key: cord-0956466-hc7efqsg authors: Yondon, Myagmarsukh; Zayat, Batsukh; Nelson, Martha I.; Heil, Gary L.; Anderson, Benjamin D.; Lin, Xudong; Halpin, Rebecca A.; McKenzie, Pamela P.; White, Sarah K.; Wentworth, David E.; Gray, Gregory C. title: Equine Influenza A(H3N8) Virus Isolated from Bactrian Camel, Mongolia date: 2014-12-03 journal: Emerg Infect Dis DOI: 10.3201/eid2012.140435 sha: 92c3491310afbd2b13368d1e64b5e46ac71934bf doc_id: 956466 cord_uid: hc7efqsg Because little is known about the ecology of influenza viruses in camels, 460 nasal swab specimens were collected from healthy (no overt illness) Bactrian camels in Mongolia during 2012. One specimen was positive for influenza A virus (A/camel/Mongolia/335/2012[H3N8]), which is phylogenetically related to equine influenza A(H3N8) viruses and probably represents natural horse-to-camel transmission. Because little is known about the ecology of influenza viruses in camels, 460 nasal swab specimens were collected from healthy (no overt illness) Bactrian camels in Mongolia during 2012. One specimen was positive for influenza A virus (A/camel/Mongolia/335/2012[H3N8]), which is phylogenetically related to equine influenza A(H3N8) viruses and probably represents natural horse-to-camel transmission. S ince the first isolation in 1963 of an avian-origin influenza A(H3N8) virus from horses (1), subtype H3N8 influenza viruses have continued to circulate panzootically among horses, causing severe outbreaks of equine influenza respiratory disease. In the United States, these viruses jumped from horses to dogs and continue to circulate among dogs (2, 3) . In Mongolia, the site of some of the world's largest epizootics of equine influenza A(H3N8) virus (EIV) infection, transmission of this virus is sustained among 2.1 million free-ranging horses, causing significant economic losses among rural herders. Major epizootics of EIV infection occurred in Mongolia during 2007-2008 (459,000 cases, 24,600 deaths) and again in 2011 (74,608 cases, 40 deaths) (4). Over previous decades in Mongolia, outbreaks of respiratory disease, thought to be influenza, among camels have been reported, In the 1980s, the virus was characterized, and researchers speculated that it was related to a reassortant influenza A(H1N1) virus vaccine strain, A/PR-8/34 + A/USSR/77, generated in a Soviet laboratory and administered to humans in Mongolia and possibly transmitted from vaccinated humans to camels in a reactivated form (5, 6) . However, only 1 genetic sequence from this outbreak among camels is available in Gen-Bank: A/camel/Mongolia/1982/H1N1. Despite reports of serologic activity against influenza A virus among camels in several African countries (7, 8) , the lack of isolated virus from these populations highlights how little is known about the ecology of influenza viruses in camels. Questions about the potential role of camels in human cases of Middle East respiratory syndrome (9) further highlight our lack of knowledge of infectious diseases in camels and the merits of increased surveillance at this unique human-animal interface. Since January 2011, surveillance of equine influenza viruses has been enhanced in 3 Mongolian aimags (provinces). Surveillance among camels was also initiated in response to anecdotal reports of signs of respiratory illness in Bactrian camels (Camelus bactrianus). We describe the isolation, full-genome sequencing, and phylogenetic characterization of an influenza A(H3N8) virus of equine lineage isolated from a Bactrian camel, thereby identifying a novel route of influenza virus interspecies transmission and raising further questions about influenza A virus ecology in understudied regions such as Mongolia. During January-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 (WHO) influenza A quantitative reverse transcription PCR (qRT-PCR) protocol (10) . Six specimens collected from camels without respiratory signs were positive for influenza A 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. 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 The phylogeny indicates that A/camel/Mongolia/ 335/2012 probably represents a relatively recent horse-tocamel 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 freeranging camel populations. A new influenza virus associated with equine respiratory disease Transmission of equine influenza virus to dogs Microevolution of canine influenza virus in shelters and its molecular epidemiology in the United States Isolation and characterization of H3N8 equine influenza A virus associated with the 2011 epizootic in Mongolia. Influenza Other Respir Viruses Previous H1N1 influenza A viruses circulating in the Mongolian population A reassortant H1N1 influenza A virus caused fatal epizootics among camels in Mongolia Detection of influenza antibody in animal sera from Kassala region, Sudan, by agar gel diffusion test Preliminary survey for antibodies against respiratory viruses among slaughter camels (Camelus dromedarius) in north-eastern Nigeria Middle East respiratory syndrome coronavirus in dromedary camels: an outbreak investigation World Health Organization. WHO information for molecular diagnosis of influenza virus in humans-update Single-reaction genomic amplification accelerates sequencing and vaccine production for classical and swine origin human influenza A viruses RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models Genome-scale evolution and phylodynamics of equine H3N8 influenza A virus A novel reassortant canine H3N1 influenza virus between pandemic H1N1 and canine H3N2 influenza viruses in Korea Emergence of fatal avian influenza in New England harbor seals We thank Badarch Darmaa for her help with validating the influenza culture work.