Emerg Infect DisEIDEmerging Infectious Diseases1080-60401080-6059Centers for Disease Control and Prevention21029540329451810-050110.3201/eid1611.100501DispatchDispatchGenome Sequence Conservation of Hendra Virus Isolates during Spillover to Horses, AustraliaConservation of Hendra Virus IsolatesMarshGlenn A.ToddShawnFoordAdamHanssonEricDaviesKellyWrightLyndaMorrissyChrisHalpinKim1MiddletonDeborahFieldHume E.DanielsPeterWangLin-FaAuthor affiliations: Australian Animal Health Laboratory, Geelong, Victoria, Australia (G.A. Marsh, S. Todd, A. Foord, E. Hansson, K. Davies, L. Wright, C. Morrissy, K. Halpin, D. Middleton, P. Daniels, L.-F. Wang);Australian Biosecurity Cooperative Research Centre for Emerging Infectious Diseases, Brisbane, Queensland, Australia (H.E. Field, L.-F. Wang);Biosecurity Queensland, Brisbane (H.E. Field)Address for correspondence: Glenn A. Marsh, CSIRO Livestock Industries, Australian Animal Health Laboratory, PO Bag 24, Geelong, Victoria 3220, Australia; email: glenn.marsh@csiro.au112010161117671769

Bat-to-horse transmission of Hendra virus has occurred at least 14 times. Although clinical signs in horses have differed, genome sequencing has demonstrated little variation among the isolates. Our sequencing of 5 isolates from recent Hendra virus outbreaks in horses found no correlation between sequences and time or geographic location of outbreaks.

Keywords: Virusesparamyxovirushenipavirusgenome sequencebat virushorsesAustraliazoonosesdispatch

Hendra virus (HeV) (family Paramyxoviridae, subfamily Paramyxovirinae, genus Henipavirus) was first isolated in 1994 during an outbreak of respiratory disease in horses at a stable in Hendra, a suburb of Brisbane, Queensland, Australia. During the outbreak, all horses at the affected property were tested. Thirteen horses died; 7 recovered with seroconversion, some without clinical signs; and 9 remained uninfected. A horse trainer and a stable worker also became infected, and the trainer subsequently died. In October 1995, HeV infection was diagnosed for a third person, who lived in Mackay, ≈1,000 km north of Brisbane. Retrospective analysis demonstrated that an outbreak had occurred in Mackay in August 1994, where the infected person had assisted with necropsies on 2 horses. At the time, he recovered from a mild undiagnosed infection. Fourteen months later, fatal encephalitis developed, suggesting either virus persistence or late onset of disease symptoms (1). Since 1994, a total of 12 outbreaks have occurred (Table 1).

Hendra virus outbreaks affecting horses and humans, Australia, August 1994–May 2010*
DateLocationHorses, no. casesHumans, no. cases/no. deaths
1994 AugMackay, QLD21/1
1994 SepHendra, QLD202/1
1999 JanTrinity Beach, QLD10/0
2004 OctGordonvale, QLD11/0
2004 DecTownsville, QLD10/0
2006 JunPeachester, QLD10/0
2006 OctMurwillumbah, NSW10/0
2007 JunPeachester, QLD10/0
2007 JulClifton Beach, QLD10/0
2008 JulRedlands, QLD52/1
2008 JulProserpine, QLD30/0
2009 JulRockhampton, QLD31/1
2009 SepBowen, QLD30/0
2010 May
Tewantin, QLD
1
0/0
Total447/4

*QLD, Queensland; NSW, New South Wales.

Serologic evidence identified flying foxes (genus Pteropus) as the likely reservoir host, and HeV was subsequently isolated from 2 species of pteropid bats (2). Serologic evidence of HeV in bats has been demonstrated along the east coast of Australia to Melbourne and west across northern Australia to Darwin. Seroprevalence can be as high as 25% (3). In recent years, the regularity of spillover events has increased. Increased monitoring of bat population sizes, virus in bat populations, and virus characterization is necessary to better learn about trigger(s) for spillover events.

Nipah virus (NiV) is the only other known species within the genus Henipavirus. NiV was first identified during a major outbreak of diseases in pigs and humans in peninsular Malaysia during 1998–99. NiV reemerged in Bangladesh in 2001, with recurrence resulting in human infection almost annually in Bangladesh and India (4). Serologic evidence of NiV or NiV-related viruses has been demonstrated in bats in Thailand (5), Indonesia (6), People’s Republic of China (7), Madagascar (8), and west Africa (9); virus has been isolated from flying foxes in Malaysia (10) and Cambodia (11).

A major characteristic of henipavirus infections is their systemic spread, with evidence of infection in multiple organ systems. HeV infection in horses typically produces an acute, febrile respiratory disease (12) with a high case-fatality rate. The 2008 Redlands outbreak was the largest in horses since the first identified outbreak in 1994. During this outbreak, infected horses showed atypical signs of HeV infections, with clinical features of a more neurologic nature (13). Before the outbreak was attributed to HeV, 2 persons became infected, resulting in 1 death and the potential exposure of >50 persons. The reason for the altered clinical picture during this spillover event is unknown.

The Study

Little is known about the genetic variation of henipaviruses because few sequences are available; most sequences came from the NiV outbreak in Malaysia and Singapore. From the NiV outbreak in Malaysia, sequences were obtained for isolates from 4 humans, 4 pigs, and 1 bat. One sequence is available for the original HeV isolate and 1 isolate each of NiV from Bangladesh and India. A limited sequence for the N and G genes for 2 isolates from Cambodia has been deposited in GenBank.

Five additional isolates from horses were obtained from outbreaks in Murwillumbah (2006), Peachester (2007), Clifton Beach (2007), Redlands (2008), and Proserpine (2008). These isolates provided us with a unique opportunity to examine HeV sequence variation over time and geographic separations.

Analysis of the sequences demonstrated the extreme conservation at the genome and protein levels. All isolates had the identical genome length of 18,234 nt, with the sequence variation across the full genome being <1%. All open reading frames (ORFs) were the same length as those of the original HeV isolate (Hendra virus/Australia/horse/1994/Hendra). Minor nucleotide changes were seen in the ORFs and the noncoding regions. The N ORF had the highest percentage of changes in both nucleotide and amino acid variation (Table 2). The number of nucleotide changes was lower than those in other RNA viruses; for example, the N gene of measles virus showed up to 7% variation in nucleotide sequence (14). The predicted size of each of the P gene products (V, W, and C) was conserved in all isolates. The small basic (SB) protein ORF identified in the original HeV isolate of HeV, but not seen in any of the NiV strains, was present in all isolates with an identical length of 65 aa. The variation in this ORF was higher than that for other ORFs, with 0–6% aa variation. Although SB is in an alternate frame within the P gene, the variation was much higher than that of the C ORF, which is also in an alternate ORF.

Nucleotide and amino acid residue changes in recent Hendra virus isolates compared with the original isolate, Australia
IsolateFull genome variation (%)Open reading frame (length in nt)
N (1,599)
P (2,124)
M (1,059)
F (1,641)
G (1,815)
L (6,735)
ntaantaantaantaantaantaa
Hendra virus/Australia/horse/2006/Murwillumbah (GenBank accession no. HM044318)171 (0.94)166546221423910
Hendra virus/Australia/horse/2007/Peachester (GenBank accession no. HM044319)87 (0.47)21134511011225711
Hendra virus/Australia/horse/2007/Clifton Beach (GenBank accession no. HM044321)139 (0.76)1246351111504811
Hendra virus/Australia/horse/2008/Redlands (GenBank accession no. HM044317)186 (1.02)164142411311105912
Hendra virus/Australia/horse/2008/Proserpine (GenBank accession no. HM044320)183 (1.00)17415472911116411

Phylogenetic analysis was performed by using both DNA and amino acid sequences for individual genes and for the complete genome. The multiple branches of the phylogenetic tree, particularly for the N gene (Figure), suggest that all these isolates branched from an ancestor earlier than 1994 when the first identified outbreak occurred. For example, the 2006 isolate from Murwillumbah shows greatest similarity to the 1994 isolate, whereas the other isolates are in a separate branch on the tree.

Phylogenetic trees based on the N open reading frame (ORF) (A, B) and the G ORF (C, D), with DNA sequences used for A and C and amino acid sequences for B and D. All sequences were compared with the reference sequences for each of the known henipavirus strains; Hendra virus/Australia/horse/1994/Hendra (GenBank accession no. AF017149), Nipah virus/Malaysia/human/1999 (GenBank accession no. AF212302), Nipah virus/Bangladesh/human/2004/Rajbari, R1 (GenBank accession no. AY988601), Nipah virus/Cambodia/bat/2004/KHM/CSUR381 (GenBank accession no. AY858110 [N ORF] and AY858111 [G ORF]) and Nipah virus/India/human/2007/GF (GenBank accession no. FJ513078). Phylogenetic trees were constructed by using the neighbor-joining algorithm in the MEGA4 software package (15). Scale bars represent substitutions per site. HeV, Hendra virus; NiV, Nipah virus.

The data reported here are consistent with each of these individual spillover events that occurred after exposure to viral variants coming from a large pool of quasispecies in the bat population in Australia. This genetic conservation in HeV isolates may suggest that HeV is genetically stable in the reservoir bat population, although because of the lack of HeV isolates from bats, the possibility cannot be excluded that this genetic similarity resulted from selection both for variants that can infect horses and selection within the infected horse. Further analysis of this sequence data and future sequence information will help with understanding of differences in the clinical picture and may provide evidence to explain the apparent increase in regularity of recent spillover events.

Conclusions

Our results demonstrated that HeV isolates from horses are genetically similar, with variation at both the nucleotide and amino acid levels, uniformly small, and <1% across the 18.2-kb genome. Furthermore, data show that different outbreaks resulted from independent spillover events from a pool of HeV quasispecies in the fruit bat populations in Australia. The genetic relatedness of HeV isolates from horses is not correlated with the time the corresponding HeV outbreaks occurred. The presence of SB ORF in all HeV isolates, but not in NiV isolates, warrants further functional analysis of this intriguing putative protein.

Suggested citation for this article: Marsh GA, Todd S, Foord A, Hansson E, Davies E, Wright L, et al. Genome sequence conservation of Hendra virus isolates during spillover to horses, Australia. Emerg Infect Dis [serial on the Internet]. 2010 Nov [date cited]. http://dx.doi.org/10.3201/eid1611.100501

Current affiliation: Life Technologies, Singapore.

Acknowledgments

We thank Biosecurity Queensland field and laboratory staff for providing samples from Hendra virus–infected horses, Tony Pye for the DNA sequencing service, and Kaylene Selleck for her technical assistance. We also thank Jackie Pallister and Mary Tachedjian for critical review of this manuscript.

This work was supported in part by a Commonwealth Scientific and Industrial Research Organisation (CSIRO) Office of the Chief Executive (OCE) postdoctoral award (to G.M) and an CSIRO OCE Science Leader Award (to L.-F.W).

Dr Marsh is a CSIRO OCE postdoctoral fellow at the Australian Animal Health Laboratory, CSIRO Livestock Industries. His research interests include viral pathogenesis and emerging viral diseases.

ReferencesHooper PT, Gould AR, Russell GM, Kattenbelt JA, Mitchell G The retrospective diagnosis of a second outbreak of equine morbillivirus infection. Aust Vet J. 1996;74:2445 10.1111/j.1751-0813.1996.tb15414.x8894044Halpin K, Young PL, Field HE, Mackenzie JS Isolation of Hendra virus from pteropid bats: a natural reservoir of Hendra virus. J Gen Virol. 2000;81:19273210900029Plowright RK, Field HE, Smith C, Divljan A, Palmer C, Tabor G, Reproduction and nutritional stress are risk factors for Hendra virus infection in little red flying foxes (Pteropus scapulatus). Proc Biol Sci. 2008;275:8619 10.1098/rspb.2007.126018198149Hsu VP, Hossain MJ, Parashar UD, Ali MM, Ksiazek TG, Kuzmin I, Nipah virus encephalitis reemergence, Bangladesh. Emerg Infect Dis. 2004;10:2082715663842Wacharapluesadee S, Lumlertdacha B, Boongird K, Wanghongasa S, Chanhome L, Rollin P, Bat Nipah virus, Thailand. Emerg Infect Dis. 2005;11:194951 10.3201/eid1112.05061316485487Sendow I, Field HE, Curran J, Darminto, Morrissy C, Meehan G, Henipavirus in Pteropus vampyrus bats, Indonesia. Emerg Infect Dis. 2006;12:7112 10.3201/eid1204.05118116715584Li Y, Wang J, Hickey AC, Zhang Y, Wu Y, Zhang H, Antibodies to Nipah or Nipah-like viruses in bats, China. Emerg Infect Dis. 2008;14:19746 10.3201/eid1412.08035919046545Iehlé C, Razafitrimo G, Razainirina J, Andriaholinirina N, Goodman SM, Faure C, Henipavirus and Tioman virus antibodies in pteropodid bats, Madagascar. Emerg Infect Dis. 2007;13:15961 10.3201/eid1301.06079117370536Hayman DT, Suu-Ire R, Breed AC, McEachern JA, Wang L, Wood JL, Evidence of henipavirus infection in West African fruit bats. PLoS ONE. 2008;3:e2739 10.1371/journal.pone.000273918648649Chua KB, Koh CL, Hooi PS, Wee KF, Khong JH, Chua BH, Isolation of Nipah virus from Malaysian Island flying foxes. Microbes Infect. 2002;4:14551 10.1016/S1286-4579(01)01522-211880045Reynes JM, Counor D, Ong S, Faure C, Seng V, Molia S, Nipah virus in Lyle's flying foxes, Cambodia. Emerg Infect Dis. 2005;11:1042716022778Hooper PT, Ketterer PJ, Hyatt AD, Russell GM Lesions of experimental equine morbillivirus pneumonia in horses. Vet Pathol. 1997;34:31222 10.1177/0300985897034004079240840Field H, Schaaf K, Kung N, Simon C, Waltisbuhl D, Middleton D, Hendra virus outbreak with novel clinical features, Australia. Emerg Infect Dis. 2010;16:3384020113576Bellini WJ, Rota PA Genetic diversity of wild-type measles viruses: implications for global measles elimination programs. Emerg Infect Dis. 1998;4:2935 10.3201/eid0401.9801059452396Tamura K, Dudley J, Nei M, Kumar S MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol. 2007;24:15969 10.1093/molbev/msm09217488738