Background
Equine influenza (EI) is a highly contagious upper respiratory disease affecting horses and other equid species [
1,
2]. EI is characterized by anorexia, marked increase in body temperature, nasal discharges, and dry cough [
3]. The disease is widespread internationally and is a major challenge for global equestrian activities [
4]. EI is caused by equine influenza virus (EIV), which is a member of the genus
Alphainfluenzavirus, and family
Orthomyxoviridae [
5]. EIV is an enveloped virus with an octameric negative sense single-stranded RNA genome that encodes at least 10 structural and non-structural proteins. Two major surface glycoproteins; hemagglutinin (HA) and neuraminidase (NA), have been described as determinants of virus infectivity and antigenicity [
3,
6].
EIV of the subtype H7N7 was first reported in various European countries in 1955–1956 [
7]. A few years later, another subtype (H3N8) emerged in the US and became the predominant subtype worldwide [
8]. Viruses of the H7N7 subtype were apparently outcompeted by the H3N8 subtype viruses and are not thought to circulate in the equine population today [
9]. In contrast, H3N8 diverged in the 1980s into two lineages; American and Eurasian [
10]. The American lineage later further diverged into three sub-lineages (Kentucky, South America, and Florida). The Florida sub-lineage continued to predominantly circulate globally to date [
11]. In the last two decades, the Florida sub-lineage was further subdivided according to the HA gene sequence into two clades; Florida clade 1 (FC1) and Florida clade 2 (FC2) [
12]. FC1 was mostly identified in the Americas, while FC2 was predominant in Asia and Europe. Intercontinental circulation was frequently documented as a result of international equine races and exhibitions [
13,
14].
Egypt is an important centre for raising and selling pure-bred Arabian horses [
15]. To the best of our knowledge, the earliest EIV study was conducted in 1983 in Egypt [
16], but it is thought that EIV had an earlier impact on the equine population in Egypt, before subtype H7N7 was identified in horses and donkeys with respiratory disease in 1989 [
17]. This was the last reported isolation of the H7N7 subtype from equids. During the winter of 2000, an EI epizootic affected large numbers of horses, donkeys, and mules in Upper Egypt. Serological evidence indicated that the circulating strain belonged to the H3N8 subtype, however no sequences were available for genetic analysis [
18]. EIV was again isolated from the equine population in nine Egyptian governorates in 2008 [
19]. Molecular characterization of partial HA gene sequences and phylogenetic analysis showed that the prevalent viruses were all members of FC1 [
20]. A concurrent isolation of another FC1 EIV, A/equine/Egypt/6066NAMRU3-VSVRI/2008 (H3N8), was used to prepare an inactivated whole virus vaccine [
15,
21]. No further data on circulating equine influenza were published during the last decade.
Vaccination is considered the most effective measure for controlling EIV worldwide [
3,
22]. However, EI is still reported in both vaccinated and non-vaccinated equine populations [
4,
14,
23,
24]. This may result from antigenic drift in the surface glycoproteins and subsequent emergence of variants, and frequent introduction and circulation of genetically diverse EI viruses due to equine trade [
6]. Therefore, continuous surveillance of the circulating strains in endemic regions to inform the choice of vaccine strains is of utmost importance according to the recommendations –World Organisation for Animal Health expert surveillance panel (OIE-ESP) [
25]. In this study, an H3 subtype of equine influenza in Florida clade 2 was identified for the first time in Egypt in three Arabian racehorses. The study reports FC2 introduction into Egyptian horse population a decade after FC1. Comparison of deduced amino acid sequences with the OIE recommended and available vaccine strains highlighted the urgency of updating the candidate(s) used in national vaccination programs.
Discussion
Influenza is the most important respiratory disease in equines worldwide. It gained its importance from the direct economic impact on equine trade and international equine events and shows. Mild EI cases may go unnoticed but complicated cases can be fatal. EIV H3N8 has shown ability to infect vaccinated horses causing low to moderate losses [
14,
33,
34], however great losses are often linked to virus introduction into naïve equine populations. For instance, introduction of EIV into the Australian equine population has resulted in infection of about 70,000 horses [
35]. Furthermore, recent emergence of EIV FCL1 into Western and Central African countries led to more than 66,000 deaths in horses and donkeys [
36,
37]. Proper vaccination against EIV in terms of timing, frequency, and relevance of vaccine antigen, stimulates a cumulative antibody response in animals to control virus spread and reduce disease outcomes. It also provides partial protection against newly introduced strains [
33]. Conversely, improper vaccination and/or poor vaccination coverage may result in new disease outbreaks, particularly with the introduction of novel strains.
The equine population in Egypt and its health status is mostly underestimated. Unofficial reports from the Egyptian ministry of agriculture estimated the equine population as approximately one million in 2017 and three million in 2021; among which horses may represent 10–25%. Data on the prevalence and distribution of EIV in Egypt over 40 years are limited to a total of four reports [
16‐
18,
20]. The most comprehensive study was conducted in 2008, when a countrywide outbreak affected all equine species in Egypt. EIV H3N8 FCL1 was detected and isolated as the causative pathogen [
20], a concurrently isolated strain (Egypt/6066NAMRU-VSVRI/2008) was used to prepare a whole inactivated virus vaccine [
15] and the Fluvac innovator® 4 (Zoetis-US) was also registered for use in Egypt. Since then, no data on the circulation pattern of the virus were retrieved although sporadic cases are frequently observed in vaccinated and non-vaccinated animals nationwide.
In this study, we aimed to investigate whether EIV H3N8 was still circulating as a cause of respiratory disease in the equine population in Egypt. Therefore, 22 samples were collected from vaccinated and non-vaccinated horses showing respiratory signs suggestive for EI between August 2017 and April 2018 from different localities in Egypt. Samples were screened by RT-qPCR and none of them was positive. The sensitivity of the assay [
26] and the successful amplification of positive controls suggest negative results were either due to improper timing of sampling or absence of the target pathogen in these particular samples. Unfortunately, no sera were available to evaluate antibody responses and possible exposure of non-vaccinated horses.
Fortunately, within the course of this study, nasal swab samples from three Arabian mares suffering from fever and gait stiffness were submitted to our lab for EIV diagnosis. These mares were unvaccinated and participating in a national horse race, which was later cancelled upon confirmation of EIV infection. Samples were screened using RT-qPCR and all of them were positive for EIV H3N8 with low C
T values [
16‐
21] suggestive of high virus load. Attempts for virus isolation in embryonated chicken eggs were unsuccessful using single, pooled, stock, or diluted samples (Data not shown). Such failure is sometimes expected due to difficult adaptation of some EIV strains in chicken embryos [
3].
The majority of the HA1 domain of all positive samples was amplified and sequenced. Sequence and phylogenetic analysis have indicated that the three viruses belong to FC2-144V subgroup, the clade that has never been reported in Egypt or in the middle east region before, compared to FC2 isolates from Algeria 2011 [
38] and Turkey 2013 [
39]. The sequence of two viruses was identical whereas the third contained 3 synonymous nucleotide changes. This variation may suggest the FC2 virus had been circulating in Egypt prior to detection as the horse race event was national where all participating horses came from Cairo and other provinces in Egypt. Furthermore, the high similarity between the Egyptian virus sequences and others identified in the UK between 2015 and 2016 [
14] suggest an earlier introduction of the virus.
Divergence of the Florida sub-lineage was first identified in 2003, when two distant outbreaks were caused by virus strains of significant HA1 sequence heterogeneity; A/equine/South Africa/03 the prototype of FC1 and A/equine/Newmarket/5/03 the prototype of FC2 [
40]. Originally, FC1 was exclusively dominant in equids of the US [
3], while both clades co-circulated in other parts of the world with apparent dominance of FC2 in Europe [
40,
41]. FC2 introduction into the US was reported in 2014 through a diseased mare imported from Germany [
42]. It is not expected that FC2 arrived in Egypt similarly as importation of horses is not a common occurrence. We assume that one or more of the Egyptian Arabian horses participated in an international event or show 2015 or later and contracted the infection before spreading the virus upon return home. This is supported by the V267I substitution which is shared between the study sequences and sequences from UK November 2015 [
14] and 2016 [
43] (data not shown). Proper vaccination of these horses might suppress the clinical disease or mild clinical signs may have been misdiagnosed as being a result of travel stress [
44]. The limited spread and late observation of FC2 in Egypt can be explained by the fact that the Arabian horses represent only 10% of the total horse population in Egypt (1% of total equids) and are mostly owned by government or private studs with high care, vaccination, and very little contact with other horses.
HA is the major determinant of pathogenicity and antigenicity in influenza viruses. It is composed of two domains; HA1 which forms the globular head and HA2 that forms the stalk [
45‐
47]. The HA1 domain carries in its structure the receptor-binding domain (RBD) and five major antigenic sites (A–E), hence it represents the principal inducer of strain specific immunity against EIV. Trivial changes in the RBD or in one of the antigenic sites can cause vaccination failure [
12,
47,
48]. On the deduced amino acid level, study HA1 sequences were identical to each other with 14 amino acid differences from the Egyptian FC1 vaccine strain, 13 amino acid differences from Kentucky/1/97 vaccine strain and 9 amino acid differences from Richmond/1/2007, the OIE-ESP recommended FC2 vaccine strain [
32]. Most of the variations observed were not linked to any of the antigenic sites in the HA1 protein [
11] except for A144V and T192K in the antigenic sites A and B respectively (Fig.
2). It was previously shown that 2 amino acid substitutions in the H3 HA1 surface were enough for antigenic drift and vaccine update [
49] but it is accepted that at least 4 amino acid changes in no-less-than two antigenic sites are required to update the strains in the vaccine [
50].
All three Egyptian sequences shared the amino acid variant A144V specific for FC2-144V subgroup isolated from Europe 2011–2015 [
14] (Fig.
1). Two unique amino acid substitutions in the Egyptian HA1 sequences (T3N and I9N) were not located in an antigenic site.
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