Lorna Melville

A new insect - specific flavivirus from northern Australia suppresses replication of West Nile virus and Murray Valley encephalitis virus in co-infected mosquito cells

Recent reports of a novel group of flaviviruses that replicate only in mosquitoes and appear to spread through insect populations via vertical transmission have emerged from around the globe. To date, there is no information on the presence or prevalence of these insect-specific flaviviruses (ISFs) in Australian mosquito species. To assess whether such viruses occur locally, we used reverse transcription-polymerase chain reaction (RT-PCR) and flavivirus universal primers that are specific to the NS5 gene to detect these viruses in mosquito pools collected from the Northern Territory. Of 94 pools of mosquitoes, 13 were RT-PCR positive, and of these, 6 flavivirus isolates were obtained by inoculation of mosquito cell culture. Sequence analysis of the NS5 gene revealed that these isolates are genetically and phylogenetically similar to ISFs reported from other parts of the world. The entire coding region of one isolate (designated 56) was sequenced and shown to have approximately 63.7% nucleotide identity and 66.6% amino acid identity with its closest known relative (Nakiwogo virus) indicating that the prototype Australian ISF represents a new species. All isolates were obtained from Coquillettidia xanthogaster mosquitoes. The new virus is tentatively named Palm Creek virus (PCV) after its place of isolation. We also demonstrated that prior infection of cultured mosquito cells with PCV suppressed subsequent replication of the medically significant West Nile and Murray Valley encephalitis viruses by 10–43 fold (1 to 1.63 log) at 48 hr post-infection, suggesting that superinfection exclusion can occur between ISFs and vertebrate-infecting flaviviruses despite their high level of genetic diversity. We also generated several monoclonal antibodies (mAbs) that are specific to the NS1 protein of PCV, and these represent the first ISF-specific mAbs reported to date.

Arboviruses recovered from sentinel livestock in Northern Australia

Over 700 arboviruses were recovered between 1981 and 1987 from the blood of sentinel livestock near Darwin. Twenty-three isolates were made from sheep, goats, swamp buffalo (Bubalus bubalis) and horses, and the remainder were from cattle. The isolates have been typed as 27 separate viruses belonging to the bluetongue, epizootic haemorrhagic disease, Palyam, Simbu, bovine ephemeral fever, Tibrogargan and alphavirus groups. Ten of these viruses have not been isolated elsewhere in Australia and four have been isolated only in Darwin. Considerable annual variations in virus activity and in the durations of detectable viraemia were observed.

Applications of a sugar-based surveillance system to track arboviruses in wild mosquito populations.

Effective arbovirus surveillance is essential to ensure the implementation of control strategies, such as mosquito suppression, vaccination, or dissemination of public warnings. Traditional strategies employed for arbovirus surveillance, such as detection of virus or virus-specific antibodies in sentinel animals, or detection of virus in hematophagous arthropods, have limitations as an early-warning system. A system was recently developed that involves collecting mosquitoes in CO2-baited traps, where the insects expectorate virus on sugar-baited nucleic acid preservation cards. The cards are then submitted for virus detection using molecular assays. We report the application of this system for detecting flaviviruses and alphaviruses in wild mosquito populations in northern Australia. This study was the first to employ nonpowered passive box traps (PBTs) that were designed to house cards baited with honey as the sugar source. Overall, 20/144 (13.9%) of PBTs from different weeks contained at least one virus-positive card. West Nile virus Kunjin subtype (WNVKUN), Ross River virus (RRV), and Barmah Forest virus (BFV) were detected, being identified in 13/20, 5/20, and 2/20 of positive PBTs, respectively. Importantly, sentinel chickens deployed to detect flavivirus activity did not seroconvert at two Northern Territory sites where four PBTs yielded WNVKUN. Sufficient WNVKUN and RRV RNA was expectorated onto some of the honey-soaked cards to provide a template for gene sequencing, enhancing the utility of the sugar-bait surveillance system for investigating the ecology, emergence, and movement of arboviruses.

Long-distance aerial dispersal modelling of Culicoides biting midges: case studies of incursions into Australia.

BackgroundPrevious studies investigating long-distance, wind-borne dispersal of Culicoides have utilised outbreaks of clinical disease (passive surveillance) to assess the relationship between incursion and dispersal event. In this study, species of exotic Culicoides and isolates of novel bluetongue viruses, collected as part of an active arbovirus surveillance program, were used for the first time to assess dispersal into an endemic region.ResultsA plausible dispersal event was determined for five of the six cases examined. These include exotic Culicoides specimens for which a possible dispersal event was identified within the range of two days – three weeks prior to their collection and novel bluetongue viruses for which a dispersal event was identified between one week and two months prior to their detection in cattle. The source location varied, but ranged from Lombok, in eastern Indonesia, to Timor-Leste and southern Papua New Guinea.ConclusionsWhere bluetongue virus is endemic, the concurrent use of an atmospheric dispersal model alongside existing arbovirus and Culicoides surveillance may help guide the strategic use of limited surveillance resources as well as contribute to continued model validation and refinement. Further, the value of active surveillance systems in evaluating models for long-distance dispersal is highlighted, particularly in endemic regions where knowledge of background virus and vector status is beneficial.

Investigations of bluetongue and other arboviruses in the blood and semen of naturally infected bulls

Small groups of bulls were exposed to natural infection with arboviruses. The bulls were bled and ejaculated regularly and the blood and semen were processed for virus isolation. Over a 5-year observation period, virus isolation and serology indicated that the 29 exposed bulls had experienced 79 viraemic episodes with the viruses of the bluetongue, epizootic haemorrhagic disease, Palyam and Simbu serogroups and an incompletely characterised rhabdovirus. In no instance was there unequivocal evidence of bluetongue virus contamination of semen, despite 18 infections in the study period.

Genomic Characterisation of Three Mapputta Group Viruses, a Serogroup of Australian and Papua New Guinean Bunyaviruses Associated with Human Disease

The Mapputta serogroup tentatively contains the mosquito-associated viruses Mapputta, Maprik, Trubanaman and Gan Gan. Interestingly, this serogroup has previously been associated with an acute epidemic polyarthritis-like illness in humans; however, there has been no ensuing genetic characterisation. Here we report the complete genome sequences of Mapputta and Maprik viruses, and a new Mapputta group candidate, Buffalo Creek virus, previously isolated from mosquitoes and detected by serology in a hospitalised patient. Phylogenetic analyses indicate that the group is one of the earliest diverged groups within the genus Orthobunyavirus of the family Bunyaviridae. Analyses show that these three viruses are related to the recently sequenced Australian bunyaviruses from mosquitoes, Salt Ash and Murrumbidgee. A notable feature of the Mapputta group viruses is the absence of the NSs (non-structural) ORF commonly found on the S segment of other orthobunyaviruses. Viruses of the Mapputta group have been isolated from geographically diverse regions ranging from tropical Papua New Guinea to the semi-arid climate of south-eastern Australia. The relevance of this group to human health in the region merits further investigation.

Distribution of Endogenous Retroviruses in Crocodilians

ABSTRACT Knowledge of endogenous retroviruses (ERVs) in crocodilians (Crocodylia) is limited, and their distribution among extant species is unclear. Here we analyzed the phylogenetic relationships of these retroelements in 20 species of crocodilians by studying the pro-pol gene. The results showed that crocodilian ERVs (CERVs) cluster into two major clades (CERV 1 and CERV 2). CERV 1 clustered as a sister group of the genus Gammaretrovirus, while CERV 2 clustered distantly with respect to all known ERVs. Interestingly, CERV 1 was found only in crocodiles (Crocodylidae). The data generated here could assist future studies aimed at identifying orthologous and paralogous ERVs among crocodilians.

Evolution of Bluetongue Virus Serotype 1 in Northern Australia over 30 Years

ABSTRACT Bluetongue virus serotype 1 (BTV 1) was first isolated in Australia from cattle blood collected in 1979 at Beatrice Hill Farm (BHF), Northern Territory (NT). From long-term surveillance programs (1977 to 2011), 2,487 isolations of 10 BTV serotypes were made. The most frequently isolated serotype was BTV 1 (41%, 1,019) followed by BTV 16 (17.5%, 436) and BTV 20 (14%, 348). In 3 years, no BTVs were isolated, and in 12 years, no BTV 1 was isolated. Seventeen BTV 1 isolates were sequenced and analyzed in comparison with 10 Australian prototype serotypes. BTV 1 showed an episodic pattern of evolutionary change characterized by four distinct periods. Each period consisted primarily of slow genetic drift which was punctuated from time to time by genetic shifts generated by segment reassortment and the introduction of new genome segments. Evidence was found for coevolution of BTV genome segments. Evolutionary dynamics and selection pressure estimates showed strong temporal and clock-like molecular evolutionary dynamics of six Australian BTV genome segments. Bayesian coalescent estimates of mean substitution rates clustered in the range of 3.5 × 10−4 to 5.3 × 10−4 substitutions per site per year. All BTV genome segments evolved under strong purifying (negative) selection, with only three sites identified as under pervasive diversifying (positive) selection. The obligate replication in alternate hosts (insect vector and vertebrate hosts) imposed strong evolutionary constraints. The dominant mechanism generating genetic diversity of BTV 1 at BHF was through the introduction of new viruses and reassortment of genome segments with existing viruses. IMPORTANCE Bluetongue virus (BTV) is the causative agent of bluetongue disease in ruminants. It is a disease of concern globally and is transmitted by biting midges (Culicoides species). Analysis of the evolutionary and selection pressures on BTV 1 at a single surveillance site in northern Australia showed strong temporal and clock-like dynamics. Obligate replication in alternate hosts of insect and vertebrate imposed strong evolutionary constraints, with all BTV genome segments evolving under strong purifying (negative) selection. Generation of genetic diversity of BTV 1 in northern Australia is through genome segment reassortment and the introduction of new serotypes.

Longitudinal study of the detection of Bluetongue virus in bull semen and comparison of real-time polymerase chain reaction assays:

Infection with Bluetongue virus (BTV) is a significant impediment to the global movement of bovine semen. Repeat testing of blood from donor animals is specified in the World Organization for Animal Health (OIE) Manual for the export of semen from regions where BTV may be present. Screening of blood or semen samples has usually been carried out by virus isolation (VI) either by inoculation of chicken embryos followed by passage onto insect and mammalian cell cultures or in vivo inoculation of sheep followed by serology to detect seroconversion. Direct testing of semen for BTV would enable earlier release of semen samples and avoid repeat testing of the donor, as well as provide an option for releasing batches of semen that were collected without certification of the donor. Quantitative (real-time) reverse transcription polymerase chain reaction (qRT-PCR) assays overcome most of the limitations of other methods and have the potential to provide higher sensitivity. The present study compared 5 qRT-PCR assays, including 2 commercially available kits, for the detection of BTV in semen serially collected from 8 bulls over a period of 90 days after experimental infection. The results of the study show that at least one of the qRT-PCR assays is extremely reproducible and has both very high sensitivity and specificity to reliably detect all available serotypes. The preferred qRT-PCR gave consistently superior results to VI, sheep inoculation, and conventional RT-PCR. Therefore, the assay can be recommended for the screening of bovine semen for freedom from BTV.

Persistent and recrudescent infection in cattle following natural infection with Middle Point orbivirus

Middle Point orbivirus (MPOV) is a recently described Australian arbovirus, related to Yunnan orbivirus from China. Analysis of genetic variation within the major serotype gene of MPOV isolates collected from sentinel cattle has identified eight co-circulating strains. The pattern of strain isolation from individual animals during the study period was consistent with an interpretation of persistent MPOV infection of up to five months, featuring episodes of quiescence (below levels required for virus isolation) followed by viral recrudescence. This is significant with regard to current interpretations of infection, persistence and recrudescence during natural infections of orbiviruses, including bluetongue virus.