Equine Influenza Virus Research Articles

Unraveling the causes of respiratory disease in the working equids of Ethiopia: A cross sectional survey

Previous research has demonstrated that respiratory disease, particularly coughing and nasal discharge are consistently ranked in the top-three health concerns for working equid owners. However, there is little existing literature on the possible aetiology and these syndromes are poorly defined. This study aimed to better understand respiratory disease, including frequency of signs, in working equids and assess exposure to the major respiratory pathogens. A cross-sectional study was conducted across 19 sites in Central Ethiopia. Owners and horses were sampled using systematic random sampling from a selected focal point in the town. A brief questionnaire was administered to drivers/owners to ascertain horse details and history of respiratory signs or disease. A clinical examination was performed and a jugular blood sample was taken. Heat-treated serum was transported to the UK for testing. Serology for antibody titres to equine influenza virus (EIV) used a multispecies Influenza A competitive Enzyme-linked Immunosorbant Assay (ELISA). Samples showing a borderline negative result ( 0.3 on ELISA. Complement fixation was used to determine serum antibody titres to equine herpes viruses (EHV-1/4) and equine rhinitis viruses (ERAV/ERBV). Over 19 sites, 350 participants were selected. Owners reported 38% animals examined had recent history of coughing (last 30 days), 8% had a history of nasal discharge and 3% had suffered other breathing problems. In addition 11% horses examined had a clinically significant nasal discharge. Serology results for 350 horses suggested recent exposure to S equi in 23%, EAV 3.4%, but exposure to influenza virus was very rare. Low antibody titres to EHV-1/4 and ERAV/ERBV were also detected. Clinical signs of respiratory disease were relatively uncommon on examination of these working horses; however owners reported higher levels of respiratory signs in the previous 30 days, particularly coughing, consistent with results from the previous participatory studies. It appears S equi is endemic in the population and is likely to be a

Analysis of codon usage bias and evolution of equine influenza viruses

Analysis of codon usage bias and evolution of equine influenza viruses

Assessment of antigenic difference of equine influenza virus by challenge study in horses

Assessment of antigenic difference of equine influenza virus by challenge study in horses

Influenza virus vaccine for neglected hosts: horses and dogs.

This study provides information regarding vaccine research and the epidemiology of influenza virus in neglected hosts (horses and dogs). Equine influenza virus (EIV) causes a highly contagious disease in horses and other equids, and outbreaks have occurred worldwide. EIV has resulted in costly damage to the horse industry and has the ability of cross the host species barrier from horses to dogs. Canine influenza is a virus of equine or avian origin and infects companion animals that live in close contact with humans; this results in possible exposure to the seasonal epizootic influenza virus. There have been case reports of genetic reassortment between human and canine influenza viruses, which results in high virulence and the ability of transmission to ferrets. This emphasizes the need for vaccine research on neglected hosts to update knowledge on current strains and to advance technology for controlling influenza outbreaks for public health.

Serological evidence of equine influenza virus in horse stables in Kaduna, Nigeria.

ABSTRACTEquine influenza virus (EIV) is a major cause of acute respiratory diseases in horses in most parts of the world that results in severe economic losses. Information on the epidemiology of EIV in tropical Africa is scanty. An enzyme-linked immunosorbent assay (ELISA) was used to detect the presence of influenza A virus nucleoprotein (NP) in 284 horse sera in Kaduna State, Northern Nigeria. The ELISA-positive sera were further examined for hemagglutination inhibition (HI) antibodies to two strains each of H3N8 and H7N3 subtypes of influenza A virus. The results showed that antibodies against influenza A virus nucleoprotein were detected in 60.9% (173 of 284) of horses examined by NP-ELISA. Equine H3 and H7 subtypes were detected in 60% (21 of 35) and 20% (7 of 35) of horse sera respectively across the stables. Adequate quarantine of all imported horses, a national equine influenza surveillance plan and an appropriate EIV control program in Nigeria are recommended to safeguard the large horse population.

Functional analysis of glyco-molecules that bind with influenza virus.

Influenza A virus (IAV) recognizes terminal sialic acid of sialoglyco-conjugates on host cells through the viral envelope glycoprotein hemagglutinin (HA), followed by initiation of entry into the cells. Molecular species of sialic acid are largely divided into two moieties: N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). A receptor for IAV infection generally means Neu5Ac. Almost all equine IAVs and some human, swine, and duck IAVs bind not only to Neu5Ac but also to Neu5Gc. In nonhuman animals, Neu5Gc has been detected in swine and equine tracheas and the duck colon, which are the main replication sites of mammalian and avian IAVs. Therefore, Neu5Gc in these sites has been suggested to be a functional receptor for IAV infection. Humans cannot synthesize Neu5Gc due to a genetic defect of the Neu5Gc-synthesizing enzyme. We evaluated the receptor function of Neu5Gc in IAV infection in human cells. Our results indicated that Neu5Gc expression on the surface of human cells is not a functional receptor for IAV infection and that it has a negative effect on infectivity of IAV possessing Neu5Gc binding ability. IAV also binds to non-sialo 3-O-sulfated galactosylceramide (sulfatide). Sulfatide has been suggested to be a functional receptor for IAV infection. However, we have shown that sulfatide is not a functional receptor for IAV infection and that the binding of HA with sulfatide enhances progeny virus production. It is expected that functions of these glyco-molecules can be used in prevention and development of new drugs against IAV.

A new safe and effective cold-adapted modified live equine influenza virus vaccine that enables the differentiation of infected from vaccinated animals

ABSTRACT An analysis of the main advantages and shortcomings of the existing inactivated and live vaccines against the equine influenza viruses (EIVs) is given in this paper. For the first time, the most important information, concerning the development of a new live modified cold-adapted (ca) equine influenza virus vaccine based on the A/HK/Otar/6:2/2010 strain is summarized. We discuss a number of unique features of the developed vaccine that have not previously been reported, and compare the new vaccine with the existing equine influenza vaccines. The properties of the developed equine vaccine include: long-lasting (12 months or more) protective immunity after a single immunization; sterile immunity after double vaccination; cross-protection against the heterologous virus at 12 months after double vaccination and the differentiation of infected from vaccinated animals.

Новая безопасная и эффективная живая модифицированная холодоадаптированная вирусная вакцина против гриппа лошадей, позволяющая дифференцировать инфицированных животных от вакцинированных

В настоящей работе дается анализ основных преимуществ и недостатков применяемых на практике инактивированных и живых вакцин против гриппа лошадей. Впервые обобщены наиболее значимые сведения, касающиеся разработки новой живой модифицированной холодоадаптированной вирусной вакцины против гриппа лошадей на основе штамма А/HK/Otar/6:2/2010. Обсуждается ряд уникальных свойств разработанной вакцины, о которых ранее не сообщалось, в сравнении с аналогичными вакцинами против гриппа лошадей. К числу подобных свойств вакцины относятся: продолжительный (не менее 12 месяцев) протективный иммунитет после однократной вакцинации; стерильный иммунитет после двукратной вакцинации; перекрестная защита от гетерологичного вируса через 12 месяцев после двукратной вакцинации; дифференциация инфицированных животных от вакцинированных.

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

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

Epidemiological and virological findings during multiple outbreaks of equine influenza in South America in 2012.

In 2012, equine influenza (EI) virus was confirmed as the cause of outbreaks of respiratory disease in horses throughout South America. In Uruguay and Argentina, hundreds of vaccinated thoroughbred horses in training and racing facilities were clinically affected. To characterise the EI viruses detected during the outbreak in Uruguay and Argentina. Virus was detected in nasopharyngeal swabs by a pan-reactive influenza type A real-time RT-PCR. The nucleotide sequence of the HA1 gene was determined and analysed phylogenetically using mega 5 software. Amino acid sequences alignments were constructed and virus was antigenically characterised with specific ferret antisera. Paired serum samples were tested by haemagglutination inhibition and single radial haemolysis. The diagnosis of EIV was confirmed by real-time RT-PCR, virus isolation and serological testing. The phylogenetic analysis of HA1 gene sequences of 18 EI viruses indicated that all of them belong to clade 1 of the Florida sublineage of the American lineage and are closely related to viruses isolated in the United States in 2012. The HA1 of viruses identified in horses in racing facilities in Maroñas, Uruguay, and in Palermo, Argentina, displayed 100% amino acid sequence identity and were identical to that of a virus isolated in Dubai in 2012, from vaccinated endurance horses recently imported from Uruguay. The surveillance data reported illustrate the international spread of EI viruses and support the recommendations of the OIE expert surveillance panel to include viruses of the Florida sublineage in vaccines.

Design and testing of multiplex RT-PCR primers for the rapid detection of influenza A virus genomic segments: Application to equine influenza virus

The avian influenza A virus causes respiratory infections in animal species. It can undergo genomic recombination with newly obtained genetic material through an interspecies transmission. However, the process is an unpredictable event, making it difficult to predict the emergence of a new pandemic virus and distinguish its origin, especially when the virus is the result of multiple infections. Therefore, identifying a novel influenza is entirely dependent on sequencing its whole genome. Occasionally, however, it can be time-consuming, costly, and labor-intensive when sequencing many influenza viruses. To compensate for the difficulty, we developed a rapid, cost-effective, and simple multiplex RT-PCR to identify the viral genomic segments. As an example to evaluate its performance, H3N8 equine influenza virus (EIV) was studied for the purpose. In developing this protocol to amplify the EIV eight-segments, a series of processes, including phylogenetic analysis based on different influenza hosts, in silico analyses to estimate primer specificity, coverage, and variation scores, and investigation of host-specific amino acids, were progressively conducted to reduce or eliminate the negative factors that might affect PCR amplification. Selectively, EIV specific primers were synthesized with dual priming oligonucleotides (DPO) system to increase primer specificity. As a result, 16 primer pairs were selected to screen the dominantly circulating H3N8 EIV 8 genome segments: PA (3), PB2 (1), PA (3), NP (3), NA8 (2), HA3 (1), NS (1), and M (2). The diagnostic performance of the primers was evaluated with eight sets composing of four segment combinations using viral samples from various influenza hosts. The PCR results suggest that the multiplex RT-PCR has a wide range of applications in detection and diagnosis of newly emerging EIVs. Further, the proposed procedures of designing multiplex primers are expected to be used for detecting other animal influenza A viruses.

Different pattern of haemagglutinin immunoreactivity of equine influenza virus strains isolated in Poland

Abstract The immunoreactivity of haemagglutinin (HA) polypeptides of equine influenza virus was compared among the strains isolated in Poland, using H3 monoclonal antibody. A stronger signal in immunoblot reaction was observed for A/equi/Pulawy/2008 HA polypeptides compared to A/equi/Pulawy/2006, despite the fact that both strains are phylogenetically closely related and belong to Florida clade 2 of American lineage. The strongest signal, observed in the case of A/equi/Pulawy/2008, seemed to be connected with the presence of G135, I213, E379, and/or V530 instead of R135, M213, G379, and I530 present in A/equi/Pulawy/2006 HA sequence. This implies that point mutations within amino acid sequences of HA polypeptides of equine influenza virus may change their immunoreactivity even when they are not located within five basic antigenic sites.

Pathology of Equine Influenza virus (H3N8) in Murine Model.

Equine influenza viruses (EIV)—H3N8 continue to circulate in equine population throughout the world. They evolve by the process of antigenic drift that leads to substantial change in the antigenicity of the virus, thereby necessitating substitution of virus strain in the vaccines. This requires frequent testing of the new vaccines in the in vivo system; however, lack of an appropriate laboratory animal challenge model for testing protective efficacy of equine influenza vaccine candidates hinders the screening of new vaccines and other therapeutic approaches. In the present investigation, BALB/c mouse were explored for suitability for conducting pathogenecity studies for EIV. The BALB/c mice were inoculated intranasally @ 2×106.24 EID50 with EIV (H3N8) belonging to Clade 2 of Florida sublineage and monitored for setting up of infection and associated parameters. All mice inoculated with EIV exhibited clinical signs viz. loss in body weights, lethargy, dyspnea, etc, between 3 and 5 days which commensurate with lesions observed in the respiratory tract including rhinitis, tracheitis, bronchitis, bronchiolitis, alveolitis and diffuse interstitial pneumonia. Transmission electron microscopy, immunohistochemistry, virus quantification through titration and qRT-PCR demonstrated active viral infection in the upper and lower respiratory tract. Serology revealed rise in serum lactate dehydrogenase levels along with sero-conversion. The pattern of disease progression, pathological lesions and virus recovery from nasal washings and lungs in the present investigations in mice were comparable to natural and experimental EIV infection in equines. The findings establish BALB/c mice as small animal model for studying EIV (H3N8) infection and will have immense potential for dissecting viral pathogenesis, vaccine efficacy studies, preliminary screening of vaccine candidates and antiviral therapeutics against EIV.

NS-gene based phylogenetic analysis of equine influenza viruses isolated in Poland

The phylogenetic analysis of influenza virus is based mainly on the variable hemagglutinin or neuraminidase genes. However, some discrete evolutionary trends might be revealed when more conservative genes are considered. We compared all available in GenBank database full length NS sequences of equine influenza virus including Polish isolates. Four nucleotides at positions A202, A237, T672 and A714 and three amino acids at positions H59, K71 and S216 which are also present in A/eq/Pulawy/2006 and A/eq/Pulawy/2008 may be discriminating for the Florida sublineage. Threonine at position 83 seems to be characteristic for EIV strains of Florida 2 isolated after 2007. There are nine common substitutions in the NS sequences of A/eq/Pulawy/2005, A/eq/Aboyne/1/2005 and A/eq/Lincolnshire/1/2006 in relation to the reference strain A/eq/Miami/63, resulting in four amino acid changes in NS1 protein (I56, E76, K140, E179) and one in NEP (R22). We grouped these strains as “Aboyne-like”. Some of the listed changes were also observed in H7N7 strains isolated between 1956 and 1966, in A/eq/Jilin/89 or in pre-divergent H3N8 strains. Two hypotheses regarding the origin of this group were postulated: three independent transfers of avian influenza viruses into the equine population or reassortation between H7N7 and H3N8 EIV. Similarities of the NS sequences of “Aboyne like” viruses to viruses isolated in the fifties or seventies can reflect a phenomenon of “frozen evolution”.

Equine influenza: literature review

Equine influenzavirus (EIV) is considered one of the major infectious agents causing respiratory outbreaks in horses in Brazil and worldwide. EIV is classified into two subtypes, H3N8 is widespread in horse population and subtype H7N7, despite being possibly detected by serological methods, is considered extinct or circulating at low levels of prevalence. The disease presents short incubation period, about 48 hours, and recovery of up to three weeks. Equine influenza is mainly characterized by high morbidity and low mortality and clinical signs vary from mild to severe, depending on the immune status of the animal, which may occur due to worsening of secondary bacterial infections. Diagnosis is based on clinical and epidemiological characteristics of the disease. It is important for the differential diagnosis mainly Rodococcus equi and Streptococcus equi. Basic therapy is supportive, including the isolation of symptomatic animals. Prophylactic measures are used as methods of health management associated with vaccination.

Case Report of Equine Influenza in Italy, in 2014

Equine influenza (EI) is a highly contagious respiratory disease presently caused by subtype H3N8. As no active surveillance is ongoing in Italy, clinical observations of veterinary practitioners would indicate the infection as endemic. This study reports the isolation and genetic characterization of an EI virus (EIV) from a stable of Thoroughbred race horses. The episode was characterized by fever and dry cough and involved eleven, 2- to 3-year-old, flu vaccinated horses, four of which were found positive for EIV, by reverse-transcriptase real-time polymerase chain reaction (RRT-PCR) targeting the matrix gene. The EIV of two of the four RRT-PCR positive samples was successfully passaged in embryonated chicken eggs. Analysis of amino acid (AA) sequences of hemagglutination (HA) residues detected seven substitutions in the Italian isolate when compared with the prototype strain A/eq/Richmond/1/2007 (H3N8 American lineage, Florida clade 2). The constructed phylogenetic tree locates the present strain within the Florida clade 2, together with viruses isolated between 2011 and 2014 in Algeria, China, Eire, Germany, Japan, Mongolia, and the United Kingdom. Within this cluster, the Italian isolate presents 100% AA identity with A/eq/North Rhine Westphalia/1/2014, responsible of an outbreak in Germany around the same period. Even if vaccinated, only two of the 11 clinically involved animals, different from the EIV-positive animals, reacted positive in HA inhibition. Monitoring of the circulating strains of EIV is essential especially for verifying the appropriateness of the vaccine virus composition, as recommended by the OIE Panel of experts, on the basis of the genetic and antigenic characteristics of emerging strains.

Effect of exercise on erythron, heart and respiratory rates in horses vaccinated against equine herpes virus 4/1 and equine influenza virus

Limited data are available for the influence of physical training in jumping horses at the background of booster vaccination. The aim of the study was to establish the effects of physical exercise applied at the background of booster vaccination against equine herpes virus 4/1 (EHV 4/1) and equine influenza virus (EIV) on changes in the erythron, breathing and pulse rates of horses. The differences were investigated between vaccinated and exercised horses and vaccinated-only controls. Fifteen Hanoverian geldings were distributed into 3 groups: group А, 3 non-vaccinated animals; group В, 6 vaccinated and group С, 6 vaccinated horses submitted to physical exercise. Horses from groups B and C were vaccinated 1 year ago against EHV 4/1 and EIV. Вlood samples were evaluated for erythron parameters following standard procedures. The pulse and breathing rates were determined during the 14-day study period. The physical training of horses resulted in most substantial increase in red blood cell count, haemoglobin and haematocrit on post exercise hour 0, day 1 and day 11; all values were within the reference ranges except for haemoglobin on hour 0. The red blood cell indices MCV and MCH were not significantly altered vs initial level except for MCHC on day 2 and day 11. Pulse and breathing rates were enhanced on post exercise hours 0 and 2 vs initial level and groups A and B. The results are important for evaluation of training effects on aerobic potential enhancing the physical performance.

Equine and Canine Influenza H3N8 Viruses Show Minimal Biological Differences Despite Phylogenetic Divergence.

The A/H3N8 canine influenza virus (CIV) emerged from A/H3N8 equine influenza virus (EIV) around the year 2000 through the transfer of a single virus from horses to dogs. We defined and compared the biological properties of EIV and CIV by examining their genetic variation, infection, and growth in different cell cultures, receptor specificity, hemagglutinin (HA) cleavage, and infection and growth in horse and dog tracheal explant cultures. Comparison of sequences of viruses from horses and dogs revealed mutations that may be linked to host adaptation and tropism. We prepared infectious clones of representative EIV and CIV strains that were similar to the consensus sequences of viruses from each host. The rescued viruses, including HA and neuraminidase (NA) double reassortants, exhibited similar degrees of long-term growth in MDCK cells. Different host cells showed various levels of susceptibility to infection, but no differences in infectivity were seen when comparing viruses. All viruses preferred α2-3- over α2-6-linked sialic acids for infections, and glycan microarray analysis showed that EIV and CIV HA-Fc fusion proteins bound only to α2-3-linked sialic acids. Cleavage assays showed that EIV and CIV HA proteins required trypsin for efficient cleavage, and no differences in cleavage efficiency were seen. Inoculation of the viruses into tracheal explants revealed similar levels of infection and replication by each virus in dog trachea, although EIV was more infectious in horse trachea than CIV. Influenza A viruses can cross species barriers and cause severe disease in their new hosts. Infections with highly pathogenic avian H5N1 virus and, more recently, avian H7N9 virus have resulted in high rates of lethality in humans. Unfortunately, our current understanding of how influenza viruses jump species barriers is limited. Our aim was to provide an overview and biological characterization of H3N8 equine and canine influenza viruses using various experimental approaches, since the canine virus emerged from horses approximately 15 years ago. We showed that although there were numerous genetic differences between the equine and canine viruses, this variation did not result in dramatic biological differences between the viruses from the two hosts, and the viruses appeared phenotypically equivalent in most assays we conducted. These findings suggest that the cross-species transmission and adaptation of influenza viruses may be mediated by subtle changes in virus biology.

Development of a real-time RT-PCR for detection of equine influenza virus

Equine influenza is a highly contagious respiratory disease in horses caused by influenza A viruses. In this work a real-time RT-PCR for fast and sensitive diagnosis of equine influenza viruses (EIV) targeting a highly conserved region of the matrix gene was developed. In addition two RT-PCR methods for the amplification of large parts of the matrix- and HA gene were adapted for molecular-epidemiological characterization of viruses. The primers of the real-time RT-PCR had homologies of 99.4% to EIV- and 97.7% to all influenza A viral sequences, whereas the minor groove binder (MGB) probe showed homologies of 99.3% and 99.6%, respectively. These high values allow application of the assay for influenza viruses in other species. Using 20 equine, 11 porcine and 2 avian samples, diagnostic suitability of the assay was confirmed. High specificity for influenza viruses was shown both experimentally and by software simulation. The assay analytical sensitivity was at 10(2)-10(3) copies of RNA and 10(0)-10(1) copies of DNA, respectively. This allows virus detection also in circumstances of minor viral shedding. All amplified EIV sequences were classified phylogenetically within the known lineages.

Inefficient transmissibility of NS-truncated H3N8 equine influenza virus in dogs.

H3N8 equine influenza virus (EIV) causes respiratory diseases in the horse population, and it has been demonstrated that EIV can transmit into dogs owing to its availability on receptors of canine respiratory epithelial cells. Recently, we isolated H3N8 EIV from an EIV-vaccinated horse that showed symptoms of respiratory disease, and which has a partially truncated nonstructural gene (NS). However, it is not clear that the NS-truncated EIV has an ability to cross the host species barrier from horses to dogs as well. Here, we experimentally infected the NS-truncated H3N8 EIV into dogs, and monitored their clinical signs and viral load in respiratory organs to determine the virus's transmissibility.