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Equine influenza (EI) is a major respiratory disease of horses, which is still causing substantial outbreaks worldwide despite several decades of surveillance and prevention. Alongside quarantine procedures, vaccination is widely used to prevent or limit spread of the disease. The panel of EI vaccines commercially available is probably one of the most varied, including whole inactivated virus vaccines, Immuno-Stimulating Complex adjuvanted vaccines (ISCOM and ISCOM-Matrix), a live attenuated equine influenza virus (EIV) vaccine and a recombinant poxvirus-vectored vaccine. Several other strategies of vaccination are also evaluated. This systematic review reports the advances of EI vaccines during the last few years as well as some of the mechanisms behind the inefficient or sub-optimal response of horses to vaccination.

Infection with equine influenza viruses in unvaccinated population could cause large-scale morbidity leading to huge economic losses compromising equine welfare in developing countries. Here we conducted a matched case-control study to identify the potential risk factors responsible for the EI epidemic in Pakistan in the year 2015-16. Total of 197 laboratory-confirmed matched pairs of cases and controls were included in the study. A predesigned questionnaire-based epidemiological data was collected for this purpose from all the matched pairs respondents. Conditional logistic regression analysis was conducted analyzing the data. Multivariable regression analysis identified local equine density and frequency of manure removal as potential risk factors for the incidence of EI. Due to lack of vaccination, the spread of EI was favored supported by many infected equine population free movements. The first case of this epidemic was imported from Afghanistan which was considered as a source case for the epidemic. Management and biosecurity practices compliance was very low due to lack of awareness amongst the respondents. These findings agree with the previous findings in various developing countries. Strategic control of infected equines free movement and vaccination of the equine population are suggested in future to control such epidemics. [ABSTRACT FROM AUTHOR]

An outbreak of respiratory disease caused by the equine-origin influenza A(H3N8) virus was first detected in dogs in 2004 and since then has been enzootic among dogs. Currently, the molecular mechanisms underlying host adaption of this virus from horses to dogs is unknown. Here, we have applied quantitative binding, growth kinetics, and immunofluorescence analyses to elucidate these mechanisms. Our findings suggest that a substitution of W222L in the hemagglutinin of the equine-origin A(H3N8) virus facilitated its host adaption to dogs. This mutation increased binding avidity of the virus specifically to receptor glycans with N-glycolylneuraminic acid (Neu5Gc) and sialyl Lewis X (SLeX) motifs. We have demonstrated these motifs are abundantly located in the submucosal glands of dog trachea. Our findings also suggest that in addition to the type of glycosidic linkage (e.g., a2,3-linkage or a2,6-linkage), the type of sialic acid (Neu5Gc or 5-N-acetyl neuraminic acid) and the glycan substructure (e.g., SLeX) also play an important role in host tropism of influenza A viruses. IMPORTANCE Influenza A viruses (IAVs) cause a significant burden on human and animal health, and mechanisms for interspecies transmission of IAVs are far from being understood. Findings from this study suggest that an equine-origin A(H3N8) IAV with mutation W222L at its hemagglutinin increased binding to canine-specific receptors with sialyl Lewis X and Neu5Gc motifs and, thereby, may have facilitated viral adaption from horses to dogs. These findings suggest that in addition to the glycosidic linkage (e.g., a2,3-linked and a2,6-linked), the substructure in the receptor saccharides (e.g., sialyl Lewis X and Neu5Gc) could present an interspecies transmission barrier for IAVs and drive viral mutations to overcome such barriers. [ABSTRACT FROM AUTHOR]

Among all the emerging and re-emerging animal diseases, influenza group is the prototype member associated with severe respiratory infections in wide host species. Wherein, Equine influenza (EI) is the main cause of respiratory illness in equines across globe and is caused by equine influenza A virus (EIV-A) which has impacted the equine industry internationally due to high morbidity and marginal morality. The virus transmits easily by direct contact and inhalation making its spread global and leaving only limited areas untouched. Hitherto reports confirm that this virus crosses the species barriers and found to affect canines and few other animal species (cat and camel). EIV is continuously evolving with changes at the amino acid level wreaking the control program a tedious task. Until now, no natural EI origin infections have been reported explicitly in humans. Recent advances in the diagnostics have led to efficient surveillance and rapid detection of EIV infections at the onset of outbreaks. Incessant surveillance programs will aid in opting a better control strategy for this virus by updating the circulating vaccine strains. Recurrent vaccination failures against this virus due to antigenic drift and shift have been disappointing, however better understanding of the virus pathogenesis would make it easier to design effective vaccines predominantly targeting the conserved epitopes (HA glycoprotein). Additionally, the cold adapted and canarypox vectored vaccines are proving effective in ceasing the severity of disease. Furthermore, better understanding of its genetics and molecular biology will help in estimating the rate of evolution and occurrence of pandemics in future. Here, we highlight the advances occurred in understanding the etiology, epidemiology and pathobiology of EIV and a special focus is on designing and developing effective diagnostics, vaccines and control strategies for mitigating the emerging menace by EIV. [ABSTRACT FROM AUTHOR]

Equine influenza is an acute respiratory infection of horses, donkeys, mules and zebras. Nowadays, the reverse transcriptase polymerase chain reaction (RT-PCR) and real-time RT-PCR (rRT-PCR) assays are being widely used in diagnostic laboratories as a more sensitive alternative to virus isolation for the detection of equine influenza virus. The purpose of this study was to standardize an rRT-PCR, based on SYBR Green-I coupled with melting curve analysis for the detection of equine influenza virus (gene M) and the typing of hemagglutinin gene (HA). Sensitivity and specificity of the assay (gene M) were evaluated. In terms of the matrix gene copy number, the detection limit was 4.5 gene copies/µl of in vitro transcribed RNA. The linear range obtained for the transcript in the assay genered a typical standard curve from 107 until 100 gene copies/µl in terms of RNA copy number. The correlation coefficient (R²) was 0.99 for the RNA copies detected in nuclease free water and the error of standard curve was 0.0139. The rRT-PCR assay showed to be specific for equine influenza virus. No specific amplification curves were obtained with any of the other viruses tested such as equine viral arteritis, equine herpesvirus-1 and equine herpesvirus-4. The assay showed amplification and specific melting curves when the second set of primers against HA gene was evaluated. Thus, the proposed SYBR Green-I based rRT-PCR assay for the detection of equine influenza virus can be used in case of a possible emergency of this agent in Cuba. [ABSTRACT FROM AUTHOR]

Background: In 2013, there was an outbreak of acute respiratory disease in racehorses in Turkey. The clinical signs were consistent with equine influenza (EI). Objective: The aim was to confirm the cause of the outbreak and characterise the causal virus. Methods: A pan‐reactive influenza type A real‐time RT‐PCR and a rapid antigen detection kit were used for confirmatory diagnosis of equine influenza virus (EIV). Immunological susceptibility to EIV was examined using single radial haemolysis and ELISA. Antigenic characterisation was completed by haemagglutinin inhibition using a panel of specific ferret antisera. Genetic characterisation was achieved by whole‐genome sequencing using segment‐specific primers with M13 tags. Results: A H3N8 EIV of the Florida clade 2 sublineage (FC2) was confirmed as the causal agent. The index cases were unvaccinated and immunologically susceptible. Phylogenetic analysis of the HA1 and NA genes demonstrated that A/equine/Ankara/1/2013 clustered with the FC2 strains circulating in Europe. Antigenic characterisation confirmed the FC2 classification and demonstrated the absence of significant drift. Whole‐genome sequencing indicated that A/equine/Ankara/1/2013 is most closely related to the viruses described as the 179 group based on the substitution I179V in HA1, for example A/equine/East Renfrewshire/2/2011, A/equine/Cambremer/1/2012 and A/equine/Saone et Loire/1/2015. The greatest diversity was observed in the NS1 segment and the polymerase complex. Conclusions: The first recorded outbreak of EI in Turkey was caused by an FC2 virus closely related to viruses circulating in Europe. Antigenic and genetic characterisation gave no indication that the current OIE recommendations for EI vaccine composition require modification. [ABSTRACT FROM AUTHOR]

Owing to the high prevalence of obesity in Crioulo horses, information allowing early diagnosis of equine metabolic syndrome (EMS) and prevention of the associated laminitis is of great value. The aim of this study was to evaluate the occurrence of EMS and the response to an oral sugar test (OST) in obese Crioulo horses. Twenty-two Crioulo horses were allocated into 3 groups according to their body condition score (BCS out of 9) and presence or absence of laminitis as follows: CON (6/22), BCS < 7; OB (8/22), BCS ≥ 7; and LAM (8/22), BCS ≥7 with clinical and/or radiographic signs of laminitis. A complete clinical history was obtained, followed by a physical examination, morphometric measurements, radiographic evaluation of front feet, and ultrasonography measurements of subcutaneous body fat. For the OST, animals were fasted overnight, and blood samples were collected for glucose and insulin concentration before and after sugar administration. Morphometric and metabolic differences ( P < 0.05) were observed between CON animals and obese ones, with horses from the LAM group presenting the highest morphometric measurements and insulin plasma concentrations. A delayed peak glucose response for OST was observed in the majority of obese animals, indicating that sampling between 60 and 90 min after sugar administration without glycemic curve follow-up, as previously used for hyperinsulinism detection, can be inadequate. The observed delay in the return to glucose baseline levels, combined with high insulin levels, supports the diagnosis of insulin dysregulation. These results indicate that there are clear obesity-related differences in the glucose and insulin responses of Crioulo horses to an OST. [ABSTRACT FROM AUTHOR]

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Participation in equestrian shows provides opportunities for contact between horses, increasing the risk of disease introduction and spread within the population. The magnitude of a potential outbreak, and the impact of disease prevention and control strategies, can be estimated using simulation modeling. The objectives of this study were to (1) examine the potential spread of equine influenza in a network of horses associated with a 2-day equestrian show in Ontario, Canada; and (2) determine the effectiveness of several interventions during a simulated outbreak. A discrete-event, continuous-time, stochastic agent-based simulation model was constructed to represent horses associated with the show, including those in attendance at the show, and those that were not in attendance but co-boarded with attending horses at their home facilities. At the beginning of each simulation run, one random horse in attendance at the show was infected with equine influenza. In the absence of interventions, the median attack rate was 0.029 (IQR: 0.016–0.056; mean: 0.043; 95% CI: 0.040–0.044) and the average outbreak duration was 19.58 days (95% CI: 19.31–19.85). The most effective intervention was the implementation of either a 5-day or 14-day quarantine period, which both resulted in the same median attack rate of 0.0026 (IQR: 0.0013–0.0039), although the mean attack rates differed (mean: 0.0043, 95% CI: 0.0039–0.0046; and mean: 0.0029, 95% CI: 0.0028–0.0029; respectively). In instances where implementing either a 5-day or 14-day quarantine period would not be feasible, quarantine for shorter time periods was effective when combined with targeted increases in initial facility-level vaccine coverage. The combined implementation of a 2-day quarantine period and an increased vaccine coverage of 75% in facilities with four or more owners resulted in a median attack rate of 0.013 (IQR: 0.0052–0.026; mean: 0.022; 95% CI: 0.020–0.024). This study demonstrates a relative comparison of intervention effectiveness during a simulated outbreak of equine influenza in a population of horses associated with an equestrian show. The results have the potential to inform and improve the current strategies used to prevent the introduction and spread of disease within the equine population. [ABSTRACT FROM AUTHOR]

Equine influenza is a highly contagious respiratory disease considered the most important respiratory disease in equids. Although influenza A virus (IAV) has caused outbreaks in equids worldwide, surveillance in these species in Spain has not been conducted. A cross-sectional study was carried out to determine the individual and herd prevalence of antibodies against H3N8 and H7N7 IAV in equids in Andalusia (southern Spain). Antibodies againsts IAV were measured by the single radial haemolysis assay. A spatial scan statistical analysis was carried out using a Bernoulli model. Risk factors associated with IAV infection were assessed by multivariate analysis. Antibodies to H3N8 IAV were detected in 241 out of 464 unvaccinated equids (51.9%; 95% CI: 47.4–56.5). Seropositivity against the H7N7 subtype IAV was not found in any of the analysed animals. Significantly higher seropositivity was found in geriatric (OR = 6.1, P  = 0.008, 95% CI = 1.6–23.1) and adult (OR = 4.8, P <  0.001, 95% CI = 2.5–9.0) equids compared to young animals. Specific antibodies against A/equine/Shropshire/2010 (H3N8) or A/equine/Newmarket/5/2003 (H3N8) only were confirmed in 11 and 45 of the animals, respectively. The spatial analysis showed a statistically significant cluster centred in the west part of Andalusia. The results confirmed widespread H3N8 subtype IAV exposure in equine species in Andalusia. Conversely, the absence of seropositivity against H7N7 IAV obtained in the present study suggests that this subtype has not circulated in southern Spain in recent years. Because of the animal health and economic consequences of IAV in equids, further surveillance and molecular studies are required to monitor and characterize the most prevalent IAV circulating in these species in Spain. [ABSTRACT FROM AUTHOR]

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

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. [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]

An outbreak of equine influenza was detected in Khyber Pakhtunkhwa during the years 2015-16. The isolated viruses were typed for the first time in Pakistan as H3N8 and grouped with the Clade 1 viruses of Florida sub-lineages on the basis of Hemagglutinin (HA) and Neuraminidase (NA) gene sequence analysis. Here we described the genetic analysis of two internal genes (Nucleoprotein and Matrix) of the Pakistani isolates. The primer pairs were designed to amplify the NP and M internal genes of the 19 equine H3N8 influenza A viruses identified and isolated in this outbreak. Phylogenetic analysis of the derived sequences was performed and compared with the contemporary available sequences online. Comparison of the nucleotide sequences of the two internal genes of the Pakistani strains showed high similarity (99.7-100%) with the avian influenza A viruses (Avian/Pakistan/H7N3/ 2004) isolated in Pakistan. Two major substitutions i.e. F63L and K243R were recorded in Matrix gene of the isolates from the closely related Chicken/Pakistan/04 H7N3 virus. Notably NP gene did not acquire any amino acid substitution on comparison with Chicken/Pakistan/04. All isolates shared 99.5-99.9% homology with the reference sequences on alignment for M1 and NP amino acid sequences, respectively. Phylogenetic analysis revealed clustering of the Pakistani isolates with the avian viruses isolated in Pakistan in previous outbreaks in commercial and backyard poultry. Epidemiological investigation of the outbreak suggested that reassortment of equine H3N8 viruses with avian Influenza A H7N3 most probably, might be due to the mix farming system here. In conclusion, Phylogenetic analysis of the internal genes reported here will be helpful in early detection and control of EI. [ABSTRACT FROM AUTHOR]

Background: There are currently no licensed vaccines available for prevention of botulism in humans. The vaccination is not desirable due to expanding therapeutic indications of botulinum toxins. The only available specific treatment for botulism is antitoxin to remove circulating toxin, thus, preventing further neuronal damage. BAT® (Botulism Antitoxin Heptavalent (A, B, C, D, E, F, G)—(Equine)) has been developed and its therapeutic efficacy evaluated against botulinum neurotoxin serotype A (BoNT/A) in Rhesus macaques. Methods and findings: In a post-exposure prophylaxis (PEP) study, animals were exposed to 4x LD50/kg of BoNT/A and administered intravenously with either BAT (1x or 0.1x scaled human dose), or placebo at 4 hours post-exposure. The animals were monitored for 14 days. For the therapeutic intervention studies, animals were exposed to a 1.7x LD50/kg of BoNT/A and treated intravenously with either placebo or BAT at a 1x scaled human dose at the onset of clinical signs. Animals were monitored on an hourly basis for 14 or 21 days. In the PEP study, all animals tolerated equine based antitoxin without any adverse clinical signs. A 100% survival was observed in groups treated with the BAT compared to 0% survival in those treated with the placebo (p<0.001, Fisher’s exact test). BAT antitoxin prevented the development of signs of neurotoxicity of botulinum toxin. In a therapeutic study, treatment with the BAT at scaled 1x human dose after the onset of clinical signs significantly enhanced survival compared to the placebo (46.6% vs. 0%, p<0.0001, Fisher’s exact test). Additionally, treatment with the BAT delayed the progression of signs (muscular weakness, respiratory distress, oral/nasal discharge) of toxin intoxication and reduced the severity of the disease. Conclusions: A single dose of BAT, when administered to symptomatic monkeys, resulted in a statistically significant survival benefit compared to the placebo. Additionally, BAT completely protected monkeys from the clinical signs of intoxication and subsequent death when administered as PEP treatment. These data in part supported the licensure of BAT under the Animal Rule in the United States by the Food and Drug Administration. [ABSTRACT FROM AUTHOR]

Equine Influenza (EI) is an important respiratory disease of horses caused by H3N8 equine influenza viruses (EIV). Vaccination is a key strategy to prevent or control this disease. However, EIV undergoes continuous antigenic drift and whilst numerous EI vaccines are commercially available worldwide, an accurate evaluation of their efficacy is frequently required through clinical trials conducted in the natural host. Room nebulisation is one of the chosen methods to challenge horses during EI vaccine studies. A potential decreased pathogenicity observed with recent Florida Clade 2 (FC2) EIV isolates have increased the heterogeneity of the clinical response and virus shedding measured after infection by room nebulisation, which reduced the statistical power of studies. Our objectives were to compare clinical and virological parameters following experimental infection with several different EIV strains and to confirm that individual nebulisation is a model refinement that prevents an increase of the number of animals per group. This study is a retrospective comparison and meta-analysis of clinical and virological results collected from 9 independent EIV infection studies in the natural host. Naïve Welsh mountain ponies were experimentally infected by room or individual nebulisation with FC2 EIV strains, including A/equine/Richmond/1/07 (R/07), A/equine/East Renfrewshire/11 (ER/11), A/equine/Cambremer/1/2012 (C/12) and A/equine/Northamptonshire/1/13 (N/1/13). The retrospective meta-analysis confirmed a decreased pathogenicity of the EIV ER/11 and C/12 strains when compared with R/07. Experimental infection by individual nebulisation improved the clinical and virological parameters induced by recent FC2 strains, when compared with conventional room nebulisation. In conclusion, individual nebulisation offers a better control of the challenge dose administered and a greater homogeneity of the response measured in control animals. This in turn, helps maintain the number of animals per group to the minimum necessary required to obtain meaningful results in vaccine efficacy studies, which adheres to the 3Rs (Replacement, Reduction and Refinement) principles. [ABSTRACT FROM AUTHOR]

If indeed globalization is an important driver of disease emergence and resurgence, we have created the conditions for increasing disease risks. Particularly in the case of animal and zoonotic diseases, network analysis has been used to understand the connections that underlie disease spread pathways. Network analysis has been used to inform disease control and surveillance (Brookes et al., 2019), and known network vulnerability to disruption can be taken advantage of within strategies to control disease spread (Rautureau et al., 2011). [Extracted from the article]

Equine influenza is an infectious and contagious disease of horses. Studies on this topic are rare in the Maghreb countries. Therefore, the aim of this work is to present the various studies conducted on serological and molecular equine influenza virus since 1972 in the Maghreb region in particular in Morocco, Algeria and Tunisia. A total of four equine influenza strains were isolated in the Maghreb Arab region. A/equine/Nador/1/1997(H3N8), A/equine/Essaouira/2/2004(H3N8), A/equine/Essaouira/3/2004(H3N8) and A/equine/Algiers/1/1972(H3N8). The highest homology of HA nucleotide sequences of A/equine/Nador/1/1997(H3N8) with European strains: A/equine/Italy/1199/1992(H3N8) and A/equine/Brescia/1999(H3N8) clearly clustered A/equine/Nador/1/1997(H3N8) with the strains belonging to the European lineage. However, A/equine/Algiers/1/1972(H3N8), A/equine/Essaouira/2/2004(H3N8) and A/equine/Essaouira/3/2004(H3N8) were placed in the predivergent lineage indicating that like-Miami/63 strains infected equids in Morocco in 2004. This finding does not corroborate the recent studies of the H3N8 subtype of equine influenza viruses which have demonstrated that the oldest equine H3N8 strains, circulating before 1990 apparently went extinct.

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.

The article focuses on the impact of Covid-19 pandemic on the Equine Health Association. Topics include considered the Memorandum of Agreement that was to be signed between the EHA and the China Horse Industry Association in 2020 was achieved via Zoom Conference with CHIA Secretary General Yue Gaofeng and signed separate documents simultaneously.

Equines in the West Indies are used for recreational purposes, tourism industry, racing and agriculture or can be found in feral populations. Little is known in the Caribbean basin about the prevalence of some major equine infectious diseases, some with zoonotic potential, listed as reportable by the OIE. Our objective was to study the prevalence of antibodies for West Nile Virus (WNV), Equine Herpes Virus-1 and 4 (EHV-1 and EHV-4), Equine Influenza (EI), Equine Viral Arteritis (EVA) and Equine Infectious Anemia Virus (EIAV) using a retrospective serological convenience study. We used 180 equine serum samples, 140 from horses and 40 from donkeys in St. Kitts, Nevis, and Sint Eustatius, collected between 2006 and 2015 that were tested with ELISA kits and virus neutralization (for WNV and EVA). Combining ELISA with virus neutralization testing, 25 (13.8%) equine sera were WNV positive (a mixture of indigenous and imported equines) and 3 sera (1.6%) showed doubtful results. For EHV-1, 41 equines (23.7%), mean age 6.7 years, were seropositive. For EHV-4, 138 equines were found seropositive (82.8%), mean age 6.3 years. For EI, 49 equines (27.2%), mean age 7.5 years, were seropositive on ELISA, some previously vaccinated horses. No antibodies against EAV were found on virus neutralization testing, although one animal (0.6%), was EAV positive on ELISA. All samples were EIAV negative. The seroprevalence for EHV-1 and EHV-4 is similar to other parts of the world. For the first time in the study location serologic evidence of antibodies against WNV and EI is reported. This was found in both indigenous and imported animals, highlighting the need for developing proper surveillance plans based on complementary methods of virus detection. Further studies will be needed to define the prevalence, rates of transmission, characterize local virus strains, and study their impact on these populations. [ABSTRACT FROM AUTHOR]

Equine influenza viruses (EIVs) are responsible for acute contagious respiratory infection in equines and the disease remains a major threat for equine population throughout the world despite vaccination strategies in place. The present study was aimed to assess the suitability of BALB/c mice as a potential small animal model for preliminary screening of EI vaccine candidates. For this, we evaluated the immunogenicity and protective efficacy of an inactivated EIV (H3N8) vaccine in BALB/c mouse model after challenge with homologous H3N8 virus (Clade 2 virus, Florida sublineage) through serology, clinical signs, gross and histopathology lesions with grading, immunohistochemistry and virus quantification. Serological responses in immunized mice were evaluated by haemagglutination inhibition assay (HAI) and antibodies were subtyped by ELISA. The vaccine induced optimum protective antibody titre on 49 dpi along with balanced Th1/Th2 responses. Immunized mice were well protected against EIV challenge as evident by significant rise in serum antibody titre which concurred with mild clinical signs, early recovery, lower gross and histopathological lesions score, less severe intensity of viral antigen distribution, restricted virus replication in respiratory tract and less virus detection in nasal washes for short duration. The duration of the viral load was also lower and only for brief period as compared to unvaccinated challenged mice. In conclusion, induction of H3N8 specific antibody response and protection against H3N8 challenge proves that egg grown inactivated H3N8 whole virus vaccine would provide an effective intercession against H3N8 virus. In addition, BALB/c mouse can serve as an attractive tool for adjudging protective efficacy of vaccine candidates prior to final testing in equines. [ABSTRACT FROM AUTHOR]

To date, vaccination is one of the most efficient methods of prevention against equine infectious diseases. The vaccinology session, which was organised during the annual meeting of the French Equine Veterinarians Association (AVEF) at Reims (France) in 2016, aimed to approach three subjects of importance for the equine industry. Vaccination against three major equine diseases were used as examples: equine influenza (equine influenza virus), rhinopneumonitis (equine herpes virus 1/4), and tetanus (Clostridium tetani neuro-toxin). (1) Emergency vaccination: while it has been very successful to reduce the impact of equine influenza epizooties and it is also recommended for tetanus in case of surgery and accident, the benefit of emergency vaccination against equine herpes virus 1/4 remains arguable; (2) Compatibility of equine vaccines from different brands: despite being a frequent concerns for equine veterinarians, little information is available about the compatibility of equine vaccines from different commercial origins. The consequence of mixing different equine vaccines targeting the same disease is believed to be limited but scientific evidences are sparse; and, (3) Laps vaccination and vaccine shortage: they could have serious consequences in terms of protection and their impact should be evaluated on a case by case basis, taking into account the risk of contact with the pathogen and the effect on herd immunity. [ABSTRACT FROM AUTHOR]

Horses are critically important for Mongolian herders' livelihoods, providing transportation and food products, and playing important cultural roles. Equine influenza virus (EIV) epizootics have been frequent among Mongolia's horses, with five occurring since 1970. We sought to estimate the prevalence for EIV infection among horses and Bactrian camels with influenza-like illness between national epizootics. In 2016-2017, active surveillance for EIV was periodically performed in four aimags (provinces). Nasal swabs were collected from 680 horses and 131 camels. Seven of the horse swabs were "positive" for qRT-PCR evidence of influenza A (Ct value ⩽ 38). Two more were "suspect positive" (Ct value > 38 and ⩽ 40). These nine specimens were collected from four aimags. None of the camel specimens had molecular evidence of infection. Despite serial blind passage in Madin-Darby Canine Kidney cells (MDCK) cells, none of the nine horse specimens yielded an influenza A virus. None of the 131 herder households surveyed had recently vaccinated their horses against EIV. It seems likely that sporadic EIV is enzootic in multiple Mongolian aimags. This finding, the infrequent use of EIV vaccination, periodic prevalence of highly pathogenic avian influenza, and the mixing of domestic and wild equid herds suggest that Mongolia may be a hot spot for novel EIV emergence. [ABSTRACT FROM AUTHOR]

Natural Horsemanship is popular among many amateur and professional trainers and as such, has been the subject of recent scientific enquiry. One method commonly adopted by Natural Horsemanship (NH) trainers is that of round pen training (RPT). RPT sessions are usually split into a series of bouts; each including two phases: chasing/flight and chasing offset/flight offset. However, NH training styles are heterogeneous. This study investigated online videos of RPT to explore the characteristics of RPT sessions and test for differences in techniques and outcomes between amateurs and professionals (the latter being defined as those with accompanying online materials that promote clinics, merchandise or a service to the public). From more than 300 candidate videos, we selected sample files for individual amateur (n = 24) and professional (n = 21) trainers. Inclusion criteria were: training at liberty in a Round Pen; more than one bout and good quality video. Sessions or portions of sessions were excluded if the trainer attached equipment, such as a lunge line, directly to the horse or the horse was saddled, mounted or ridden. The number of bouts and duration of each chasing and non-chasing phase were recorded, and the duration of each RPT session was calculated. General weighted regression analysis revealed that, when compared with amateurs, professionals showed fewer arm movements per bout (p<0.05). Poisson regression analysis showed that professionals spent more time looking up at their horses, when transitioning between gaits, than amateurs did (p<0.05). The probability of horses following the trainer was not significantly associated with amount of chasing, regardless of category. Given that, according to some practitioners, the following response is a goal of RPT, this result may prompt caution in those inclined to give chase. The horses handled by professionals showed fewer conflict behaviours (e.g. kicking, biting, stomping, head-tossing, defecating, bucking and attempting to escape), and fewer oral and head movements (e.g. head-lowering, licking and chewing) than those horses handled by amateurs Overall, these findings highlight the need for selectivity when using the internet as an educational source and the importance of trainer skill and excellent timing when using negative reinforcement in horse training. [ABSTRACT FROM AUTHOR]

Background: Equine influenza is a major health problem of equines worldwide. The polymerase genes of influenza virus have key roles in virus replication, transcription, transmission between hosts and pathogenesis. Hence, the comprehensive genetic and codon usage bias of polymerase genes of equine influenza virus (EIV) were analyzed to elucidate the genetic and evolutionary relationships in a novel perspective. Results: The group - specific consensus amino acid substitutions were identified in all polymerase genes of EIVs that led to divergence of EIVs into various clades. The consistent amino acid changes were also detected in the Florida clade 2 EIVs circulating in Europe and Asia since 2007. To study the codon usage patterns, a total of 281,324 codons of polymerase genes of EIV H3N8 isolates from 1963 to 2015 were systemically analyzed. The polymerase genes of EIVs exhibit a weak codon usage bias. The ENc-GC3s and Neutrality plots indicated that natural selection is the major influencing factor of codon usage bias, and that the impact of mutation pressure is comparatively minor. The methods for estimating host imposed translation pressure suggested that the polymerase acidic (PA) gene seems to be under less translational pressure compared to polymerase basic 1 (PB1) and polymerase basic 2 (PB2) genes. The multivariate statistical analysis of polymerase genes divided EIVs into four evolutionary diverged clusters - Pre-divergent, Eurasian, Florida sub-lineage 1 and 2. Conclusions: Various lineage specific amino acid substitutions observed in all polymerase genes of EIVs and especially, clade 2 EIVs underwent major variations which led to the emergence of a phylogenetically distinct group of EIVs originating from Richmond/1/07. The codon usage bias was low in all the polymerase genes of EIVs that was influenced by the multiple factors such as the nucleotide compositions, mutation pressure, aromaticity and hydropathicity. However, natural selection was the major influencing factor in defining the codon usage patterns and evolution of polymerase genes of EIVs. [ABSTRACT FROM AUTHOR]

Background A widespread epidemic of equine influenza (EI) occurred in nonvaccinated equine population across multiple districts in Khyber Pakhtunkhwa Province of Pakistan during 2015–2016. Objectives and methods An epidemiological surveillance study was conducted from Oct 2015 to April 2016 to investigate the outbreak. EI virus strains were isolated in embryonated eggs from suspected equines swab samples and were subjected to genome sequencing using M13 tagged segment specific primers. Phylogenetic analyses of the nucleotide sequences were concluded using Geneious. Haemagglutinin (HA), Neuraminidase (NA), Matrix (M) and nucleoprotein (NP) genes nucleotide and amino acid sequences of the isolated viruses were aligned with those of OIE recommended, FC-1, FC-2, and contemporary isolates of influenza A viruses from other species. Results HA and NA genes amino acid sequences were very similar to Tennessee/14 and Malaysia/15 of FC-1 and clustered with the contemporary isolates recently reported in the USA. Phylogenetic analysis showed that these viruses were mostly identical (with 99.6% and 97.4% nucleotide homology) to, and were reassortants containing chicken/Pakistan/14 (H7N3) and Canine/Beijing/10 (H3N2) like M and NP genes. Genetic analysis indicated that A/equine/Pakistan/16 viruses were most probably the result of several re-assortments between the co-circulating avian and equine viruses, and were genetically unlike the other equine viruses due to the presence of H7N3 or H3N2 like M and NP genes. Conclusion Epidemiological data analysis indicated the potential chance of mixed, and management such as mixed farming system by keeping equine, canine and backyard poultry together in confined premises as the greater risk factors responsible for the re-assortments. Other factors might have contributed to the spread of the epidemic, including low awareness level, poor control of equine movements, and absence of border control disease strategies. [ABSTRACT FROM AUTHOR]

Canine influenza viruses (CIVs) cause a contagious respiratory disease in dogs. CIV subtypes include H3N8, which originated from the transfer of H3N8 equine influenza virus (EIV) to dogs; and the H3N2, which is an avian-origin virus adapted to infect dogs. Only inactivated influenza vaccines (IIVs) are currently available against the different CIV subtypes. However, the efficacy of these CIV IIVs is not optimal and improved vaccines are necessary for the efficient prevention of disease caused by CIVs in dogs. Since live-attenuated influenza vaccines (LAIVs) induce better immunogenicity and protection efficacy than IIVs, we have combined our previously described H3N8 and H3N2 CIV LAIVs to create a bivalent vaccine against both CIV subtypes. Our findings show that, in a mouse model of infection, the bivalent CIV LAIV is safe and able to induce, upon a single intranasal immunization, better protection than that induced by a bivalent CIV IIV against subsequent challenge with H3N8 or H3N2 CIVs. These protection results also correlated with the ability of the bivalent CIV LAIV to induce better humoral immune responses. This is the first description of a bivalent LAIV for the control and prevention of H3N8 and H3N2 CIV infections in dogs. [ABSTRACT FROM AUTHOR]

Background: Influenza virus isolation in embryonated chicken eggs (ECEs) is not applicable for rapid diagnosis, however it allows the recovery and propagation of the viable virus. A low number of infectious virus particles in the swabs, poor quality of samples or individual strain properties can lead to difficulties during the virus isolation process. We propose to utilize chorioallantoic membranes (CAM) of ECEs with the assistance of real-time RT PCR to facilitate equine influenza virus isolation. Methods: Real-time RT PCR was used to detect influenza virus genetic material in amniotic/allantoic fluids (AF) and CAM of ECEs. Haemagglutination assay was used for AF. We used highly diluted virus as a substitute of clinical specimen for ECEs inoculation. Results: Our study demonstrated that real-time RT PCR testing of CAM homogenates was more useful than testing of AF for EIV detection in ECEs. Positive results from CAM allowed to select the embryos from those with haemagglutination assay (HA) - and real-time RT PCR-negative AF for further passages. Using homogenates of CAM for subsequent passages, we finally obtained HA-positive AF, which confirmed virus replication. Conclusion: We postulate that real-time RT PCR testing of CAM homogenates and their subsequent passages may facilitate the isolation of equine influenza viruses. [ABSTRACT FROM AUTHOR]

Equine influenza virus (EIV) is a major respiratory pathogen of horses despite the availability of equine influenza vaccines. This study aimed to determine genetic evolution of EIV strains in France between 1967 to present. A whole genome comparative analysis was also conducted on recent French strains in order to identify potential factors of pathogenicity. Comparison of French EIV sequences with vaccine and worldwide epidemic strains revealed amino acid substitutions in both haemagglutinin (HA) and neuraminidase, especially within the antigenic sites and/or close to receptor binding sites (HA). Amino acid substitutions were also identified in other genes, mainly the polymerase complex proteins and PB1-F2. Viruses belonging to Eurasian and American lineages have circulated until 2003 and Florida sub-lineage Clade 2 strains predominates since 2005. The last French strain (2015) displayed several specificities in HA suggesting the occurrence of antigenic drift with presence of pathogenic markers in the PA and PB1-F2 genes. [ABSTRACT FROM AUTHOR]

Japan established a vaccine selection system, in which a committee evaluates veterinary influenza vaccines to determine if the vaccine should be updated. In 2013, it was concluded that the present equine influenza vaccine strains did not have to be updated, but clade 2 (Fc2) viruses of the Florida sublineage should be included. We collected three Fc2 viruses as candidates and conducted comparative tests. Results indicated that A/equine/Carlow/2011 (H3N8) is not suitable, because of its unstable antigenic characteristics. A comparison between A/equine/Richmond/1/2007 (H3N8) (Richmond/07) and A/equine/Yokohama/aq13/2010 (H3N8) (Yokohama/10) in eggs showed that they shared equal growth properties. Immunogenicity test in mice showed that Yokohama/10 induced higher HI antibody titers than Richmond/07. Therefore, we concluded that Yokohama/10 was the most suitable strain. [ABSTRACT FROM AUTHOR]

Equine influenza viruses (EIV) are a major cause of acute respiratory disease in horses worldwide and occasionally also affect vaccinated animals. Like other influenza A viruses, they undergo antigenic drift, highlighting the importance of both surveillance and virus characterisation in order for vaccine strains to be kept up to date. The aim of the work reported here was to monitor the genetic and antigenic changes occurring in EIV circulating in the UK from 2013 to 2015 and to identify any evidence of vaccine breakdown in the field. Virus isolation, reverse transcription polymerase chain reaction (RT-PCR) and sequencing were performed on EIV-positive nasopharyngeal swab samples submitted to the Diagnostic Laboratory Services at the Animal Health Trust (AHT). Phylogenetic analyses were completed for the haemagglutinin-1 (HA1) and neuraminidase (NA) genes using PhyML and amino acid sequences compared against the current World Organisation for Animal Health (OIE)-recommended Florida clade 2 vaccine strain. Substitutions between the new isolates and the vaccine strain were mapped onto the three-dimensional structure protein structures using PyMol. Antigenic analyses were carried out by haemagglutination inhibition assay using a panel of post-infection ferret antisera. Sixty-nine outbreaks of equine influenza in the UK were reported by the AHT between January 2013 and December 2015. Forty-seven viruses were successfully isolated in eggs from 41 of the outbreaks. Only three cases of vaccine breakdown were identified and in each case the vaccine used contained a virus antigen not currently recommended for equine influenza vaccines. Nucleotide sequencing of the HA and NA genes revealed that all of the viruses belonged to the Florida clade 2 sub-lineage of H3N8 EIV. Phylogenetic and sequence analyses showed that the two sub-populations, previously identified within clade 2, continued to circulate and had accrued further amino acid substitutions. Antigenic characterisation using post-infection ferret antisera in haemagglutination inhibition assays however, failed to detect any marked antigenic differences between the isolates. These findings show that Florida clade 2 EIV continue to circulate in the UK and support the current OIE recommendation to include an example of Florida clade 2 in vaccines. [ABSTRACT FROM AUTHOR]

Sarcoids are the most common equine skin tumours Although they do not metastasize, they can be locally aggressive and cause significant clinical symptoms in affected horses. Despite being common, very little is known about the host immune response and the biological mechanisms underlying persistence and recurrence of equine sarcoids. The latter reflects the need for further research in this field. This in-vitro study used sarcoid explants from horses with naturally occurring sarcoids (n = 12) to evaluate the induction of a humoral immune response directed against equine sarcoid-derived bovine papilloma-virus (BPV)− 1 infected fibroblasts using a flow cytometric crossmatch assay. The presence of antibodies against exogenous bovine serum albumin (BSA) and fibroblast-like mesenchymal stromal cells (MSCs) was also evaluated by ELISA and flow cytometry, respectively. The viral load in the sarcoid explants, the corresponding cultured sarcoid fibroblasts, and matched peripheral blood mononuclear cells (PBMCs) from affected horses were determined by quantitative BPV-1/− 2 PCR analysis. Antibodies against autologous sarcoid cells were present in six out of twelve sarcoid-affected horses. Serum from all horses showed cross reactivity with allogeneic sarcoid cells, while only a part reacted with BSA or MSCs. Screening of host PBMCs demonstrated the absence of BPV E1 nucleic acids. Statistical analysis revealed a significantly higher mean viral load in the parental sarcoid tissue compared to the low passage fibroblasts (P < 0.001). These results support the hypothesis that sarcoid-affected horses may develop antibodies recognizing tumour-specific antigens. In contrast to sarcoid explants, equine PBMCs do not seem to contain complete BPV genomes. These results provide a basis for future investigations on the clinical relevance of these antibodies. [ABSTRACT FROM AUTHOR]

The paper presents the findings of specific antibodies in the blood sera of donkeys against the following viruses: equine infectious anemia virus (EIAV), African horse sickness virus (AHSV), equine herpesvirus type 1 (EHV-1), equine influenza virus subtype H3N8 (EIV) and equine arteritis virus (EAV). The analyses were conducted during the year 2022. From a total of 199 donkeys bred in "Zasavica", blood was sampled from 53 animals (2 male donkeys and 51 female donkeys), aged 3 to 10 years. Specific antibodies against EIAV were not detected in any of the tested animals using the agar-gel immunodiffusion (AGID) assay. No specific antibodies against AHSV, tested by enzyme-linked immunosorbent assay (ELISA), or antibodies against EAV, tested by the virus neutralization test (VNT) and ELISA were detected in any of these animals. A positive serological result for EHV-1 was determined by the VNT in all animals, with antibody titer values ranging from 1:2 to 1:128, while a very low antibody titer value for EIV (subtype H3N8) of 1:16 was determined in 18 donkeys using the hemagglutination inhibition test (HI test).

ANP32 proteins, which act as influenza polymerase cofactors, vary between birds and mammals. In mammals, ANP32A and ANP32B have been reported to serve essential but redundant roles to support influenza polymerase activity. The well-known mammalian adaptation PB2-E627K enables influenza polymerase to use mammalian ANP32 proteins. However, some mammalian-adapted influenza viruses do not harbor this substitution. Here, we show that alternative PB2 adaptations, Q591R and D701N, also allow influenza polymerase to use mammalian ANP32 proteins, whereas other PB2 mutations, G158E, T271A, and D740N, increase polymerase activity in the presence of avian ANP32 proteins as well. Furthermore, PB2-E627K strongly favors use of mammalian ANP32B proteins, whereas D701N shows no such bias. Accordingly, PB2-E627K adaptation emerges in species with strong pro-viral ANP32B proteins, such as humans and mice, while D701N is more commonly seen in isolates from swine, dogs, and horses, where ANP32A proteins are the preferred cofactor. Using an experimental evolution approach, we show that the passage of viruses containing avian polymerases in human cells drove acquisition of PB2-E627K, but not in the absence of ANP32B. Finally, we show that the strong pro-viral support of ANP32B for PB2-E627K maps to the low-complexity acidic region (LCAR) tail of ANP32B. IMPORTANCE Influenza viruses naturally reside in wild aquatic birds. However, the high mutation rate of influenza viruses allows them to rapidly and frequently adapt to new hosts, including mammals. Viruses that succeed in these zoonotic jumps pose a pandemic threat whereby the virus adapts sufficiently to efficiently transmit human-to-human. The influenza virus polymerase is central to viral replication and restriction of polymerase activity is a major barrier to species jumps. ANP32 proteins are essential for influenza polymerase activity. In this study, we describe how avian influenza viruses can adapt in several different ways to use mammalian ANP32 proteins. We further show that differences between mammalian ANP32 proteins can select different adaptive changes and are responsible for some of the typical mutations that arise in mammalian-adapted influenza polymerases. These different adaptive mutations may determine the relative zoonotic potential of influenza viruses and thus help assess their pandemic risk., Competing Interests: The authors declare no conflict of interest.

We previously reported that horse antiserum against the Japanese equine influenza vaccine virus, A/equine/La Plata/1993 ( LP93) exhibited reduced cross-neutralization against some Florida sublineage Clade (Fc) 2 viruses, for example, A/equine/Carlow/2011 ( CL11). As a result, Japanese vaccine manufacturers will replace LP93 with A/equine/Yokohama/aq13/2010 (Y10, Fc2). To assess the benefit of updating the vaccine, five horses vaccinated with inactivated Y10 vaccine and five vaccinated with inactivated LP93 were challenged by exposure to a nebulized aerosol of CL11. The durations of pyrexia (≥38.5°C) and other adverse clinical symptoms experienced by the Y10 group were significantly shorter than those of the LP93 group. [ABSTRACT FROM AUTHOR]