Technical report | Characterisation and Next-generation Sequencing Analysis of Unknown Arboviruses

Abstract

 n the last few years there has been an increased incidence of neurological and encephalitic syndromes in Australia that have not been linked to a known pathogen. Viruses from the families Rhabdoviridae and Bunyaviridae are postulated to play a role. This report describes the characterisation of two unknown bunyaviruses, designated Maprik (MPKV) and Buffalo Creek (BCV), and two unknown rhabdoviruses, designated Harrison Dam (HARDV) and Holmes Jungle (HOJV), using techniques such as PCR-select subtraction and next-generation sequencing. Preliminary analysis of the four sequenced viruses has shown that they are all novel, and phylogenetic studies will now be performed to further classify these organisms. In addition, the report details the success in using a novel method to prepare MPKV and BCV RNA for next-generation sequencing. The use of this method allows for an increased level of efficiency when sequencing unknown viruses and has now been incorporated into our standard viral preparation protocols.

Executive Summary

The term arbovirus is used to describe viruses transmitted via blood-feeding arthropods, such as mosquitoes, biting midges and ticks. Two families of arboviruses, the Rhabdoviridae and Bunyaviridae, are suspected to play some role in the incidence of encephalitis and neurological disease occurring in northern regions of Australia, for which there is no known cause. Significant numbers of Australian Defence Force (ADF) personnel are posted to these areas, and military exercises are frequently conducted. In addition, bunyaviruses such as Rift Valley fever, Crimean Congo haemorrhagic fever and hantavirus, and rhabdoviruses such as rabies virus and Australian bat lyssavirus have been implicated as potential bioterrorism agents. This is due to their infectivity, ability to induce a fatal or seriously incapacitating illness, lack of adequate control measures, and the ease of production of large quantities of virus. Characterisation by sequencing is therefore an important step in identification and detection of these viruses. It also assists in determining their role in disease, which may lead to the implementation of intervention strategies by the ADF.This report focuses on characterisation of two unknown rhabdoviruses, Holmes Jungle virus (HOJV) and Harrison Dam virus (HARDV), and two unknown bunyaviruses, Buffalo Creek Virus (BCV) and Maprik virus (MPKV). It describes the techniques such PCR select subtraction, Rapid Amplification of cDNA Ends (RACE), and next-generation high throughput sequencing, which are used to obtain the full genetic sequence of these viruses. While PCR select subtraction is a gold standard method of obtaining unknown viral sequence, it is a relatively complicated, time consuming process. Next-generation sequencing is a new powerful technology which can be utilised to identify and characterise unknown viruses with greater speed and at lower cost. The rapid advancement of new generation sequencing techniques allows for highly specific acquisition of gigabases of sequence information in just a few days.

This report also describes a new method to prepare the virus samples for next-generation sequencing analysis, which our results show is equally as effective, yet considerably faster, simpler and cheaper than the current standard preparation methods. Preliminary analysis of the four sequenced viruses has also shown that they are all novel viruses, and we are currently in the process of preparing collaborative manuscripts for publication in international scientific journals with the Berrimah Veterinary Laboratory and the CSIRO Australian Animal Health Laboratory describing the novel findings.