Figure 1

Figure 1. The 3 aimags from which nasal swab specimens were collected from healthy Bactrian camels, for influenza A virus testing, Mongolia, 2012. 1, Töv; 2, Khentii; 3, Dundgovi.

During January 2012–January 2013, a total of 460 nasal swab specimens were collected through active surveillance of horses and camels in 3 Mongolian aimags (Figure 1) known for high densities of free-ranging horses and camels (Table). Specimens were collected monthly, as weather permitted, from 50 free-ranging horses and 20 free-ranging Bactrian camels that were safely and carefully restrained with halters, ropes, and by hand, according to a protocol approved by the Department of Veterinary and Animal Breeding, Government of Mongolia. During sampling, camels were in a crouched or take-down position. Horse and camel specimens were carefully stored and shipped in separate containers; to prevent cross-contamination with EIV, specimens were separated during laboratory analyses.

All specimens were first screened at the Institute of Veterinary Medicine laboratory (Ulaanbaatar, Mongolia) by using the World Health Organization influenza A quantitative reverse transcription PCR (qRT-PCR) protocol (10). Six specimens collected from camels without respiratory signs were positive for influenza A virus and were double-blind passaged in embryonated chicken eggs. Subsequent testing revealed hemagglutination activity in all 6 specimens. Allantoic fluid of the 6 cultured specimens was then shipped to the University of Florida for confirmation testing and sequencing. Only 1 specimen was confirmed positive by influenza A virus–specific qRT-PCR (cycle threshold [C_t]<35), suggesting possible virus degradation during shipment, despite specimens being shipped on dry ice and carefully handled upon receipt (10). The original swab specimen from a camel was later shared with the Mongolia National Influenza Center for confirmation in an anonymized panel of 10 camel swab specimens. Using World Health Organization qRT-PCR procedures, staff identified the specimen as having the strongest evidence (by C_t) of influenza A virus. Staff further studied the specimen with conventional RT-PCR primers and probes for the hemagglutinin and neuraminidase genomes. These reactions yielded amplicons of the expected size, which were sequenced and found to be 100% identical to the corresponding portions of the J. Craig Venter Institute (Rockville, MD, USA) sequences described below.

Sanger sequence data for the hemagglutinin and neuraminidase genes of this isolate demonstrated extremely high levels of identity with recent EIV from Asia isolated under this project in 2011 (4). Full-genome sequencing of the 8 genome segments amplified by multisegment RT-PCR (11) was performed at the J. Craig Venter Institute by using Ion Torrent PGM technology (Life Technologies, Grand Island, NY, USA) with a 314v2 chip, and the sequences were validated by using the MiSeq platform (Illumina, Inc., San Diego, CA, USA). Full-genome sequence data for this virus, named A/camel/Mongolia/335/2012(H3N8), were deposited in GenBank (accession nos. CY164120.1, CY164121.1, CY164122.1, CY164123.1, CY164124.1, CY164125.1, CY164126.1 and CY164127.1). The isolate should soon (summer 2014) become available for other research use through BEI Resources (Manassas, VA, USA).

Figure 2

Figure 2. Evolutionary relationships of 155 full-length hemagglutinin sequences from equine A(H3N8)viruses collected globally and A/camel/Mongolia/335/2012 (arrow). The 2 clades associated with most recent equine influenza A(H3N8) viruses, Florida clade 1 and Florida...

For each of the 8 viral genome segments, phylogenetic trees were inferred separately by use of the maximum-likelihood methods available in RAxML version 7.2.6 (12), a general time-reversible model of nucleotide substitution, and a gamma-distributed rate variation among sites, with a bootstrapping resampling process (500 replicates). All 8 viral genome segments (polymerase basic protein [PB] 2, PB1, polymerase acidic protein (PA), hemagglutinin (HA) , nucleocapsid protein (NP), neuraminidase (NA), matrix protein (MP), and nonstructural protein (NS) are closely related to the equine influenza A(H3N8) viruses that have recently been circulating among the horse population in Asia; this lineage is evolutionarily distinct from the influenza A(H3N8) viruses circulating among birds in Asia (Technical Appendix Figures 1–7). A more detailed phylogenetic analysis of the hemagglutinin segment performed by using the additional background sequence data representing the global diversity of influenza A(H3N8) viruses in horses indicates that A/camel/Mongolia/335/2012 is positioned within Florida clade 2 (FC2) (Figure 2) (13) and, more specifically, within a bootstrap-supported clade that contains 3 influenza A(H3N8) viruses isolated from horses in Mongolia in 2011: A/equine/Mongolia/6/2011 (100% nt similarity), A/equine/Mongolia/56/2011 (99.9% nt similarity), and A/equine/Mongolia/3/2011 (100% nt similarity). A/camel/Mongolia/335/2012 also contains an insertion of 2 aa (I and F) near the beginning of the hemagglutinin sequence (hemagglutinin positions 8–9, Technical Appendix) that was first detected among FC2 equine viruses in 2005 (A/equine/Bari/2005/H3N8) and has been detected in most FC2 viruses, including all viruses that are closely related to A/camel/Mongolia/335/2012.

The phylogeny indicates that A/camel/Mongolia/335/2012 probably represents a relatively recent horse-to-camel transmission event. Without additional isolates from camels or corresponding epidemiologic data, and given the close genetic relationship between A/camel/Mongolia/335/2012 and related equine viruses, it is impossible to determine at this time whether the virus has been successfully transmitted from camel to camel.

In recent years, enhanced surveillance has detected influenza A viruses across a wider range of mammalian hosts, including horses, swine, dogs (14), seals (15), cats, and now camels, providing a more complete picture of the ecology of influenza A viruses beyond their presence in birds. How influenza A viruses successfully jump from 1 host species to another and what the constraints on interspecies transmission are remain key questions about influenza virus ecology and assessments of pandemic threats. Our findings highlight the need to further elucidate the ecology of influenza viruses and other pathogens in free-ranging camel populations.