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Background: Dexamethasone is routinely administered to horses but its effect on the antibody response to a commercial EIV/EHV vaccine is unclear. Hypothesis: Horses receiving dexamethasone will have lower postvaccination antibody levels against EIV and EHV‐1 than vaccinated controls. Animals: Fifty‐five healthy adult research horses. Methods: Randomized cohort study. Control (no vaccine, group 1), vaccination only (EIV/EHV‐1/EHV‐4, Prestige 2, Merck Animal Health, group 2), vaccination and concurrent single intravenous dose of dexamethasone (approximately.05 mg/kg, group 3), vaccination and 3 intravenous doses of dexamethasone at 24 hours intervals (group 4). Serum SAA levels were measured on day 1 and day 3. Antibody levels against EIV (hemagglutination inhibition assay, Kentucky 2014 antigen) and EHV‐1 (multiplex ELISA targeting total IgG and IgG 4/7) were measured on day 1 and day 30. Results: Significantly increased mean antibody titers after vaccination were only noted against EIV and only after the vaccination alone (n = 14, prevaccine mean [prvm] 166.9, SD 259.6, 95% CI 16.95‐316.8; postvaccine mean [povm] 249.1, SD 257.2, 95% confidence interval [CI] 100.6‐397.6, P =.02) and the single dose dexamethasone (n = 14, prvm 93.14, SD 72.2, CI 51.45‐134.8; povm 185.1, SD 118, CI 116.7‐253.6, P =.01), but not after multiple doses of dexamethasone (n = 14, prvm 194.3, SD 258.3, CI 45.16‐343.4; povm 240.0, SD 235.7, CI 103.9‐376.1, P >.05). Conclusion: The effect of dexamethasone on the postvaccine antibody response varies depending on the dosing frequency and the antigen‐specific antibody type. [ABSTRACT FROM AUTHOR]

Cross-species virus transmission events can lead to dire public health emergencies in the form of epidemics and pandemics. One example in animals is the emergence of the H3N8 equine influenza virus (EIV), first isolated in 1963 in Miami, FL, USA, after emerging among horses in South America. In the early 21st century, the American lineage of EIV diverged into two 'Florida' clades that persist today, while an EIV transferred to dogs around 1999 and gave rise to the H3N8 canine influenza virus (CIV), first reported in 2004. Here, we compare CIV in dogs and EIV in horses to reveal their host-specific evolution, to determine the sources and connections between significant outbreaks, and to gain insight into the factors controlling their different evolutionary fates. H3N8 CIV only circulated in North America, was geographically restricted after the first few years, and went extinct in 2016. Of the two EIV Florida clades, clade 1 circulates widely and shows frequent transfers between the USA and South America, Europe and elsewhere, while clade 2 was globally distributed early after it emerged, but since about 2018 has only been detected in Central Asia. Any potential zoonotic threat of these viruses to humans can only be determined with an understanding of its natural history and evolution. Our comparative analysis of these three viral lineages reveals distinct patterns and rates of sequence variation yet with similar overall evolution between clades, suggesting epidemiological intervention strategies for possible eradication of H3N8 EIV. [ABSTRACT FROM AUTHOR]

Antibodies to influenza D virus (IDV) have been detected in horses, but no evidence of disease in the field has been reported. To determine whether IDV is infectious, immunogenic, and pathogenic in horses, four 2-year-old horses seronegative for both influenza A (H3N8) and D viruses were intranasally inoculated with 6.25 × 107 TCID50/animal of D/bovine/California/0363/2019 (D/CA2019) virus, using a portable equine nebulizer system. Horses were observed daily for clinical signs including rectal temperature, nasal discharge, coughing, lung sounds, tachycardia, and tachypnea. No horses exhibited clinical signs of disease. Nasopharyngeal swabs collected from 1–8 days post-infection demonstrated virus shedding by qRT-PCR. The horses showed evidence of seroconversion as early as 13 days post-infection (dpi) and the geometric mean of the antibody titers (GMT) of all four horses ranged from 16.82–160 as demonstrated by the microneutralization assay. Further, deep RNA sequencing of the virus isolated in embryonated chicken eggs revealed no adaptive mutations indicating that IDV can replicate in horses, suggesting the possibility of interspecies transmission of IDV with bovine reservoir into equids in nature. [ABSTRACT FROM AUTHOR]

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Background: Worldwide, horses play critical roles in recreation, food production, transportation, and as working animals. Horses' roles differ by geographical region and the socioeconomic status of the people, but despite modern advances in transportation, which have in some ways altered humans contact with horses, potential risks for equine zoonotic pathogen transmission to humans occur globally. While previous reports have focused upon individual or groups of equine pathogens, to our knowledge, a systematic review of equine zoonoses has never been performed. Methods: Using PRISMA's systematic review guidelines, we searched the English literature and identified 233 previous reports of potential equine zoonoses found in horses. We studied and summarized their findings with a goal of identifying risk factors that favor disease transmission from horses to humans. Results: These previous reports identified 56 zoonotic pathogens that have been found in horses. Of the 233 articles, 13 involved direct transmission to humans (5.6%).The main potential routes of transmission included oral, inhalation, and cutaneous exposures. Pathogens most often manifest in humans through systemic, gastrointestinal, and dermatological signs and symptoms. Furthermore, 16.1% were classified as emerging infectious diseases and thus may be less known to both the equine and human medical community. Sometimes, these infections were severe leading to human and equine death. Conclusions: While case reports of zoonotic infections directly from horses remain low, there is a high potential for underreporting due to lack of knowledge among health professionals. Awareness of these zoonotic pathogens, their disease presentation in horses and humans, and their associated risk factors for cross-species infection are important to public health officials, clinicians, and people with recreational or occupational equid exposure. [ABSTRACT FROM AUTHOR]

Equine herpesvirus-1 (EHV-1) is one of the most important and prevalent viral pathogens of horses and a major threat to the equine industry throughout most of the world. EHV-1 primarily causes respiratory disease but viral spread to distant organs enables the development of more severe sequelae; abortion and neurologic disease. The virus can also undergo latency during which viral genes are minimally expressed, and reactivate to produce lytic infection at any time. Recently, there has been a trend of increasing numbers of outbreaks of a devastating form of EHV-1, equine herpesviral myeloencephalopathy. This review presents detailed information on EHV-1, from the discovery of the virus to latest developments on treatment and control of the diseases it causes. We also provide updates on recent EHV-1 research with particular emphasis on viral biology which enables pathogenesis in the natural host. The information presented herein will be useful in understanding EHV-1 and formulating policies that would help limit the spread of EHV-1 within horse populations. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 thisH3N8 isolate shared the highest percentage identity toA/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. [ABSTRACT FROM AUTHOR]

The article discusses a study which examined the capability of one-step TaqMan real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assays targeting the nucleoprotein, matrix and hemagglutinin genes of H3N8 Equine influenza virus in detecting Canine influenza virus (CIV). It also evaluated assays using tissue culture fluid (TCF) or allantoic fluid samples containing CIV. Results showed that none of the assays detected the H1N1 Swine influenza virus strain in current circulation.

Please cite this paper as: Lin et al. (2010) Infectivity of equine H3N8 influenza virus in bovine cells and calves. Influenza and Other Respiratory Viruses 4(6), 357-361. Serological evidence for influenza A, subtype H1 and H3 virus infections of bovines, associated with respiratory disease and decreased milk production, has been reported. Equine H3N8 influenza virus circulates widely and was responsible for the introduction of H3N8 influenza into canines. To explore the possibility that equine H3N8 influenza might also infect bovines. To assess the incidence of seroconversion in the field, a retrospective survey of bovine serum samples was carried out. Also, primary cultures of bovine nasal turbinate cells, and live beef calves, were studied for their permissiveness to infection. We found serological evidence of exposure of bovines in Kentucky to H3 influenza. We demonstrate that cultured bovine respiratory epithelium is permissive for the growth of equine H3N8 influenza virus in vitro, but this virus does not replicate extensively or produce disease in experimentally inoculated cattle. [ABSTRACT FROM AUTHOR]

Equine influenza virus (EIV) is a constantly evolving viral pathogen that is responsible for yearly outbreaks of respiratory disease in horses termed equine influenza (EI). There is currently no evidence of circulation of the original H7N7 strain of EIV worldwide; however, the EIV H3N8 strain, which was first isolated in the early 1960s, remains a major threat to most of the world's horse populations. It can also infect dogs. The ability of EIV to constantly accumulate mutations in its antibody-binding sites enables it to evade host protective immunity, making it a successful viral pathogen. Clinical and virological protection against EIV is achieved by stimulation of strong cellular and humoral immunity in vaccinated horses. However, despite EI vaccine updates over the years, EIV remains relevant, because the protective effects of vaccines decay and permit subclinical infections that facilitate transmission into susceptible populations. In this review, we describe how the evolution of EIV drives repeated EI outbreaks even in horse populations with supposedly high vaccination coverage. Next, we discuss the approaches employed to develop efficacious EI vaccines for commercial use and the existing system for recommendations on updating vaccines based on available clinical and virological data to improve protective immunity in vaccinated horse populations. Understanding how EIV biology can be better harnessed to improve EI vaccines is central to controlling EI. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background Equine influenza virus (EIV) is considered enzootic in North America and experimental studies have documented human EIV infections. Study design This cross-sectional study examined 94 horse-exposed and 34 non-exposed controls for serological evidence of EIV infection. Sera were evaluated for antibodies against three EIV and two human H3N2 viruses using microneutralization (MN), neuraminidase inhibition (NI), enzyme-linked lectin (ELLA), and hemagglutination inhibition (HI) serological assays. Risk factor analyses were conducted using logistic regression and proportional odds modeling. Results There was evidence of previous infection by MN assay against A/equine/Ohio/2003(H3N8) but not the other 2 EIVs. Eleven (11.7%, maximum titer 1:320) horse-exposed and 2 (5.9%, maximum titer 1:160) control subjects had MN titers ≥1:80. Among the horse-exposed, 18 (19.1%) were positive by NI assay and 8 (8.5%) had elevated ELLA titers ≥1:10. Logistic regression modeling among horse-exposed revealed that having an elevated MN or ELLA titer (≤1:10) was associated with having a positive NI titer (OR = 4.9; 95% CI = 1.3–18.7, and OR = 53.2; 95% CI = 5.9–478.5, respectively). Upon proportional odds modeling, having worked as an equine veterinarian (OR = 14.0; 95% CI = 2.6–75.9), having a history of smoking (OR = 3.1; 95% CI = 1.2–7.7), and receipt of seasonal influenza vaccine between 2000 and 2005 (OR = 2.3; 95% CI = 1.1–5.0) were important independent risk factors for elevations in MN assay. Conclusions While we cannot rule out confounding exposures, these data support the premise that occupational exposure to EIV may lead to human infection. [ABSTRACT FROM AUTHOR]

Equid herpesvirus 1 (EHV-1) is a viral pathogen of horse populations worldwide spread by the respiratory route and is known for causing outbreaks of neurologic syndromes and abortion storms. Previously, we demonstrated that an EHV-1 strain of the neuropathogenic genotype, T953, downregulates the beta interferon (IFN-β) response in vitro in equine endothelial cells (EECs) at 12 h postinfection (hpi). In the present study, we explored the molecular correlates of this inhibition as clues toward an understanding of the mechanism. Data from our study revealed that EHV-1 infection of EECs significantly reduced both Toll-like receptor 3 (TLR3) and TLR4 mRNA expression at 6 hpi and 12 hpi. While EHV-1 was able to significantly reduce IRF9 mRNA at both 6 hpi and 12 hpi, the virus significantly reduced IFN regulatory factor 7 (IRF7) mRNA only at 12 hpi. EHV-1 did not alter the cellular level of Janus-activated kinase 1 (JAK1) at any time point. However, EHV-1 reduced the cellular level of expression of tyrosine kinase 2 (TYK2) at 12 hpi. Downstream of JAK1-TYK2 signaling, EHV-1 blocked the phosphorylation and activation of signal transducer and activator of transcription 2 (STAT2) when coincubated with exogenous IFN, at 12 hpi, although not at 3 or 6 hpi. Immunofluorescence staining revealed that the virus prevented the nuclear translocation of STAT2 molecules, confirming the virus-mediated inhibition of STAT2 activation. The pattern of suppression of phosphorylation of STAT2 by EHV-1 implicated viral late gene expression. These data help illuminate how EHV-1 strategically inhibits the host innate immune defense by limiting steps required for type I IFN sensitization and induction. IMPORTANCE To date, no commercial vaccine label has a claim to be fully protective against the diseases caused by equid herpesvirus 1 (EHV-1), especially the neurologic form. The interferon (IFN) system, of which type I IFN is of great importance, still remains a viable immunotherapeutic option against EHV-1 infection. The type I IFN system has been exploited successfully to treat other viral infections, such as chronic hepatitis B and C in humans. The current state of research on how EHV-1 interferes with the protective effect of type I IFN has indicated transient induction of type I IFN production followed by a rapid shutdown in vitro in equine endothelial cells (EECs). The significance of our study is the identification of certain steps in the type I IFN signaling pathway targeted for inhibition by EHV-1. Understanding this pathogen-host relationship is essential for the long-term goal of developing effective immunotherapy against EHV-1. [ABSTRACT FROM AUTHOR]

H3N8 equine influenza virus (EIV) is an important and significant respiratory pathogen of horses. EIV is enzootic in Europe and North America, mainly due to the suboptimal efficacy of current vaccines. We describe, for the first time, the generation of a temperature sensitive (ts) H3N8 EIV live-attenuated influenza vaccine (LAIV) using reverse-genetics approaches. Our EIV LAIV was attenuated (att) in vivo and able to induce, upon a single intranasal administration, protection against H3N8 EIV wild-type (WT) challenge in both a mouse model and the natural host, the horse. Notably, since our EIV LAIV was generated using reverse genetics, the vaccine can be easily updated against drifting or emerging strains of EIV using the safety backbone of our EIV LAIV as master donor virus (MDV). These results demonstrate the feasibility of implementing a novel EIV LAIV approach for the prevention and control of currently circulating H3N8 EIVs in horse populations. [ABSTRACT FROM AUTHOR]

Abstract: Equine influenza A (H3N8) virus infection is a leading cause of respiratory disease in horses, resulting in widespread morbidity and economic losses. As with influenza in other species, equine influenza strains continuously mutate, often requiring the development of new vaccines. Current inactivated (killed) vaccines, while efficacious, only offer limited protection against diverse subtypes and require frequent boosts. Research into new vaccine technologies, including gene-based vaccines, aims to increase the neutralization potency, breadth, and duration of protective immunity. Here, we demonstrate that a DNA vaccine expressing the hemagglutinin protein of equine H3N8 influenza virus generates homologous and heterologous immune responses, and protects against clinical disease and viral replication by homologous H3N8 virus in horses. Furthermore, we demonstrate that needle-free delivery is as efficient and effective as conventional parenteral injection using a needle and syringe. These findings suggest that DNA vaccines offer a safe, effective, and promising alternative approach for veterinary vaccines against equine influenza. [Copyright &y& Elsevier]

Horses are the third major mammalian species, along with humans and swine, long known to be subject to acute upper respiratory disease from influenza A virus infection. The viruses responsible are subtype H7N7, which is believed extinct, and H3N8, which circulates worldwide. The equine influenza lineages are clearly divergent from avian influenza lineages of the same subtypes. Their genetic evolution and potential for interspecies transmission, as well as clinical features and epidemiology, are discussed. Equine influenza is spread internationally and vaccination is central to control efforts. The current mechanism of international surveillance and virus strain recommendations for vaccines is described., (Copyright © 2022 Cold Spring Harbor Laboratory Press; all rights reserved.)

The use of a hemagglutination inhibition (HI) assay to assess humoral immune response to equine influenza virus (EIV) vaccines from various manufacturers administered to previously immunized adult horses was investigated. Subjects were allocated into one of 3 groups and vaccinated with various commercially available vaccines. Groups were subdivided into subjects that received 1 dose of a particular vaccine and those that received a second dose, 30 d later. Serum was collected at various times to assess antibody responses to contemporary EIV Florida sub-lineage strains. Statistical significance was set at P < 0.05 and all groups had a significant increase in antibody titers pre- and post-administration of the first dose. In contrast, there was no significant difference between day 30 titers and titers at subsequent time points, regardless of protocol. We concluded that administration of various commercial influenza vaccines containing a different sub-lineage clade stimulated equivalent HI antibody titers after 1 booster vaccination., (Copyright and/or publishing rights held by the Canadian Veterinary Medical Association.)

Background: Worldwide, horses play critical roles in recreation, food production, transportation, and as working animals. Horses' roles differ by geographical region and the socioeconomic status of the people, but despite modern advances in transportation, which have in some ways altered humans contact with horses, potential risks for equine zoonotic pathogen transmission to humans occur globally. While previous reports have focused upon individual or groups of equine pathogens, to our knowledge, a systematic review of equine zoonoses has never been performed. Methods: Using PRISMA's systematic review guidelines, we searched the English literature and identified 233 previous reports of potential equine zoonoses found in horses. We studied and summarized their findings with a goal of identifying risk factors that favor disease transmission from horses to humans. Results: These previous reports identified 56 zoonotic pathogens that have been found in horses. Of the 233 articles, 13 involved direct transmission to humans (5.6%).The main potential routes of transmission included oral, inhalation, and cutaneous exposures. Pathogens most often manifest in humans through systemic, gastrointestinal, and dermatological signs and symptoms. Furthermore, 16.1% were classified as emerging infectious diseases and thus may be less known to both the equine and human medical community. Sometimes, these infections were severe leading to human and equine death. Conclusions: While case reports of zoonotic infections directly from horses remain low, there is a high potential for underreporting due to lack of knowledge among health professionals. Awareness of these zoonotic pathogens, their disease presentation in horses and humans, and their associated risk factors for cross-species infection are important to public health officials, clinicians, and people with recreational or occupational equid exposure.

Serologic tests for equine influenza virus (EIV) antibodies are used for many purposes, including retrospective diagnosis, subtyping of virus isolates, antigenic comparison of different virus strains, and measurement of immune responses to EIV vaccines. The hemagglutination inhibition (HI) assay, single radial hemolysis (SRH), and serum micro-neutralization tests are the most widely used for these purposes and are described here. The presence of inhibitors of hemagglutination in equine serum complicates interpretation of HI assay results, and there are alternative protocols (receptor-destroying enzyme, periodate, trypsin-periodate) for their removal. With the EIV H3N8 strains in particular, equine antibody titers may be magnified by pre-treating the HI test antigen with Tween-80 and ether. The SRH assay offers stronger correlations between serum antibody titers and protection from disease. Other tests are sometimes used for specialized purposes such as the neuraminidase-inhibition assay for subtyping, or ELISA for measuring different specific antibody isotypes, and are not described here.

Equine influenza viruses are cultured in embryonated chicken eggs or in mammalian cells, generally Madin-Darby canine kidney (MDCK) cells, using methods much the same as for other influenza A viruses. Mutations associated with host adaptation occur in both eggs and MDCK cells, but the latter show greater heterogeneity and eggs are the generally preferred host. Both equine-1 H7N7 and equine-2 H3N8 viruses replicate efficiently in 11-day-old eggs, but we find that equine-1 viruses kill the embryos whereas equine-2 viruses do not.

In horses, presumptive diagnosis of equine influenza is commonly made on the basis of clinical signs. This alone is insufficient for confirmation of equine influenza, because other equine infectious respiratory diseases can in some degree have similar clinical presentations. Surveillance and control of equine influenza also necessitate detection of subclinical cases. Effective diagnosis of equine influenza virus infection is critically dependent on obtaining adequate specimens of virus-containing respiratory secretions for testing. These specimens are also valuable as sources for isolation of virus strains for antigenic characterization and potential inclusion in vaccines. Both nasal swabs and nasopharyngeal swabs are employed with horses. These differ little in their invasiveness, but nasopharyngeal swabs typically yield more virus than nasal swabs and are superior diagnostic specimens. Methods for obtaining nasopharyngeal swab specimens are described.