in 1997 and 2004. The antigenic and genetic characterizations of the equine influenza virus H3N8 are reported here. The highest similarity between the HA1 nucleotide sequences of A/equine/Nador/1/1997 and those of A/equine/Rome/5/1991 and A/equine/Italy/1199/1992 demonstrate that A/equine/Nador/1/1997 belongs to the European lineage. On the other hand, A/equine/Essaouira/2/2004 and A/equine/Essaouira/3/2004 were classified in the predivergent lineage. The present work emphasizes the importance of a national influenza survey program, which requires a collaborative laboratory network to promote the collection and characterization (antigenic and genetic) of equine influenza viruses in real time.

Equine influenza vaccines in Europe: A view from the animal health industry

Equine influenza vaccines in <scp>E</scp>urope: A view from the animal health industry

Influenza vaccine strains: Licensing perspectives

Influenza vaccine strains: Licensing perspectives

Expression of the hemagglutinin HA1 subunit of the equine influenza virus using a baculovirus expression system

Equine influenza virus is a leading cause of respiratory disease in horses worldwide. Disease prevention is by vaccination with inactivated whole virus vaccines. Most current influenza vaccines are generated in embryonated hens’ eggs. Virions are harvested from allantoic fluid and chemically inactivated. Although this system has served well over the years, the use of eggs as the substrate for vaccine production has several well-recognized disadvantages (cost, egg supply, waste disposal and yield in eggs). The aim of this study was to evaluate a baculovirus system as a potential method for producing recombinant equine influenza hemagglutinin to be used as a vaccine. The hemagglutinin ectodomain (HA1 subunit) was cloned and expressed using a baculovirus expression vector. The expression was determined by SDS-PAGE and immunoblotting. A high yield, 20μg/ml of viral protein, was obtained from recombinant baculovirus-infected cells. The immune response in BALB/c mice was examined following rHA1 inoculation. Preliminary results show that recombinant hemagglutinin expressed from baculovirus elicits a strong antibody response in mice; therefore it could be used as an antigen for subunit vaccines and diagnostic tests.

Equine Influenza and Equine Herpesvirus Vaccination: Evaluation of Immune Response and Safety of Concurrent Administration of Equip® FT and Equip® EHV 1,4

AimsThe concurrent administration of EHV1,4 and EIV vaccines is not currently recommended. This study aimed to evaluate safety and immunogenicity of concurrent administration of Equip® FT and Equip® EHV1,4 (treatment group T04) as compared with vaccination with Equip® FT (treatment group T02) or Equip® EHV1,4 (treatment group T03) alone.MethodsEach treatment group included 10 ponies vaccinated on study Days 0 and 42. Immune responses to vaccine antigens were measured on Day 56. Tetanus antibody titres were determined with the toxin binding inhibition test (ToBI). Immunity to equine influenza virus (EIV) induced by vaccination was measured by single radial haemolysis (SRH; antibody response) and IFN gamma assay (a marker of cell‐mediated immunity; CMI). Immune responses to Herpesvirus 1,4 were measured by complement fixation (CF) and IFN gamma assay.ResultsConcurrent administration of EIV and EHV1,4 vaccines was well tolerated. Concurrent administration was proven to be efficacious against tetanus with similar serological immune response of treatment group T04 as compared with T02. Mean SRH results for ponies that received both vaccines were above 85 mm2 for A/eq/Borlange/91 and A/eq/Kentucky/98 antigens, a threshold associated with clinical protection against closely related EIV strains. The highest levels of IFN gamma response were measured in group T02. Although concurrent vaccination seemed to have an effect on both equine herpesvirus (EHV) serology and EHV CMI, EHV results obtained during this study were not conclusive and may have been influenced by a natural EHV‐4 infection occurring prior to study start.Conclusion and practical relevanceConcurrent administration of Equip® FT and Equip® EHV1,4 was well tolerated and induced mean serum antibody levels consistent with clinical protection for tetanus and EIV. However, it was not possible to gather relevant information about the EHV immune response under the conditions of this study.Ethical animal researchThe study was conducted under the Animal Health Trust Home Office Project License and with approval of the Zaventem Ethics Review Assessment team (Zoetis). Sources of funding: The study was sponsored by Pfizer/Zoetis Animal Health. Competing interests: Equip® FT and Equip® EHV1,4 are owned by Zoetis, who sponsored this study. Romain Paillot reports no conflict of interest. All other authors are employed by the study sponsor.

Equine Influenza and Tetanus Immunisation: Evaluation of Antibody Response and Safety of Equip® FT and Equip® F Administration after Reduction of the Primary Course Interval to 3 Weeks

AimsA 6‐week interval is currently recommended during the primary course of the equine influenza (EI) vaccines Equip® FT or Equip® F. This study aimed to evaluate immunogenicity and safety of a 3‐week accelerated interval in order to improve flexibility and to reduce the period of susceptibility to EI virus (EIV) infection.MethodsThree groups of 10 ponies were respectively vaccinated with the vaccine Equip® FT according to the recommended 6‐week V1–V2 interval (T02), Equip® F or Equip® FT according to an experimental accelerated 3‐week interval (T03 and T04, respectively). These groups were boosted (V3) with Equip® F 5 months after V2. Ten ponies in a control group were injected with saline (T01). The antibody response to the vaccine antigens (tetanus toxoid and EIV) were measured 2 weeks after V2 and V3 with the toxin binding inhibition test (ToBI) and single radial haemolysis (SRH), respectively. All animal work received ethical approval.ResultsThe accelerated schedule of immunisation was well tolerated. At both time points, in all vaccinated groups, SRH least squares means for A/eq/Borlange/91 and A/eq/Kentucky/98 antigens were well above 85 mm2, a level described previously to provide clinical protection against homologous EIV strains. Non inferiority of SRH least square means were demonstrated at 2 weeks post V2 for both antigens for T03 and T04, and for A/eq/Kentucky/98 antigen in group T04 after V3. For tetanus,ToBI least squares means were 86.3 iu/mI for T02 and 45.9 iu/ml for T04 after V2 and 2.6 iu/ml for T02 and 2.2 iu/ml for T04 after V3. A level of 0.02 iu/ml has been described as being protective in various publications.Conclusion and practical relevanceA 3‐week accelerated primary course interval was well tolerated and serology results suggested good immunogenicity.Ethical animal researchThe study was conducted under the Animal Health Trust Home Office Project License and with approval of the Zaventem Ethics Review Assessment team (Zoetis). Sources of funding: The study was sponsored by Pfizer/Zoetis Animal Health. Competing interests: Equip® FT is owned by Zoetis, who sponsored this study. Romain Paillot reports no conflict of interest. All other authors are employed by the study sponsor.

Complete Genomic Sequences of an H3N8 Equine Influenza Virus Strain Isolated in China

We report the complete genomic sequence of A/equine/Heilongjiang/1/2010, a strain of Florida sublineage clade 2 of H3N8 subtype equine influenza virus (EIV) isolated in northern China. This is the first announcement of a complete genomic sequence of EIV of such a clade in China.

Sparse evidence for equine or avian influenza virus infections among Mongolian adults with animal exposures

In recent years, Mongolia has experienced recurrent epizootics of equine influenza virus (EIV) among its 2·1 million horses and multiple incursions of highly pathogenic avian influenza (HPAI) virus via migrating birds. No human EIV or HPAI infections have been reported. In 2009, 439 adults in Mongolia were enrolled in a population-based study of zoonotic influenza transmission. Enrollment sera were examined for serological evidence of infection with nine avian, three human, and one equine influenza virus strains. Seroreactivity was sparse among participants suggesting little human risk of zoonotic influenza infection.

Mutations and phylogenetic analysis of the equine influenza virus (H3N8) nucleoprotein isolated in Morocco

The equine influenza (EI) is a highly contagious respiratory viral disease of equines. The aim of the present study was to determine the amino acid mutation sequences of the partial nucleoprotein (NP), which includes four epitopes of the equine leucocyte antigen (ELA) of A/equine/Nador/1/1997 (H3N8). These epitopes are critical for their binding to major histocompatibility molecule complex (MHC) class I and recognition by specific Cytotoxic T Lymphocytes (CTLs). The isolate was subjected to RT-PCR amplifications followed by sequencing analysis. Phylogenetic analysis showed that Moroccan isolate belongs to equine host-specific lineage and more closely related to Italian strains A/equine/Rome/5/1991 and A/equine/Italy/1062/1991. Amino acid sequence comparison of the NP showed that the strain A/equine/Nador/1/1997 has twelve substitutions at the residues T/284/A, A/286/T, R/293/K, I/299/V, V/312/I, N/319/K, S/344/L, V/353/I, M/374/I, C/377/N, N/397/S and R/452/K. All substitutions concerned both the interaction domains NP–NP and NP–PB2. However, the mutation N319 K enhances the NP–PB2 interaction and polymerase activity in mammalian infected cells. S/344/L mutation was located on the FEDLRVSNFI epitope (aa 338–347), this substitution is likely to help the virus to overcome the barrier of cell-mediated immunity of the host. The identified mutations were grouped into two groups, one included residues that facilitate the adaptation and evolution of influenza viruses within the equine lineage such as A/286/T, R/293/K, S/344/L, V/353/I and R/452/K, while the second contained the substitutions which enhance the virulence as polymerase activity (N319K) and mutations that affect CTL epitopes, resulting in an escape from immune surveillance by specific CTLs (S/344/L).

Surveillance of equine influenza viruses through the RESPE network in France from November 2005 to October 2010

The Réseau d'Epidémio-Surveillance en Pathologie Equine (RESPE, the French epidemiological network for equine diseases) is a network for epidemio-surveillance of major equine diseases based around sentry veterinarians in France. The aim of this study was to evaluate the contribution of RESPE to efficient surveillance of equine influenza virus (EIV) in France. Retrospective cross-sectional study. From November 2005 to October 2010, epidemiological and phylogenetic studies were performed on 1426 nasopharyngeal swabs received at the Frank Duncombe Laboratory. Detection was performed by real-time reverse transcription polymerase chain reaction using original primers and probes designed in the matrix protein gene. Phylogenetic analysis was carried out on the HA1 part of haemagglutinin gene amplified from 47 positive-testing samples. Epidemiological information was provided with the majority of samples submitted through RESPE. Of the 920 samples submitted by RESPE-associated veterinarians, 121 (13.1%) from 42 premises were positive for EIV, compared to 26 (5.1%) of the 607 samples received from non-RESPE associated veterinarians. The most extensive outbreak was observed between February and May 2009, affecting 70 horses on 23 premises, 15 of which were managed by RESPE-associated veterinarians. All strains belonged to the American lineage, Florida sublineage, Clade 1 and Clade 2. Clade 1 was identified only during the Grosbois episode. RESPE improved detection of EIV in France, enabled characterisation of the virus strains, yielded valuable information relating to the epidemiology of the disease and identified vaccine breakdown. Implementation of a similar surveillance network in other countries may reduce the economic losses associated with outbreaks of EIV.

Isolation and genetic characterization of naturally NS-truncated H3N8 equine influenza virus in South Korea

Equine influenza virus (EIV) causes a highly contagious respiratory disease in equids, with confirmed outbreaks in Europe, America, North Africa, and Asia. Although China, Mongolia, and Japan have reported equine influenza outbreaks, Korea has not. Since 2011, we have conducted a routine surveillance programme to detect EIV at domestic stud farms, and isolated H3N8 EIV from horses showing respiratory disease symptoms. Here, we characterized the genetic and biological properties of this novel Korean H3N8 EIV isolate. This H3N8 EIV isolate belongs to the Florida sublineage clade 1 of the American H3N8 EIV lineage, and surprisingly, possessed a non-structural protein (NS) gene segment, where 23 bases of the NS1-encoding region were naturally truncated. Our preliminary biological data indicated that this truncation did not affect virus replication; its effect on biological and immunological properties of the virus will require further study.

Genetic analysis of the PB1-F2 gene of equine influenza virus

Amino acid and phylogenetic analysis showed that PB1-F2 of H7N7 and H3N8 equine influenza virus (EIV) evolved into two and nine clades, respectively. The PB1-F2 gene of H7N7 EIVstrains isolated after 1973 was identified as being replaced by that of H3N8 EIV circulating simultaneously. All H7N7 EIVstrains before 1973 contained PB1-F2 of 34 aa, while 90 aa after 1973. Meanwhile, most H3N8 EIVstrains contained PB1-F2 of 90 or 81 aa. Evolutionary rate of PB1-F2 of H3N8 EIV was similar with that of PB1 in previous study. Meanwhile, PB1-F2 of H7N7 EIVevolved in significantly higher rate when compared with PB1-F2 of H3N8EIV. Codon usage analysis revealed that PB1-F2 gene of EIV was less biased, which was identified as being determined by three main factors: mutational bias, selection pressure, and gene length. Our studies first in details report the genetic evolution, evolutionary rate, and the factors influencing codon usage bias of PB1-F2 of EIV.

Genetic Analysis of the Neuraminidase (NA) Gene of Equine Influenza Virus (H3N8) from Epizootic of 2008–2009 in India

The neuraminidase (NA) gene sequences of four Indian equine influenza viruses (EIVs) isolated from epizootic in 2008 and 2009 were analyzed. The phylogenetic relationship and selection pressure of NA genes were established in comparison to other EIVs circulating worldwide along with the domains and motifs of the encoded protein to find out the significance of mutational changes. Among Indian isolates, two amino acid (aa) changes each in Mysore/12/08 (Asn67Tyr & Asp396Gly), Gopeshwar/1/09 (Ile49Val & Asp396Gly), and Uttarkashi/1/09 (Ile49Val & Asp396Gly) isolates were observed in respect to Jammu-Katra/06/08 isolate. Amino acid (aa) sequence analysis also revealed five consistent aa residue changes viz, Gly/Arg40Glu, Tyr66His, Val191Ile, Val209Ile and Asp235Asn in Asian including Indian isolates, Spain/07 and Spain/09 isolates in comparison to other EIVs circulating worldwide. The topology of the phylogenetic tree revealed that the Indian, Chinese, Mongolian and Kazakhstan isolates together formed a subgroup with Yokohama/10 isolate. Spain/07 & Spain/09 isolates showed closest clustering with Asian isolates. This indicates that non-synonymous mutations in Asian isolates with temporal pattern originating from Spain/07, led to the subgroup of the Asian isolates within Florida clade 2 sublineage. The analysis of the predicted secondary structure has not shown any significant difference in the NA proteins of all Indian isolates. Fixed-effects likelihood (FEL) analysis of the selection pressure revealed three codons (43, 355 & 434) under positive selection pressure. The overall evolutionary changes (ω value) of 3.4 indicates NA gene to be under strong selection pressure. Further, seven putative N-glycosylation sites were observed in the NA protein. The mapping of specific aa changes, their mutational and functional analysis need to be carried out to ascertain their role in pathogenecity of the virus.

Duration of equine influenza virus shedding and infectivity in immunised horses after experimental infection with EIV A/eq2/Richmond/1/07

Equine influenza (EI) is a major respiratory disease of horses. Recent outbreaks of EI have demonstrated the ease with which EI virus (EIV) can be transmitted internationally. This study aimed to improve our understanding of EIV shedding after infection of vaccinated horses, which would inform possible changes to current quarantine requirements. Our objectives were to compare commonly used diagnostic tests and to evaluate the relative merits of nasal and nasopharyngeal swabs for detection of EIV in vaccinated and unvaccinated ponies following EIV infection and to use these data to inform optimal quarantine procedures for the safe international movement of horses. Five ponies vaccinated against EI were infected experimentally with A/eq/Richmond/1/07 (Florida clade 2), 11 weeks after V2. Nasal and nasopharyngeal swabs were taken daily for 14 days and every 2 days for another 2 weeks. The 5 vaccinates were introduced sequentially for 48h to 3 groups of 2 naïve sentinel ponies each on days 2, 4 and 6 post-challenge respectively. Clinical signs of disease and EIV shedding were monitored for 14 days after co-mingling. EIV was detected by 3 different methods of detection (EIV nucleoprotein ELISA, EIV nucleoprotein qRT-PCR and isolation/titration in embryonated hens' eggs). Directigen™ EZ Flu A+B tests were also performed on samples from the vaccinated ponies for 6 days after infection. Results show that nasopharyngeal swabs were superior to nasal swabs, with increased frequency and amount of virus detected. The average mean duration of shedding was 6-8 days in naïve animals. All 3 sentinel groups were infected successfully with EIV after commingling with vaccinates, indicating up to 6 days of transmission. EI protection induced by vaccination is a dynamic process, naturally fluctuating and dependent on the time since last immunisation, with periods of high immunity (peak of immunity shortly after boost immunisation) and periods of susceptibility to EIV infection. This result indicates that vaccinated horses may actively transmit EIV if the immunity gap (a usual period of susceptibility between V2 and V3) is not adequately closed by immunisation. In infected sentinels EIV was detectable up to 12 days after commingling. Results also suggest that tests such as qRT-PCR may be a suitable substitute for time spent in pre-export quarantine.

Replication and immunogenicity of swine, equine, and avian h3 subtype influenza viruses in mice and ferrets.

Since it is difficult to predict which influenza virus subtype will cause an influenza pandemic, it is important to prepare influenza virus vaccines against different subtypes and evaluate the safety and immunogenicity of candidate vaccines in preclinical and clinical studies prior to a pandemic. In addition to infecting humans, H3 influenza viruses commonly infect pigs, horses, and avian species. We selected 11 swine, equine, and avian H3 influenza viruses and evaluated their kinetics of replication and ability to induce a broadly cross-reactive antibody response in mice and ferrets. The swine and equine viruses replicated well in the upper respiratory tract of mice. With the exception of one avian virus that replicated poorly in the lower respiratory tract, all of the viruses replicated in mouse lungs. In ferrets, all of the viruses replicated well in the upper respiratory tract, but the equine viruses replicated poorly in the lungs. Extrapulmonary spread was not observed in either mice or ferrets. No single virus elicited antibodies that cross-reacted with viruses from all three animal sources. Avian and equine H3 viruses elicited broadly cross-reactive antibodies against heterologous viruses isolated from the same or other species, but the swine viruses did not. We selected an equine and an avian H3 influenza virus for further development as vaccines.

First Report on Molecular Detection of Equine Upper Respiratory Infectious Viruses in Republic of Korea

The prevalence of equine respiratory virus infections among a suspected population of race horses was examined using polymerase chain reaction (PCR). One or more of five equine respiratory viruses were detected in the nasal swabs of 45 of 89 horses (50.6%), and the detection rate of equine herpesvirus type 1 (EHV-1), equine herpesvirus type 4 (EHV-4), equine herpesvirus type 5 (EHV-5), equine rhinitis A virus (ERAV) and equine rhinitis B virus (ERBV) were 5.6%, 7.9%, 39.0%, 2.2%, and 6.7%, respectively. Among the 45 infected horses, 7 were co-infected with EHV and/or equine rhinitisvirus (ERV). Equine influenzavirus and equine arteritisvirus were not detected in any samples. Specific antibodies to EHV-1 and/or EHV-4 were detected in 59 of 73 tested sera (80.8%), using a virus neutralization test. This investigation suggests that equine respiratory viruses are endemic at Seoul Race Park and that the impact of viral infections on race horses’ health in Republic of Korea should be evaluated.

Study on risk factors and serologic prevalence of antibodies against equine influenza virus in the south of Iran

Six hundred horse serum samples were analyzed for equine influenza virus-specific antibodies using competitive ELISA in Fars Province (south of Iran) (2011–2012). In our study, 2.5 % of horses had a positive reaction which was proved to be due to natural exposure to equine influenza virus. Of the horses, 1.94 % were positive in autumn and 5.47 % were positive in winter. No significant difference was found among the prevalences of the disease in different breeds. It was concluded that the seroprevalence of equine influenza is quite low in the south of Iran. In view of no vaccination coverage for equine influenza in the country, the necessity of surveillance should be considered.