Phylogenetic Analysis and Characterization of a Sporadic Isolate of Equine Influenza A H3N8 from an Unvaccinated Horse in 2015

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 this H3N8 isolate shared the highest percentage identity to A/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.


Introduction
Equine influenza epizootics, which affect horses, zebra, mules, and donkeys all over the world, are characterized by an acute dry cough, high body temperature, mucopurulent nasal discharge, lethargy and anorexia [1][2][3][4][5]. Vaccination failure, the mobility of unvaccinated horses and insufficient quarantine measures The aim of this study was to characterize an equine H3N8 virus isolate, obtained from a 3-year-old unvaccinated gelding showing respiratory disease, from Montana, USA, in 2015. Phylogenetically distinct clades/lineages of EIV have been co-circulating globally, undergoing gene reassortments, thereby posing a serious challenge in the selection of vaccine strains. Hence, understanding the evolutionary profile of EIV is imperative to estimate the phylogenetic diversity and distribution of equine influenza viruses. Here, we describe the phylogenetic characterization of all the eight gene segments of this recently isolated EIV. Our phylogenetic analysis inferred that the polymerase basic protein 1 (PB1), polymerase acidic (PA), and nucleoprotein (NP) segments of A/equine/Montana/9564-1/2015 clustered with A/equine/Tennessee/29A/2014 and hemagglutinin (HA), polymerase basic protein 2 (PB2), neuraminidase (NA), matrix (M), and non-structural (NS) segments clustered with A/equine/Malaysia/M201/2015 and A/equine/Tennessee/29A/2014, both belong to Clade 1 (FC1) viruses of Florida sub-lineage [45]. To test the in vitro cross-species susceptibility and zoonotic potential, we infected A/equine/Montana/9564-1/2015 on airway derived primary cells from different mammalian species such as swine, bovine, and equine, along with human lung epithelial A549 cells and MDCK cells from canine origin. Our results showed that A/equine/ Montana/9564-1/2015 productively replicated in these cell lines, suggestive of its broad host spectrum and possible cross-species transmissibility.

Case Description
In May 2015, nasal swabs were sent to the Animal Disease and Research Diagnostic Laboratory (ADRDL), South Dakota State University from three horses showing respiratory disease from Montana, USA. Among the three horses, only one tested positive for equine influenza A by Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) and one of the other two animals tested positive for Streptococcus equi. The influenza A positive horse was an unvaccinated 3-year-old gelding and the case was reported as a sporadic case of natural infection. The virus was isolated by passaging on MDCK cells and designated as A/equine/Montana/9564-1/2015.

Whole Genome Sequencing and Phylogenetic Analysis
Whole genome sequencing of A/equine/Montana/9564-1/2015 propagated on MDCK cells was performed using the Illumina MiSeq instrument and Nextera XT library preparation kit (San Diego, CA, USA), as described previously [46]. Whole genome sequencing confirmed it as a case of equine influenza A, H3N8 subtype. The sequences were submitted to the NCBI GenBank (Accession #s MG198996-MG199003). A BLASTn search analysis, optimized for highly similar sequences (megablast), was conducted for all the eight gene segments of A/equine/Montana/9564-1/2015 (H3N8) [47].
Both canine and equine nucleotide sequences of H3N8 subtype were acquired from the influenza virus resource (https://www.ncbi.nlm.nih.gov/genomes/FLU/Database/nph-select.cgi?go=database, accessed 5 January 2018) and phylogenetic analyses were performed using MEGA 7.0 [45,48]. Nucleotide sequences were aligned using MUltiple Sequence Comparison by Log-Expectation (MUSCLE) and the evolutionary history of each segment was inferred by constructing maximum likelihood trees, using the best nucleotide substitution models, suggested by the 'test for best DNA/ protein fitness' in MEGA 7.0 [48,49]. The best nucleotide substitution models inferred for maximum likelihood trees for the gene segments were general time-reversible with gamma distributed with invariant sites (GTR+G+I) for PB2, PB1, NP; Tamurai-Nei with gamma distributed with invariant sites (TN93+G+I) for PA, HA; general time-reversible with gamma distributed (GTR+G) for NA; Hasegawa-Kishino-Yano with gamma distributed with invariant sites (HKY+G+I) for M; Hasegawa-Kishino-Yano with gamma distributed (HKY+G) for NS [50][51][52]. All nucleotide positions containing gaps and missing data were partially deleted and very strong branch filters were applied to run the analysis. For each taxon, the bootstrap value was determined from 1000 replicates to verify the tree topology.

Virus and Cell Culture
A/equine/Montana/9564-1/2015 virus, isolated from a nasal swab and propagated on MDCK cells, was a generous gift from the ADRDL. MDCK cells, maintained in Dulbecco's Modified Eagle medium, supplemented with 10% fetal bovine serum (FBS) (PAA Laboratories Inc., Dartmouth, MA, USA) and penicillin-streptomycin (Life Technologies, Carlsbad, CA, USA) (100 U/mL) were used in this study. MDCK cells, cultured in the T-75 flask, were inoculated with the virus inoculum at 0.01 multiplicity of infection (MOI) and incubated at 37 • C in 5% CO 2 for 1 h. Following infection, the virus growth medium, consisting of fresh DMEM with 0.3% bovine serum albumin (BSA), 1 µg/mL tolylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin (Sigma, Saint Louis, MO, USA) and penicillin-streptomycin (100 U/mL) (Life Technologies, Carlsbad, CA, USA), was added for further incubation at 37 • C in 5% CO 2 for 48-72 h. The infected cell cultures were freeze-thawed. The supernatant was spun at 500× g for 10 min at 4 • C to remove the cellular debris. Determination of virus titers in MDCK cells was done according to the Reed and Muench method [53].

BLAST Analysis
Whole genome sequencing was performed, and the virus was identified as EIV subtype

Phylogenetic Analysis
To estimate the evolutionary history of A/equine/Montana/9564-1/2015, we performed phylogenetic analyses of all the eight gene segments, with the currently available sequences of both canine and equine H3N8 subtypes in the influenza virus resource database [45,54]. The EIV sequences we analyzed, included the sequences from pre-divergence, Eurasian and American lineages (Kentucky and South American sub-lineages) and Clades 1 and 2 of the Florida sub-lineage. The total number of canine and equine sequences of H3N8 subtype, used to construct the phylogenetic trees for each segment were The evolutionary history of the eight viral gene segments obtained by the phylogenetic analyses was in complete agreement with the percent identity score obtained by BLASTn, clustering with equine influenza A H3N8 viruses and not with canine H3N8 viruses. The evolutionary history of HA and NA segments of A/equine/Montana/9564-1/2015, was shown as complete trees, in which the different phylogenetic groups of EIV were color-coded and grouped to describe the pre-divergence, Eurasian and American lineages (strains from Kentucky and Argentina/South American sub-lineages), the Florida sub-lineage and Clade 1 and Clade 2 viruses (Figures 1 and 2).

Viral Replication Kinetics
A/equine/Montana/9564-1/2015 was propagated in MDCK cells and the infectivity of the virus was determined by hemagglutination assay (HA) and by 50% tissue culture infective dose (TCID50/mL). To explore the in vitro cross-species susceptibility of this virus isolate, we used a panel of six cells, which included the airway primary cells derived from equine, swine and bovine species and A549 from human lung epithelium to determine the infectivity of A/equine /Montana/9564-1/2015. An MOI of 1.0 was used for infection of these cell types because lower MOIs failed to produce measurable replication kinetics. For MDCK cells, a cell line routinely used to replicate equine influenza viruses, only 0.01 MOI of the virus was used for infection. Interestingly, A/equine/Montana/9564-1/2015 productively replicated in swine primary tracheal epithelial cells

Viral Replication Kinetics
A/equine/Montana/9564-1/2015 was propagated in MDCK cells and the infectivity of the virus was determined by hemagglutination assay (HA) and by 50% tissue culture infective dose (TCID 50 /mL). To explore the in vitro cross-species susceptibility of this virus isolate, we used a panel of six cells, which included the airway primary cells derived from equine, swine and bovine species and A549 from human lung epithelium to determine the infectivity of A/equine /Montana/9564-1/2015. An MOI of 1.0 was used for infection of these cell types because lower MOIs failed to produce measurable replication kinetics. For MDCK cells, a cell line routinely used to replicate equine influenza viruses, only 0.01 MOI of the virus was used for infection. Interestingly, A/equine/Montana/9564-1/2015 productively replicated in swine primary tracheal epithelial cells (SPTrE), swine primary lung epithelial cells (SPLE) and bovine primary nasal turbinate (BPT), yielding peak titers of 5.03 (12 hpi), 5.21 (48 hpi) and 4.67 log 10 TCID 50 /mL (12 hpi), respectively (Figure 4). Equine primary tracheal myofibroblasts also yielded a comparable peak titer of 4.8 log 10 TCID 50 /mL at 24 hpi, whereas A549, the human lung epithelial cell line, supported A/equine/Montana/9564-1/2015 with a peak titer of 4.68 logs at 12 hpi. Infected MDCK cells demonstrated a peak titer of 6.8 log 10 TCID 50 /mL, which was 2 logs higher than those seen in primary cells, in spite of the low MOI used for infection. Overall, A/equine/Montana/9564-1/2015 productively replicated in all the six types of cells derived from swine, bovine, equine, human and canine to very appreciable titers (Figure 4).

Discussion
EIV H3N8 epidemics have been reported worldwide, on a large scale, since the first reported case of H3N8 occurred in Florida in 1963. Just like any other influenza epidemic in the past, EIV epidemics occur in vaccinated and immunologically naïve populations, in particular, H3N8 EIV epidemics can happen in horses of all ages, regardless of vaccination status [7,[56][57][58]. Vaccination breakdowns have been associated with EIV outbreaks in the past, as documented in thoroughbred yearlings in Kentucky, and several other parts of the world, including France and Ireland and horses imported into South Arabia and Japan [58]. The report we presented here was a sporadic case, which occurred in an unvaccinated 3-year-old gelding, characterized by clinical manifestations. The two other horses from the ranch exhibiting respiratory symptoms tested negative for influenza by qRT-PCR. As such, we concluded that this could be a sporadic infection. There is no information available pertaining to the movement or involvement of any social events/shows. Such cases of sporadic infection usually occur due to the mobility of inadequately quarantined, or sub-clinically infected, vaccinated or unvaccinated horses, into unvaccinated or inadequately vaccinated herds with little or no immunity [16,32].
According to the OIE Expert Surveillance Panel on Equine Influenza Vaccine Composition, the year 2015 witnessed an increased activity in H3N8 EIV cases, reported from 46 premises over 23 states in the USA [43]. In 2016, 30 confirmed EIV cases were reported from 16 states [43]. Unfortunately, there is no vaccination data available on these outbreaks from the USA. In 2015-2016, EIV outbreaks were reported in other parts of the world, such as Ireland, Sweden, and the UK. The outbreaks reported in the UK occurred in unvaccinated animals. As per the report, no cases have been reported from Asia and South America during this period [43].The OIE report also concluded that EIV H3N8

Discussion
EIV H3N8 epidemics have been reported worldwide, on a large scale, since the first reported case of H3N8 occurred in Florida in 1963. Just like any other influenza epidemic in the past, EIV epidemics occur in vaccinated and immunologically naïve populations, in particular, H3N8 EIV epidemics can happen in horses of all ages, regardless of vaccination status [7,[56][57][58]. Vaccination breakdowns have been associated with EIV outbreaks in the past, as documented in thoroughbred yearlings in Kentucky, and several other parts of the world, including France and Ireland and horses imported into South Arabia and Japan [58]. The report we presented here was a sporadic case, which occurred in an unvaccinated 3-year-old gelding, characterized by clinical manifestations. The two other horses from the ranch exhibiting respiratory symptoms tested negative for influenza by qRT-PCR. As such, we concluded that this could be a sporadic infection. There is no information available pertaining to the movement or involvement of any social events/shows. Such cases of sporadic infection usually occur due to the mobility of inadequately quarantined, or sub-clinically infected, vaccinated or unvaccinated horses, into unvaccinated or inadequately vaccinated herds with little or no immunity [16,32].
According to the OIE Expert Surveillance Panel on Equine Influenza Vaccine Composition, the year 2015 witnessed an increased activity in H3N8 EIV cases, reported from 46 premises over 23 states in the USA [43]. In 2016, 30 confirmed EIV cases were reported from 16 states [43]. Unfortunately, there is no vaccination data available on these outbreaks from the USA. In 2015-2016, EIV outbreaks were reported in other parts of the world, such as Ireland, Sweden, and the UK. The outbreaks reported in the UK occurred in unvaccinated animals. As per the report, no cases have been reported from Asia and South America during this period [43].The OIE report also concluded that EIV H3N8 viruses isolated from the USA in 2016 were homologous to the isolates from 2015 and belonged to the Florida sub-lineage Clade 1 (FC1). On the other hand, viruses detected from the UK in 2015-2016 were Florida sub-lineage Clade 2 (FC2) viruses. The data we obtained from phylogenetic analysis were in complete agreement with this observation.
The BLASTn analysis of all viral genome segments, except PB2, NA, and NS, showed the highest percentage identity to six H3N8 EIV strains isolated from Tennessee in 2014. Considering the percentage identity between these six highly homologous strains, we used A/equine/Tennessee/29A/2014 as a representative strain for our phylogenetic analysis. It was very difficult to conclude that this sporadic infection occurred from the contaminated premises, as there were no other horses on the premises reported to have EIV infection, according to available information to us. We also checked the influenza virus database (https://www.ncbi.nlm.nih.gov/genomes/FLU/Database/nph-select.cgi?go=database, Equine H3N8 viruses are known to have jumped the species barrier and have successfully established infections in canines, causing canine influenza epidemics and epizootics. Experimental infection of EIV has been demonstrated in vitro [59,60] and in vivo in calves, and cats [33,34]. An in vitro study by Feng et al. also demonstrated robust infection in MDCK, fibroblast cells from the dog (A72) and Norden Laboratories feline kidney (NLFK), moderate infection in Mustela putorius furo (Mpf) cell line from ferrets and poor infection in A549 and equine kidney cells (EQKD) [61]. Further, two H3N8 viruses of equine origin have been isolated from pigs in China during 2004-2006 and these strains were found to be closely related to the European H3N8 strains from the early 1990s [35]. Previous studies have shown that pigs are the 'mixing vessels'-harboring avian, swine and human influenza viruses-which facilitate gene reassortment, giving rise to novel reassortant influenza viruses with high transmissibility to humans [62]. Hence, the isolation of H3N8 viruses of equine origin from pigs in China demands attention, as the establishment and dissemination of the reassortant mammalian-adapted viruses can happen elsewhere in the world. The zoonotic potential of EIV has not been documented, even though there has been occasional serological evidence in occupational workers [39]. Nevertheless, EIV H3N8 has the potential to infect humans, as demonstrated in the experimental infection of human volunteers with EIV H3N8 and influenza-like symptoms were detected in this cohort [36][37][38]. In this study, we used species-specific primary respiratory epithelial cells, of swine, bovine and equine origin, to evaluate the replication competency of A/equine /Montana/9564-1/2015 and this was compared to MDCK, a cell line widely used for influenza studies [63]. The airway primary cells we used were non-transformed cells and can mimic the normal physiological environment. To represent respiratory cells from human origin, we used human lung epithelial cell line A549, which is yet another cell line used for influenza studies [64,65]. The increase in the viral titer demonstrated in the MDCK cells by 2 logs, at a lower MOI of 0.01, could be due to the adaptation of this EIV isolate to grow in MDCK cells, as the virus was initially isolated in MDCK cells [63]. Compared to MDCK cells, we used 100 times more MOI on non-transformed primary cells from bovine, swine and equine and also on the human lung epithelial cell line, A549, because lower MOIs failed to produce measurable replication kinetics. The explanation for the low virus titer in these primary cells and A549, despite using a high MOI, could be attributed to the differences in receptor preference or replication competence in different cell types and species-specific innate immune responses of the cells, posing a barrier to restrict the replication of this H3N8 virus [63,66]. Our data suggested the potential for cross-species susceptibility. However, these results should only be interpreted after taking into consideration the inherent limitations of translating in vitro findings to in vivo conditions.
Overall, this study provided us insights about the evolutionary relationship and in vitro cross-species infectivity of A/equine /Montana/9564-1/2015 (H3N8) virus. A comprehensive genome-scale analysis of new isolates is essential to understand the molecular evolution and phylodynamics of EIV, which in turn would help in the strategic selection of vaccine strains, effective surveillance, and control. Antigenic and genetic variations caused by evolutionary processes play a critical role in determining the dynamics of host range and tropism of influenza viruses. Further in vivo studies are needed to evaluate the cross-species transmissibility of EIV H3N8 and its ability to cause infections and respiratory diseases in other mammalian hosts, including humans.