Mass spectrometry-based identification and whole-genome characterisation of the first pteropine orthoreovirus isolated from monkey faeces in Thailand

Kosoltanapiwat et al. BMC Microbiology (2018) 18:135 https://doi.org/10.1186/s12866-018-1302-9 RESEARCH ARTICLE Open Access Mass spectrometry-based identification and whole-genome characterisation of the first pteropine orthoreovirus isolated from monkey faeces in Thailand Nathamon Kosoltanapiwat1* , Onrapak Reamtong2, Tamaki Okabayashi3,4, Sumate Ampawong5, Amporn Rungruengkitkun1, Tipparat Thiangtrongjit2, Narin Thippornchai1, Pornsawan Leaungwutiwong1, Aongart Mahittikorn6, Hirotake Mori6, Thanada Yoohanngoa1 and Prechaya Yamwong1 Abstract Background: The pteropine orthoreovirus (PRV) was isolated from monkey (Macaca fascicularis) faecal samples collected from human-inhabited areas in Lopburi Province, Thailand. These samples were initially obtained to survey for the presence of hepatitis E virus (HEV). Results: Two virus isolates were retrieved by virus culture of 55 monkey faecal samples. Liquid chromatographytandem mass spectrometry (LC-MS/MS) was successfully used to identify the viruses as the segmented dsRNA orthoreovirus. Phylogenetic analysis of the Lopburi orthoreovirus whole-genomes revealed relationships with the well-characterised PRVs Pulau (segment L1), Cangyuan (segments L2, M3 and S3), Melaka (segments L3 and M2), Kampar (segments M1 and S2) and Sikamat (segments S1 and S4) of Southeast Asia and China with nucleotide sequence identities of 93.5–98.9%. RT-PCR showed that PRV was detected in 10.9% (6/55) and HEV was detected in 25.5% (14/55) of the monkey faecal samples. Conclusions: PRV was isolated from monkey faeces for the first time in Thailand via viral culture and LC-MS/MS. The genetic diversity of the virus genome segments suggested a re-assortment within the PRV species group. The overall findings emphasise that monkey faeces can be sources of zoonotic viruses, including PRV and HEV, and suggest the need for active virus surveillance in areas of human and monkey co-habitation to prevent and control emerging zoonotic diseases in the future. Keywords: Monkey, Macaque, Macaca fascicularis, Orthoreovirus, Hepatitis E virus, Mass spectrometry Background In some areas in Lopburi Province, Thailand, humans and monkeys, mostly macaques, live in close contact. In these areas, faeces excreted by the animals can unavoidably contaminate the human environment. Faeces are sources of pathogens, including bacteria, parasites and viruses. Most enteric viruses typically present in faeces are non-enveloped viruses that can exist outside the host * Correspondence: 1 Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, 420/6 Ratchawithi Road, Ratchathewi, Bangkok 10400, Thailand Full list of author information is available at the end of the article for several days [1, 2]. Some of these are zoonotic viruses that can infect both humans and animals. Hence, in areas in which humans and animals co-inhabit, the potential for zoonotic transmission is elevated, requiring observation and control. Pteropine orthoreovirus (PRV) is a member of the genus Orthoreovirus and family Reoviridae. Reoviridae is a large family of non-enveloped, icosahedral, segmented dsRNA viruses that infect a wide range of hosts, i.e. fungi, plants, insects, molluscs, fish, reptiles, birds and mammals, including humans [3]. Orthoreovirus, which contains 10 genome segments (three large, three medium and four small segments), consists of five © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kosoltanapiwat et al. BMC Microbiology (2018) 18:135 species: mammalian orthoreovirus (MRV), avian orthoreovirus (ARV), PRV [formerly known as Nelson Bay orthoreovirus (NBV)], baboon orthoreovirus and reptilian orthoreovirus [4, 5]. Of these, MRV is the only species that does not exert a syncytial cytopathic effect (CPE) in cell culture; therefore, it is called a non-fusogenic orthoreovirus. MRV is the prototypic orthoreovirus that causes diseases in mammals, including humans and monkeys [3]. The first isolation of PRV occurred in 1970 from a grey-headed flying fox (Pteropus poliocephalus) in Nelson Bay, Australia [6]. Its characteristics are intermediate between MRV and ARV, in which it is a fusogenic orthoreovirus that infects mammals but does not kill chicken embryos [3]. It is therefore called a mammalian fusogenic orthoreovirus. Later, other PRVs were isolated from bats and humans. Pulau virus, the second member of the NBV species group, was isolated in 1999 from pooled urine samples of the fruit bat Pteropus hypomelanus in Tioman Island, Malaysia [7]. Bat-associated Melaka and Kampar orthoreoviruses with genome sequences related to NBV were isolated in 2006 in Malaysia from patients with acute respiratory disease. The reports of these two viruses suggested the ability of PRV to cause disease in humans and evidenced its human-to-human transmission potential [8, 9]. Subsequently, new strains of PRV were consecutively isolated from humans and bats, including patients with respiratory tract infection from Hong Kong [10, 11], Japan [12] and Malaysia [5] and bats from China [13, 14], Italy [15] and, most recently, the Philippines [16]. It must be noted that the infected patients from Hong Kong and Japan had histories of travel to Indonesia, and the PRV-positive bats in Italy were also imported from Indonesia. Thus far, excluding NBV, which originated from Australia, PRV has only been isolated in Southeast Asia (Malaysia, Indonesia and the Philippines) and China. Meanwhile, mass spectrometric techniques have been increasingly utilised in virus studies. Mass spectrometry (MS), peptide mass fingerprinting and protein profiling via time-of-flight mass spectrometry (TOF MS) have been applied in studies of both human and plant viruses such as poliovirus, rhinovirus, tobacco mosaic virus and brome mosaic virus [17–19]. However, the use of peptide mass fingerprinting to identify unknown viruses is restricted by limitations of viral peptide mass fingerprint databases and the capability of TOF MS to identify small amounts of viral peptides when disturbed by mammalian proteins from cell culture. To overcome these limitations, protein separation techniques such as gel electrophoresis and liquid chromatography (LC) in combination with MS are applied. MS, in particular tandem MS (MS/MS), is a sensitive method for analysing protein mixtures [20]. Two-dimensional (2D) electrophoresis in combination Page 2 of 14 with high-perfor (...truncated)