Primary oral vaccination followed by a vaginal pull protects mice against genital HSV-2 infection.

PROBLEM: HSV-2 infected more than 491 million people aged 15-49 world-wide in 2016. The morbidity associated with recurrent infections and the increased risk of HIV infection make this a major health problem. To date there is no effective vaccine. Because HSV-2 ascends to the dorsal route ganglion within 12-18 h of infection, an effective vaccine will need to elicit a strong local resident CD8+ T cell response to prevent the infection from becoming life-long. METHOD OF STUDY: Using a mouse model we investigated the potential of oral immunization with a novel lipid adjuvant (LiporaleTM ) followed by local vaginal application of an inflammatory agents to protect against primary HSV-2 infections. RESULTS: Oral vaccination of mice with live-attenuated HSV-2 in Liporale followed by vaginal application of DNFB or CXCL9/10 led to recruitment of tissue-resident CD8+ memory cells into the genital epithelia. This prime and pull vaccination strategy provided complete protection against wild-type HSV-2 challenge and prevented viral dissemination to the spinal cords. CONCLUSIONS: Activation of mucosal immunity by oral immunization, combined with induction of transient local genital inflammation can recruit long-lived tissue resident CD8+ T cells into the genital epithelium, providing significant protection against primary HSV-2 infection.

An "Uncharacterized" Australian Virus Is the Earliest Known Example of Ross River Virus with Changes in the nsP3 Protein Associated with the Explosive Outbreak of Ross River Virus Infection in the Pacific Region from 1979 to 1980.

Ross River virus recovered from a South Australian patient during an outbreak of epidemic polyarthritis in 1971 is the earliest known genome sequence with the duplicated 12-amino-acid motif in the nsP3 protein that was found in strains responsible for the outbreak of epidemic polyarthritis in the Pacific region from 1979 to 1980.

Circulation of 2 Barmah Forest Virus Lineages in Military Training Areas, Australia.

During 2017-2018, Barmah Forest virus was recovered from mosquitoes trapped in military training areas in Australia and from a soldier infected at 1 of these areas. Phylogenies of the nucleotide sequences of the envelope glycoprotein gene E2 and the 3' untranslated region suggest that 2 lineages are circulating in eastern Australia.

Australian Aedes aegypti mosquitoes are susceptible to infection with a highly divergent and sylvatic strain of dengue virus type 2 but are unlikely to transmit it.

BACKGROUND: Humans are the primary hosts of dengue viruses (DENV). However, sylvatic cycles of transmission can occur among non-human primates and human encroachment into forested regions can be a source of emergence of new strains such as the highly divergent and sylvatic strain of DENV2, QML22, recovered from a dengue fever patient returning to Australia from Borneo. The objective of the present study was to evaluate the vector competence of Australian Aedes aegypti mosquitoes for this virus. METHODS: Four- to five-day-old mosquitoes from two strains of Ae. aegypti from Queensland, Australia, were fed a meal of sheep blood containing 108 50% cell culture infectious dose per ml (CCID50/ml) of either QML22 or an epidemic strain of DENV serotype 2 (QML16) isolated from a dengue fever patient in Australia in 2015. Mosquitoes were maintained at 28 °C, 75% relative humidity and sampled 7, 10 and 14 days post-infection (dpi). Live virions in mosquito bodies (abdomen/thorax), legs and wings and saliva expectorates from individual mosquitoes were quantified using a cell culture enzyme-linked immunosorbent assay (CCELISA) to determine infection, dissemination and transmission rates. RESULTS: The infection and dissemination rates of the sylvatic DENV2 strain, QML22, were significantly lower than that for QML16. While the titres of virus in the bodies of mosquitoes infected with either of these viruses were similar, titres in legs and wings were significantly lower in mosquitoes infected with QML22 at most time points although they reached similar levels by 14 dpi. QML16 was detected in 16% (n = 25) and 28% (n = 25) of saliva expectorates at 10 and 14 dpi, respectively. In contrast, no virus was detected in the saliva expectorates of QML22 infected mosquitoes. CONCLUSIONS: Australia urban/peri-urban Ae. aegypti species are susceptible to infection by the sylvatic and highly divergent DENV 2 QML22 but replication of QML22 is attenuated relative to the contemporary strain, QML16. A salivary gland infection or escape barrier may be acting to prevent infection of saliva and would prevent onward transmission of this highly divergent virus in Australia.

Wolbachia strain wAlbB blocks replication of flaviviruses and alphaviruses in mosquito cell culture.

BACKGROUND: Wolbachia pipientis are bacterial endosymbionts of arthropods currently being implemented as biocontrol agents to reduce the global burden of arboviral diseases. Some strains of Wolbachia, when introduced into Aedes aegypti mosquitoes, reduce or block the replication of RNA viruses pathogenic to humans. The wAlbB strain of Wolbachia was originally isolated from Aedes albopictus, and when transinfected into Ae. aegypti, persists in mosquitoes under high temperature conditions longer than other strains. The utility of wAlbB to block a broad spectrum of RNA viruses has received limited attention. Here we test the ability of wAlbB to reduce or block the replication of a range of Flavivirus and Alphavirus species in cell culture. METHODS: The C6/36 mosquito cell line was stably infected with the wAlbB strain using the shell-vial technique. The replication of dengue, West Nile and three strains of Zika (genus Flavivirus), and Ross River, Barmah Forest and Sindbis (genus Alphavirus) viruses was compared in wAlbB-infected cells with Wolbachia-free controls. Infectious virus titres were determined using either immunofocus or plaque assays. A general linear model was used to test for significant differences in replication between flaviviruses and alphaviruses. RESULTS: Titres of all viruses were significantly reduced in cell cultures infected with wAlbB versus Wolbachia-free controls. The magnitude of reduction in virus yields varied among virus species and, within species, also among the strains utilized. CONCLUSION: Our results suggest that wAlbB infection of arthropods could be used to reduce transmission of a wide range of pathogenic RNA viruses.

Infection of Western Gray Kangaroos (Macropus fuliginosus) with Australian Arboviruses Associated with Human Infection.

More than 75 arboviruses (arthropod-borne viruses) have been identified in Australia. While Alfuy virus (ALFV), Barmah Forest virus (BFV), Edge Hill virus (EHV), Kokobera virus (KOKV), Murray Valley encephalitis virus (MVEV), Sindbis virus (SINV), Ross River virus (RRV), Stratford virus (STRV), and West Nile virus strain Kunjin (KUNV) have been associated with human infection, there remains a paucity of data regarding their respective transmission cycles and any potential nonhuman vertebrate hosts. It is likely that these viruses are maintained in zoonotic cycles involving native animals rather than solely by human-to-human transmission. A serosurvey (n = 100) was undertaken to determine the prevalence of neutralizing antibodies against a panel of Australian arboviruses in western gray kangaroos (Macropus fuliginosus) obtained from 11 locations in the midwest to southwest of Western Australia. Neutralizing antibodies against RRV were detected in 25%, against BFV in 14%, and antibodies to both viruses in 34% of serum samples. The prevalence of antibodies against these two viruses was the same in males and females, but higher in adult than in subadult kangaroos (p < 0.05). Twenty-one percent of samples had neutralizing antibodies against any one or more of the flaviviruses ALFV, EHV, KOKV, MVEV, and STRV. No neutralizing antibodies against SINV and KUNV were detected. If this sample of kangaroo sera was representative of the broader Australian population of macropods, it suggests that they are common hosts for RRV and BFV. The absence or low seroprevalence of antibodies against the remaining arboviruses suggests that they are not prevalent in the region or that kangaroos are not commonly infected with them. The detection of neutralizing antibodies to MVEV requires further investigation as this virus has not been identified previously so far south in Western Australia.

Neglected Australian Arboviruses Associated With Undifferentiated Febrile Illnesses.

Infections with commonly occurring Australian arthropod-borne arboviruses such as Ross River virus (RRV) and Barmah Forest virus (BFV) are diagnosed routinely by pathology laboratories in Australia. Others, such as Murray Valley encephalitis (MVEV) and Kunjin (KUNV) virus infections may be diagnosed by specialist reference laboratories. Although Alfuy (ALFV), Edge Hill (EHV), Kokobera (KOKV), Sindbis (SINV), and Stratford (STRV) viruses are known to infect humans in Australia, all are considered 'neglected.' The aetiologies of approximately half of all cases of undifferentiated febrile illnesses (UFI) in Australia are unknown and it is possible that some of these are caused by the neglected arboviruses. The aims of this study were to determine the seroprevalence of antibodies against several neglected Australian arboviruses among residents of Queensland, north-east Australia, and to ascertain whether any are associated with UFI. One hundred age- and sex-stratified human plasma samples from blood donors in Queensland were tested to determine the prevalence of neutralising antibodies against ALFV, BFV, EHV, KOKV, KUNV, MVEV, RRV, SINV, and STRV. The seroconversion rates for RRV and BFV infections were 1.3 and 0.3% per annum, respectively. The prevalence of antibodies against ALFV was too low to enable estimates of annual infection rates to be determined, but the values obtained for other neglected viruses, EHV (0.1%), KOKV (0.05%), and STRV (0.05%), indicated that the numbers of clinical infections occurring with these agents are likely to be extremely small. This was borne out by the observation that only 5.7% of a panel of 492 acute phase sera from UFI patients contained IgM against any of these arboviruses, as detected by an indirect immunofluorescence assay. While none of these neglected arboviruses appear to be a cause of a significant number of UFIs in Australia at this time, each has the potential to emerge as a significant human pathogen if there are changes to their ecological niches.

Localized Outbreaks of Epidemic Polyarthritis among Military Personnel Caused by Different Sublineages of Ross River Virus, Northeastern Australia, 2016-2017.

Two outbreaks of epidemic polyarthritis occurred among Australian Defence Force personnel during and following short military exercises in the Shoalwater Bay Training Area, northeastern Australia, in 2016 and 2017. Ross River virus (RRV) IgM was detected in acute-phase serum samples from most patients (28/28 in 2016 and 25/31 in 2017), and RRV was recovered from 4/38 serum samples assayed (1/21 in 2016 and 3/17 in 2017). Phylogenetic analyses of RRV envelope glycoprotein E2 and nonstructural protein nsP3 nucleotide sequences segregated the RRV isolates obtained in 2016 and 2017 outbreaks into 2 distinct sublineages, suggesting that each outbreak was caused by a different strain of RRV. The spatiotemporal characteristics of the 2016 outbreak suggested that some of the infections involved human-mosquito-human transmission without any intermediate host. These outbreaks highlight the importance of personal protective measures in preventing vectorborne diseases for which no vaccine or specific prophylaxis exists.

Identification of the source of blood meals in mosquitoes collected from north-eastern Australia.

BACKGROUND: More than 70 arboviruses have been identified in Australia and the transmission cycles of most are poorly understood. While there is an extensive list of arthropods from which these viruses have been recovered, far less is known about the non-human hosts that may be involved in the transmission cycles of these viruses and the relative roles of different mosquito species in cycles of transmission involving different hosts. Some of the highest rates of human infection with zoonotic arboviruses, such as Ross River (RRV) and Barmah Forest (BFV) viruses, occur in coastal regions of north-eastern Australia. METHODS: Engorged mosquitoes collected as a part of routine surveillance using CO2-baited light traps in the Rockhampton Region and the adjoining Shire of Livingstone in central Queensland, north-eastern Australia, were analysed for the source of their blood meal. A 457 or 623 nucleotide region of the cytochrome b gene in the blood was amplified by PCR and the amplicons sequenced. The origin of the blood was identified by comparing the sequences obtained with those in GenBank®. RESULTS: The most common hosts for the mosquitoes sampled were domestic cattle (26/54) and wild birds (14/54). Humans (2/54) were an infrequent host for this range of mosquitoes that are known to transmit arboviruses causing human disease, and in an area where infections with human pathogens like RRV and BFV are commonly recorded. The blood meals identified in the most abundant vector analysed, Culex annulirostris, were from 10 different vertebrate hosts. The notable detection of chimpanzee blood in two mosquitoes, presumably obtained from a nearby zoo, extends the known range of hosts for this species. Culex quinquefasciatus and Cx. sitiens fed almost exclusively on a variety of bird species. CONCLUSIONS: While human-mosquito-human transmission of arboviruses like RRV can occur, this study highlights the potential importance of zoonotic cycles of transmission, including avian species, of arboviruses that are indigenous to Australia. Further studies on larger samples of blood-engorged mosquitoes are required to validate the trends observed herein. Moreover, serological and virological evidence that the hosts on which the mosquitoes are feeding are being infected with arboviruses of interest are required.

Complete Genomic Sequence of an Australian Sindbis Virus Isolated 44 Years Ago Reveals Unique Indels in the E2 and nsP3 Proteins.

The complete genome sequence of a Sindbis virus (SINV) strain (SINV_AUS_1975_18953) isolated in Australia in 1975 from Culex annulirostris mosquitoes revealed unique deletions in amino acid positions 182 to 184 and 201 to 228 of the E2 envelope protein and multiple indels in the nonstructural protein 3 (nsP3).

The new European invader Aedes (Finlaya) koreicus: a potential vector of chikungunya virus.

Arthropod-borne disease outbreaks, facilitated by the introduction of exotic mosquitoes, pose a significant public health threat. Recent chikungunya virus (CHIKV) epidemics in Europe highlight the importance of understanding the vector potential of invading mosquitoes. In this paper we explore the potential of Aedes koreicus, a mosquito new to Europe, to transmit CHIKV. Mosquitoes were challenged with CHIKV and maintained at two temperatures: 23 °C and a fluctuating temperature. Total CHIKV infection rates at 3, 10 and 14 days post-feeding were low for both temperature treatments (13.8% at 23 °C; 6.2% at fluctuating T). A low percentage (6.1%, n = 65) of mosquitoes maintained at a constant 23 °C showed dissemination of the virus to the wings and legs. Infection of mosquito saliva, with live virus, occurred in 2 mosquitoes. No dissemination was noted under the fluctuating temperature regime. Based on these results we conclude that CHIKV transmission by this species is possible.

Fitness peaks of dengue virus populations.

The role of intra-host genetic diversity in dengue viral populations remains a topic of debate, particularly the impact on transmission of changes in this diversity. Several approaches have been taken to increasing and decreasing the genetic diversity of populations of RNA viruses and have drawn what appear to be contradictory conclusions. A 2-6 fold increase in genetic diversity of a wild type population of dengue virus serotype 1(DENV1) and of an infectious clone population derived from the wild type population, produced by treatment with nucleotide analogue 5 fluorouracil (5FU), drove the populations to extinction. Removal of 5FU immediately prior to extinction, resulted in a return to pre-treatment levels of fitness and genetic diversity, albeit with novel single nucleotide polymorphisms. These observations support the concept that DENV populations exist on fitness peaks determined by their transmission requirements and either an increase or a decrease in genetic diversity may result in a loss of fitness.

Neglected Australian Arboviruses and Undifferentiated Febrile Illness: Addressing Public Health Challenges Arising From the 'Developing Northern Australia' Government Policy.

The Australian Government is currently promoting the development of Northern Australia, with an associated increase in the local population. Consequent to this is the public health threat posed by heightened human exposure to many previously neglected arboviruses that are indigenous to the region. This initiative to support economic activity in the tropical north of the continent is leading to the accelerated expansion of an infection-naïve human population into hitherto un-encountered ecosystems inhabited by reservoir animals and vectors for these arboviruses. Combined with an apparent rise in the number and impact of dramatic climate events, such as tropical cyclones and floods caused by torrential monsoonal rainfall, this heightens the potential for viral transmission to humans. More than 75 arboviruses have been identified in Australia, some of which are associated with human disease but for which routine tests are not available to diagnose infection. Here, we describe briefly the neglected Australian arboviruses that are most likely to emerge as significant agents of human disease in the coming decades. We also advocate the establishment of a thorough surveillance and diagnostic protocol, including developing new pan-viral rapid tests for primary care use to assist in the early diagnosis and correct treatment of affected patients. We propose that the implementation of these activities will enhance our understanding of the geographical range, prevalence, identification and control of neglected Australian arboviruses. This would minimise and limit the possibility of large-scale outbreaks with these agents as population and economic growth expands further into Australia's tropical north.

Neglected Australian arboviruses: quam gravis?

At least 75 arboviruses have been identified from Australia. Most have a zoonotic transmission cycle, maintained in the environment by cycling between arthropod vectors and susceptible mammalian or avian hosts. The primary arboviruses that cause human disease in Australia are Ross River, Barmah Forest, Murray Valley encephalitis, Kunjin and dengue. Several other arboviruses are associated with human disease but little is known about their clinical course and diagnostic testing is not routinely available. Given the significant prevalence of undifferentiated febrile illness in Australia, investigation of the potential threat to public health presented by these viruses is required.

Laboratory colonization of the European invasive mosquito Aedes (Finlaya) koreicus.

BACKGROUND: Aedes (Finlaya) koreicus (Edwards) is a mosquito that has recently entered Europe from Asia. This species is considered a potential threat to newly colonized territories, but little is known about its capacity to transmit pathogens or ability to compete with native mosquito species. The establishment of a laboratory colony is a necessary first step for further laboratory studies on the biology, ecology and vector competence of Ae. koreicus. RESULTS: A self-mating colony was established at QIMR Berghofer Medical Research Institute (Brisbane, Australia) from eggs of the F1 progeny of individuals collected as free-living larvae in northeastern Italy (Belluno province). Mosquitoes are currently maintained on both defibrinated sheep blood provided via an artificial membrane system and human blood from volunteers. Larvae are maintained in rain water and fed with Tetramin® fish food (©2015 Spectrum Brands - Pet, Home and Garden Division, Tetra-Fish). Morphometric measurements related to body size were taken and a fecundity index, based on wing length, was calculated. An in vivo technique for differentiating male and female pupae has been optimized. Our findings provide the basis for further studies on the ecology and physiology of Ae. koreicus. CONCLUSION: We describe the establishment of an Ae. koreicus colony in the laboratory and identify critical requirements for the maintenance of this mosquito species under artificial conditions. The laboratory colony will facilitate studies investigating the vector potential of this species for human pathogens.

Highly Divergent Dengue Virus Type 2 in Traveler Returning from Borneo to Australia.

Dengue virus type 2 was isolated from a tourist who returned from Borneo to Australia. Phylogenetic analysis identified this virus as highly divergent and occupying a basal phylogenetic position relative to all known human and sylvatic dengue virus type 2 strains and the most divergent lineage not assigned to a new serotype.

Dengue viruses cluster antigenically but not as discrete serotypes.

The four genetically divergent dengue virus (DENV) types are traditionally classified as serotypes. Antigenic and genetic differences among the DENV types influence disease outcome, vaccine-induced protection, epidemic magnitude, and viral evolution. We characterized antigenic diversity in the DENV types by antigenic maps constructed from neutralizing antibody titers obtained from African green monkeys and after human vaccination and natural infections. Genetically, geographically, and temporally, diverse DENV isolates clustered loosely by type, but we found that many are as similar antigenically to a virus of a different type as to some viruses of the same type. Primary infection antisera did not neutralize all viruses of the same DENV type any better than other types did up to 2 years after infection and did not show improved neutralization to homologous type isolates. That the canonical DENV types are not antigenically homogeneous has implications for vaccination and research on the dynamics of immunity, disease, and the evolution of DENV.

Dengue virus in sub-tropical northern and central Viet Nam: population immunity and climate shape patterns of viral invasion and maintenance.

Dengue virus transmission occurs in both epidemic and endemic cycles across tropical and sub-tropical regions of the world. Incidence is particularly high in much of Southeast Asia, where hyperendemic transmission plagues both urban and rural populations. However, endemicity has not been established in some areas with climates that may not support year-round viral transmission. An understanding of how dengue viruses (DENV) enter these environments and whether the viruses persist in inapparent local transmission cycles is central to understanding how dengue emerges in areas at the margins of endemic transmission. Dengue is highly endemic in tropical southern Vietnam, while increasingly large seasonal epidemics have occurred in northern Viet Nam over the last decade. We have investigated the spread of DENV-1 throughout Vietnam to determine the routes by which the virus enters northern and central regions of the country. Phylogeographic analysis of 1,765 envelope (E) gene sequences from Southeast Asia revealed frequent movement of DENV between neighboring human populations and strong local clustering of viral lineages. Long-distance migration of DENV between human population centers also occurred regularly and on short time-scales, indicating human-mediated viral invasion into northern Vietnam. Human populations in southern Vietnam were found to be the primary source of DENV circulating throughout the country, while central and northern Vietnam acted as sink populations, likely due to reduced connectedness to other populations in the case of the central regions and to the influence of temperature variability on DENV replication and vector survival and competence in the north. Finally, phylogeographic analyses suggested that viral movement follows a gravity model and indicates that population immunity and physical and economic connections between populations may play important roles in shaping patterns of DENV transmission.

Community-based control of Aedes aegypti by using Mesocyclops in southern Vietnam.

We previously reported a new community-based mosquito control strategy that resulted in elimination of Aedes aegypti (Linn.) in 40 of 46 communes in northern and central Vietnam, and with annual recurrent total costs (direct and indirect) of only $0.28-$0.89 international dollars per person. This control strategy was extended to four provinces in southern Vietnam in Long An and Hau Giang (2004-2007) and to Long An, Ben Tre, and Vinh Long (2005-2010). In a total of 14 communes with 124,743 residents, the mean ± SD of adult female Ae. aegypti was reduced from 0.93 ± 0.62 to 0.06 ± 0.09, and the reduction of immature Ae. aegypti averaged 98.8%. By the final survey, no adults could be collected in 6 of 14 communes, and one commune, Binh Thanh, also had no immature forms. Although the community-based programs also involved community education and clean-up campaigns, the prevalence of Mesocyclops in large water storage containers > 50 liters increased from 12.77 ± 8.39 to 75.69 ± 9.17% over periods of 15-45 months. At the conclusion of the study, no confirmed dengue cases were detected in four of the five communes for which diagnostic serologic analysis was performed. The rate of progress was faster in communes that were added in stages to the program but the reason for this finding was unclear. At the completion of the formal project, sustainability funds were set up to provide each commune with the financial means to ensure that community-based dengue control activities continued.

Safety and immunogenicity of an inactivated whole virus Vero cell-derived Ross River virus vaccine: a randomized trial.

BACKGROUND: Ross River virus (RRV) is endemic in Australia and several South Pacific Islands. Approximately 5000 cases of RRV disease, which is characterized by debilitating polyarthritis, are recorded each year in Australia. This study describes the first clinical trial of a candidate RRV vaccine. METHODS: An inactivated whole-virus Vero cell-derived RRV vaccine was tested in 382 healthy, RRV-naïve adults in a phase 1/2 dose-escalation study at ten sites in Austria, Belgium and The Netherlands. Subjects were equally randomized to receive 1.25 µg, 2.5 µg, 5 µg, or 10 µg aluminum hydroxide-adjuvanted or non-adjuvanted RRV vaccine, with a second dose after three weeks and a booster at six months. Vaccine immunogenicity was determined by measurements of serum IgG and neutralizing antibody titers. Vaccine tolerability and safety were monitored over the entire study period. RESULTS: The optimal vaccine formulation was the adjuvanted 2.5 µg dose, as calculated using a repeated mixed model analysis of covariance comparing log-transformed RRV-specific IgG titers between different dose groups. Geometric means of RRV-specific serum antibodies measured 21 days after the third vaccination with the 2.5 µg adjuvanted formulation were 520.9 (90% CI 377.2-719.4) as determined by IgG ELISA and 119.9 (82.6-173.9) as determined by virus neutralization assay, resulting in seropositivity rates of 92.9% (82.6-98.0) and 92.7% (82.2-98.0), respectively. All vaccine formulations and doses were well tolerated after the first, second and third vaccination. CONCLUSIONS: The adjuvanted, inactivated whole-virus Vero cell-derived Ross River virus vaccine is highly immunogenic in RRV-naïve adults and well tolerated at all dose levels.