Equine Influenza Virus Research Articles

Mutated influenza A virus exhibiting reduced susceptibility to baloxavir marboxil from an experimentally infected horse.

Baloxavir marboxil (BXM), an inhibitor of the cap-dependent endonuclease of the influenza virus polymerase acidic protein (PA), exerts an antiviral effect against influenza A virus. It has been available in Japan since March 2018. This study evaluated the antiviral efficacy of BXM against equine influenza A virus (EIV) by an experimental challenge study using horses. Six horses were experimentally inoculated with EIV, and BXM was administered to the three horses at 2 days post inoculation. Horses treated with BXM showed milder clinical signs than horses without treatment and shed less virus. These results suggest that BXM is effective against EIV. The PA gene of viruses present in the nasopharyngeal swabs collected from horses treated with BXM was sequenced. Two mutations have been detected in viruses recovered from horses treated with BXM. These mutations were the substitution of isoleucine with threonine at position 38 (PA-I38T) and that of asparagine with aspartic acid at position 675 in PA (PA-N675D). A mutated virus with PA-I38T was less susceptible to BXM than viruses with PA-N675D or without mutation. A PA-I38T mutation has also been detected in viruses recovered from humans treated with BXM and is responsible for the reduction in susceptibility to BXM. This suggests that we should not unthinkingly use BXM for the treatment of EI. BXM is likely to easily induce resistance in influenza A viruses, not only in humans but also in horses.

Host-range shift of H3N8 canine influenza virus: a phylodynamic analysis of its origin and adaptation from equine to canine host

Prior to the emergence of H3N8 canine influenza virus (CIV) and the latest avian-origin H3N2 CIV, there was no evidence of a circulating canine-specific influenza virus. Molecular and epidemiological evidence suggest that H3N8 CIV emerged from H3N8 equine influenza virus (EIV). This host-range shift of EIV from equine to canine hosts and its subsequent establishment as an enzootic CIV is unique because this host-range shift was from one mammalian host to another. To further understand this host-range shift, we conducted a comprehensive phylodynamic analysis using all the available whole-genome sequences of H3N8 CIV. We found that (1) the emergence of H3N8 CIV from H3N8 EIV occurred in approximately 2002; (2) this interspecies transmission was by a reassortant virus of the circulating Florida-1 clade H3N8 EIV; (3) once in the canine species, H3N8 CIV spread efficiently and remained an enzootic virus; (4) H3N8 CIV evolved and diverged into multiple clades or sublineages, with intra and inter-lineage reassortment. Our results provide a framework to understand the molecular basis of host-range shifts of influenza viruses and that dogs are potential “mixing vessels” for the establishment of novel influenza viruses.

A Bivalent Live-Attenuated Vaccine for the Prevention of Equine Influenza Virus.

Vaccination remains the most effective approach for preventing and controlling equine influenza virus (EIV) in horses. However, the ongoing evolution of EIV has increased the genetic and antigenic differences between currently available vaccines and circulating strains, resulting in suboptimal vaccine efficacy. As recommended by the World Organization for Animal Health (OIE), the inclusion of representative strains from clade 1 and clade 2 Florida sublineages of EIV in vaccines may maximize the protection against presently circulating viral strains. In this study, we used reverse genetics technologies to generate a bivalent EIV live-attenuated influenza vaccine (LAIV). We combined our previously described clade 1 EIV LAIV A/equine/Ohio/2003 H3N8 (Ohio/03 LAIV) with a newly generated clade 2 EIV LAIV that contains the six internal genes of Ohio/03 LAIV and the HA and NA of A/equine/Richmond/1/2007 H3N8 (Rich/07 LAIV). The safety profile, immunogenicity, and protection efficacy of this bivalent EIV LAIV was tested in the natural host, horses. Vaccination of horses with the bivalent EIV LAIV, following a prime-boost regimen, was safe and able to confer protection against challenge with clade 1 (A/equine/Kentucky/2014 H3N8) and clade 2 (A/equine/Richmond/2007) wild-type (WT) EIVs, as evidenced by a reduction of clinical signs, fever, and virus excretion. This is the first description of a bivalent LAIV for the prevention of EIV in horses that follows OIE recommendations. In addition, since our bivalent EIV LAIV is based on the use of reverse genetics approaches, our results demonstrate the feasibility of using the backbone of clade 1 Ohio/03 LAIV as a master donor virus (MDV) for the production and rapid update of LAIVs for the control and protection against other EIV strains of epidemiological relevance to horses.

Multifocal outbreak of equine influenza in vaccinated horses in Argentina in 2018: Epidemiological aspects and molecular characterisation of the involved virus strains.

Equine influenza is an important cause of respiratory disease of horses worldwide. The equine influenza virus (EIV) undergoes antigenic drift through the accumulation of amino acid substitutions in the viral proteins, which may lead to vaccine breakdown. To describe the epidemiological findings and the molecular characteristics of the EIV detected during the multifocal outbreak that occurred in Argentina between March and July 2018 and evidence a vaccine breakdown. Observational, descriptive study. Virus was detected in nasopharyngeal swabs using real-time reverse transcriptase PCR (RT-PCR). Nucleotide and deduced amino acid sequences of the haemagglutinin (HA) and neuraminidase (NA) genes were obtained from EIV positive nasopharyngeal swabs, and phylogenetic analysis was undertaken. Amino acid sequences were compared against the current World Organisation for Animal Health (OIE)-recommended Florida clade 1 vaccine strain and strain components of vaccines used in Argentina. Serum samples were tested using haemagglutination inhibition test. Equine influenza virus infection was confirmed using real-time RT-PCR and serological testing. The phylogenetic analysis of the HA and NA genes revealed that all the EIV identified during the outbreak belong to the H3N8 subtype, Florida clade 1. Multiple amino acid changes, some of them at antigenic sites, were observed in the circulating virus when compared with the strains included in the most commonly used vaccine in Argentina. Seventy-six percent of the affected horses had been vaccinated with this vaccine, suggesting the occurrence of vaccine breakdown. The study does not include antigenic characterisation and full genome sequencing of Argentinian strains, that could provide additional information. The occurrence of this multifocal equine influenza outbreak in regularly vaccinated horses is a field evidence of vaccine breakdown, reinforcing the necessity of keeping vaccine strains updated according to OIE recommendations. It also underlines the importance of the implementation of appropriate quarantine measures and restriction of horse movement in the face of disease.

The Effects of Biannual Equine Influenza Vaccine on Performance in Adult Horses

<p><strong>PICO question</strong></p><p>In three day event horses, does biannual routine influenza vaccination compared to annual routine influenza vaccination reduce performance levels?</p><p><strong>Clinical bottom line</strong><strong><br /> </strong></p><p>There is no evidence that biannual equine influenza vaccination compared to annual booster vaccination in three day event horses is associated with reduced performance.</p><p>A group of five studies published over an 11 year period from one were evaluated. In adult warmblood horses there is weak evidence that exercise in the 28 day period post booster vaccination for equine influenza and equine herpes virus 1 and 4 (EHV1&4), is associated with changes in physical and clinical pathophysiological parameters including total red blood cell (RBC) count, neutrophil and lymphocyte count, fibrinogen concentration and serum proteins. These changes occurred at variable time points in the 14 days post exercise and values were not outside the published reference ranges for the reporting laboratories where published. Athletic performance of the horses was not evaluated.</p><p>No recommendations for equine influenza vaccination protocols in three day event horses can be made from the evidence.</p><p> </p><p><img src="https://www.veterinaryevidence.org/rcvskmod/icons/oa-icon.jpg" alt="Open Access" /> <img src="https://www.veterinaryevidence.org/rcvskmod/icons/pr-icon.jpg" alt="Peer Reviewed" /></p>

A single amino acid change in hemagglutinin reduces the cross-reactivity of antiserum against an equine influenza vaccine strain.

Equine influenza virus is an important pathogen for the horse industry because of its economic impact, and vaccination is a key control measure. Our previous work suggested that a mutation at position 144 in the hemagglutinin of Florida sublineage clade 2 viruses reduces the cross-neutralizing activity of antiserum against a former vaccine strain. To confirm this suggestion, here, we generated viruses by reverse genetics. Antibody titers against the mutated viruses were one-tenth to one-sixteenth of those against the former vaccine strain. Our findings confirm that this single amino acid substitution reduces the cross-reactivity of antiserum against this former Japanese vaccine.

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.

Equine Influenza Virus—A Neglected, Reemergent Disease Threat

Equine influenza virus (EIV) is a common, highly contagious equid respiratory disease. Historically, EIV outbreaks have caused high levels of equine illness and economic damage. Outbreaks have occurred worldwide in the past decade. The risk for EIV infection is not limited to equids; dogs, cats, and humans are susceptible. In addition, equids are at risk from infection with avian influenza viruses, which can increase mortality rates. EIV is spread by direct and indirect contact, and recent epizootics also suggest wind-aided aerosol transmission. Increased international transport and commerce in horses, along with difficulties in controlling EIV with vaccination, could lead to emergent EIV strains and potential global spread. We review the history and epidemiology of EIV infections, describe neglected aspects of EIV surveillance, and discuss the potential for novel EIV strains to cause substantial disease burden and subsequent economic distress.

Isolation and characterization of equine influenza virus (H3N8) from an equine influenza outbreak in Malaysia in 2015.

SummaryEquine influenza is a major cause of respiratory infections in horses and can spread rapidly despite the availability of commercial vaccines. In this study, we carried out molecular characterization of Equine Influenza Virus (EIV) isolated from the Malaysian outbreak in 2015 by sequencing of the HA and NA gene segments using Sanger sequencing. The nucleotide and amino acid sequences of HA and NA were compared with representative Florida clade 1 and clade 2 strains using phylogenetic analysis. The Florida clade 1 viruses identified in this outbreak revealed numerous amino acid substitutions in the HA protein as compared to the current OIE vaccine strain recommendations and representative strains of circulating Florida sub‐lineage clade 1 and clade 2. Differences in HA included amino acids located within antigenic sites which could lead to reduced immune recognition of the outbreak strain and alter the effectiveness of vaccination against the outbreak strain. Detailed surveillance and genetic information sharing could allow genetic drift of equine influenza viruses to be monitored more effectively on a global basis and aid in refinement of vaccine strain selection for EIV.

Whole Genome Sequencing of the First H3N8 Equine Influenza Virus Identified in Malaysia.

In August 2015, Malaysia experienced an outbreak of acute respiratory disease in racehorses. Clinical signs observed were consistent with equine influenza (EI) infection. The index cases were horses recently imported from New Zealand. Rapid control measures, including temporary cancellation of racing, were implemented to minimize the impact of the outbreak. By November, the disease outbreak was resolved, and movement restrictions were lifted. The aim of this study was to confirm the clinical diagnosis and characterize the causal virus. A pan-reactive influenza type A real-time RT-PCR was used for confirmatory diagnosis. Antigenic characterization by haemagglutinin inhibition using a panel of specific ferret antisera indicated that the causal virus belonged to clade 1 of the H3N8 Florida sub-lineage. The genetic characterization was achieved by the whole genome sequencing of positive nasal swabs from clinically affected animals. Pylogenetic analysis of the haemagglutinin (HA) and neuraminidase (NA) genes demonstrated ≥99% homology with several EI strains that had recently circulated in the USA and Japan. The antigenic and genetic characterization did not indicate that the current World Organisation for Animal Health (OIE) recommendations for EI vaccine composition required modification.

Prediction of genetic mutations of equine influenza virus related to adaptive determination nuclear export ribonucleoprotein complex

BackgroundSegment eight (08) of equine influenza virus strain (H3N8) encodes for two non-structural proteins: NS1 (non-structural protein number 1) and NS2, also called NEP (nuclear export protein), via alternative splicing of the pre-messenger RNA transcripts. The NS1 inhibits the cell defense system, while the NEP activates the translocation of the ribonucleoprotein complex to the cytoplasm, a key step in viral assembly. Mutations in this protein could influence viral multiplication.ResultsIn this present study, we used computational and in silico tools to investigate the impact of mutations on the three-dimensional structure of the CRM1 of NEP proteins of three equine influenza strains (H3N8) isolated in Morocco (A/equine/Nador/1/1997 (H3N8), A/equine/Essaouira/2/2004 (H3N8), and A/equine/Essaouira/3/2004 (H3N8)). Compared to the reference strain, the two strains of Essaouira showed no mutations in the domain-binding CRM1 of the NEP protein, while the A/equine/Nador/1/1997 strain (H3N8) had two mutations at the T/33/I and Q/34/R residues. These two mutations corresponded to adaptation phenomenon of the equine cellular environment, since they enhance the interaction between the protein NEP and CRM1 proteins with a greater affinity compared to the reference strain.ConclusionThe three-dimensional modification of the NEP protein of the A/equine/Nador/1/1997 strain further facilitates nucleocytoplasmic trafficking in equine species.

Frequency of molecular detection of equine coronavirus in faeces and nasal secretions in 277 horses with acute onset of fever.

Due to the inconsistent development of enteric signs associated with ECoV infection in adult horses, many practitioners collect nasal secretions rather than feces for the molecular diagnostic work-up of such horses. ECoV infection should be considered in horses presenting with acute onset of fever, especially when nasal discharge is absent as one of the cardinal clinical sign. A total of 277 adult horses with acute onset of fever were enrolled in this study. Feces were tested for ECoV and nasal secretions for common respiratory pathogens (equine herpesvirus (EHV)-1, EHV-4, equine influenza virus (EIV), equine rhinitis viruses (ERVs) and Streptococcus equi ss. equi) and ECoV by qPCR. Each submission was accompanied by a questionnaire requesting information pertaining to signalment, use, recent transportation, number of affected horses on the premise and presence of clinical signs at the time of sample collection. The total number of horses testing qPCR-positive for ECoV in feces was 20 (7.2%), 4 of which also tested qPCR-positive for ECoV in nasal secretions. In the same population 9.0% of horses tested qPCR-positive for EHV-4, 6.1% for EIV, 4.3% for Streptococcus equi ss. equi, 3.2% for ERVs and 0.7% for EHV-1. Draft horses, pleasure use, multiple horses affected on a premise and lack of nasal discharge were significantly associated with ECoV qPCR-positive horses. The present study results showed that 7.2% of horses with acute onset of fever tested qPCR-positive for ECoV in feces, highlighting the importance of testing such horses for ECoV in feces. The various prevalence factors associated with ECoV qPCR-positive status likely relate to the high infectious nature of ECoV and breed-specific differences in management and husbandry practices. ECoV infection should be suspected and tested for in horses presenting with acute onset of fever, lethargy and anorexia with no respiratory signs. A two-step approach should be consider in which respiratory secretions and feces should be collected from such horses and submitted to a diagnostic laboratory. If the respiratory secretions test negative by qPCR for a panel of respiratory pathogens, feces already submitted to the laboratory should be tested for ECoV.

Choosing an equine influenza vaccine

Equine influenza (EI) vaccination has been mandatory in the UK for most competitive pursuits for several decades, but failure to update vaccine products at the same rate as EI virus...

Application of Novel Sialoglyco Particulates Enhances the Detection Sensitivity of the Equine Influenza Virus by Real-Time Reverse Transcriptase Polymerase Chain Reaction.

Sialoglyco particulates carrying an N-glycolylneuraminyl-α-(2 → 3)-N-acetyllactosamine (Neu5Gcα2,3LacNAc) residue that displays a high level of affinity for the equine influenza virus (EIV) were generated using sialoglycopolypeptide and hexyl-containing hybrid silica particulates. The particulates were spherical with a diameter of approximately 950 nm and found to have good dispersibility in aqueous solution. Interaction between the sialoglyco particulates and the EIV was investigated by real-time reverse transcriptase polymerase chain reaction (rRT-PCR) of the EIV genome captured on the particulates. The number of EIV-specific genes detected by rRT-PCR on a nasal swab obtained from infected horses clearly increased when the sample was treated with sialoglyco particulates. Our results show these novel sialoglyco particulates can be used as a highly sensitive tool for detecting low levels of EIV that were previously undetectable in the early or late stage of infection.

Validation of two multiplex real-time PCR assays based on single nucleotide polymorphisms of the HA1 gene of equine influenza A virus in order to differentiate between clade 1 and clade 2 Florida sublineage isolates.

We validated 2 multiplex real-time PCR (rtPCR) assays based on single nucleotide polymorphisms (SNPs) of the hemagglutinin-1 ( HA1) gene of H3N8 equine influenza A virus (EIV) to determine clade affiliation of prototype and field isolates. Initial validation of the 2 multiplex rtPCR assays (SNP1 and SNP2) was performed using nucleic acid from 14 EIV Florida sublineage clade 1 and 2 prototype strains. We included in our study previously banked EIV rtPCR-positive nasal secretions from 341 horses collected across the United States in 2012-2017 to determine their clade affiliation. All 14 EIV prototype strains were identified correctly as either Florida sublineage clade 1 or clade 2 using the 2 SNP target positions. Of 341 EIV rtPCR-positive samples, 337 (98.8%) and 4 (1.2%) isolates were classified as belonging to clade 1 and 2 Florida sublineage EIV, respectively. All clade 1 Florida sublineage EIV strains were detected in domestic horses, three clade 2 Florida sublineage EIV strains originated from horses recently imported into the United States, and one clade 2 Florida sublineage EIV strain originated from a healthy horse recently vaccinated with a modified-live intranasal EIV vaccine containing the American lineage strain A/eq/Kentucky/1991. EIV Florida sublineage clade differentiation using a fast and reliable multiplex rtPCR platform will help monitor the introduction of clade 2 Florida sublineage EIV strains into North America via international transportation.

Protective efficacy of inactivated reverse genetics based equine influenza vaccine candidate adjuvanted with MontanideTM Pet Gel in murine model.

Equine influenza is a leading cause for respiratory illness in equines. Major control measures involve vaccination which requires continuous harmonization owing to antigenic drift. The present study focused on assessing the protective efficacy of an inactivated recombinant equine influenza virus (rgEIV) vaccine candidate adjuvanted with MontanideTM Pet Gel in murine model. The rgEIV was generated using reverse genetics by incorporating HA and NA segments from EIV/H3N8, clade 2-Florida sublineage in an A/WSN/33 /H1N1 backbone and inactivated by formalin. The vaccine was prepared by mixing inactivated rgEIV with MontanideTM Pet Gel adjuvant followed by intranasal inoculation into BALB/c mice intranasally. The immune responses and protective efficacy of the vaccine was evaluated by measurement of antibody titer, immunoglobulin subtyping, cytokines, clinical signs and pathological lesions after immunization and challenge with wild EIV. Serology and cytokine expression pattern indicated that the vaccine activated mixed Th1- and Th2-like responses of vaccine. Booster immunization stimulated strong antibody responses (HAI titre: 192 ± 28.6) at 42 days post immunization and the predominant antibody subtype was IgG1. Upregulation of interferon (IFN)-gamma, interleukin (IL)-12 and IL-2 levels indicates effective induction of Th1 type response. We found that vaccination has protected mice against equine influenza virus challenge as adjudged through a lack of nonappearance of visible clinical signs of disease, no loss of body weight loss, reduced pathology in the lungs and markedly reduced virus shedding from the respiratory tract. Therefore, we conclude that recombinant EIV vaccine candidate adjuvanted with MontanideTM Pet Gel could aid in quick harmonization of the vaccines through replacement of HA and NA genes for control of EIV outbreaks.

Chemical Composition and Antiviral Effect of Extracts of <i>Origanum vulgare</i>

This study aimed determine the activity of aqueous and ethanolic extracts of Origanum vulgare against some viruses of veterinary importance (bovine viral diarrhea virus (BVDV), equine arteritis virus (EAV), equine influenza virus (EIV), feline calicivirus (FCV), canine distemper virus (CDV), canine adenovirus (CAV), and canine cororavirus (CCoV) by evaluating the possibility of inhibition of viral particles production. The aqueous extract from 1600 μg/mL did not show cytotoxicity for all cellular lineages evaluated, Madin Darby bovine kidney cells (MDBK), Rabbit kidney cells (RK 13), Madin Darby canine kidney cells (MDCK) and Crandell feline kidney cells (CRFK), and the ethanolic extract of Origanum vulgare was not toxic at 600 μg/mL. The addition of aqueous extract of Origanum vulgare in media resulted in a significant reduction of the EAV titer from 105.42 infecting dose for cellular culture at 50% (TCID50) to 102.09 TCID50/100 μL while in the presence of the ethanolic extract of Origanum vulgare in media resulted in a significant reduction of the EAV titer from 105.42 TCID50 to 100.79 TCID50/100 μL. To CDV the addition of aqueous extract resulted in a reduction from 102.00 TCID50 to 100.00 TCID50/100 μL while in the presence of the ethanolic extract titers were reduced from 102.00 TCID50 to 101.50 TCID50/100 μL. No significant differences in titers regarding the others analyzed viruses were detected. With respect to chemical analysis of the extracts of Origanum vulgare, were identified in the ethanol extract phenolics rosmarinic acid, caffeic acid, carnosol, p-coumaric acid, carnosic acid, luteolin, apigenin, kaempferol and quercetin. In aqueous extracts of Origanum vulgare were detected rosmarinic acid, p-coumaric acid carnosic acid, luteolin, apigenin, kaempferol and quercetin. The data obtained stimulate other biological assays in order to determine which compounds are responsible for the antiviral activity as well as which are the mechanisms involved. The results presented and the considerations we were able to draw from them allowed us to conclude that the ethanolic extract of Origanum vulgare demonstrated lower cell viability than the aqueous extract and has significant antiviral activity against EAV and the both aqueous and ethanolic extracts have antiviral action against CDV.

Application of phylogeography to reconstruct Equine influenza virus migration on a global and a US scale

Application of phylogeography to reconstruct Equine influenza virus migration on a global and a US scale

Genetic Analysis of the M Gene of Equine Influenza Virus Strains Isolated in Poland, in the Context of the Asian-like Group Formation.

IntroductionTraditionally, evolutionary analysis of equine influenza virus (EIV) is based on the HA gene. However, the specificity of the influenza virus enables the classification of viral strains into different phylogenetic groups, depending on the gene being analysed. The aim of the study was to analyse phylogenetic paths of EIV based on M gene with reference to the HA gene.Material and MethodsM gene of Polish isolates has been sequenced and analysed along with all M sequences of EIV available in GenBank database. Phylogenetic analysis was performed using BioEdit, ClustalW, and MEGA7 softwares.ResultsThe clustering of the strains isolated not only from Asia but also from Europe into one common Asian-like group of EIV was observed. Twelve nucleotide substitutions in the M gene of strains from the Asian-like group were crucial for the evolutionary analysis. We also observed homology in the M gene of the Asian-like and H7N7 strains.ConclusionsM gene specific for the Asian-like group is present in strains recently isolated in Europe and Asia, which were classified previously in the Florida 2 clade based on HA. Therefore, Asian-like group does not seem to be assigned to a specific geographical region. Traces of H7N7 strains in more conservative genes like M of some contemporary EIV strains may indicate the link between the old phylogenetic group and recent H3N8 strains. Analysis of conservative genes may be more useful in tracking the direction of virus evolution than in the genes where the high variability rate may blur the original relationships.

Development and evaluation of a multiplex reverse-transcription real-time PCR assay for detection of equine respiratory disease viruses

We developed a multiplex reverse-transcription real-time PCR (RT-rtPCR) assay for the simultaneous detection of the main equine respiratory viruses: equid alphaherpesviruses 1 and 4 (EHV-1, -4) and equine influenza virus (EIV; species Influenza A virus). The primers and probes amplified only the targeted viruses, and there were no inter-assay cross-amplifications or nonspecific interactions. The multiplex assay efficiencies were 92.5%, 97%, and 90% for EHV-1, EHV-4, and EIV, respectively. The R2 values of the monoplex and multiplex assays were ⩾0.990, and the slopes were -3.37 to -3.59. The performance of the assay was evaluated by analyzing 152 samples from clinically infected horses. EHV-1 DNA was detected in 12 samples, EHV-4 DNA in 9 samples, and both EHV-1 and EHV-4 in 4 samples. The accuracy of the assay was confirmed by comparing these results using commercial rtPCR and RT-rtPCR kits. Our multiplex RT-rtPCR was a sensitive, specific, accurate, and cost-effective method for the detection of the target viruses whether they occur alone or as part of coinfections.