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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 (Liporale TM ) 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., (© 2022 The Authors. American Journal of Reproductive Immunology published by John Wiley & Sons Ltd.)

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.

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.

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 10 8 50% cell culture infectious dose per ml (CCID 50 /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.

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.

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.

Infections with commonly occurring Australian ar thropod- bo rne 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., (Copyright © 2019 Gyawali, Taylor-Robinson, Bradbury, Pederick, Faddy and Aaskov.)

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.

The complete genome sequence of a Sindbis virus (SINV) strain (SINV&#95;AUS&#95;1975&#95;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)., (Copyright © 2019 Pickering et al.)

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 CO 2 -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.

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.

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.

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.

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., (Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

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.

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.

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., (Copyright © 2015, American Association for the Advancement of Science.)

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.

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.

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

We examined the structure and extent of genetic diversity in intrahost populations of Ross River virus (RRV) in samples from six human patients, focusing on the nonstructural (nsP3) and structural (E2) protein genes. Strikingly, although the samples were collected from contrasting ecological settings 3,000 kilometers apart in Australia, we observed multiple viral lineages in four of the six individuals, which is indicative of widespread mixed infections. In addition, a comparison with previously published RRV sequences revealed that these distinct lineages have been in circulation for at least 5 years, and we were able to document their long-term persistence over extensive geographical distances.

Ross River Virus has caused reported outbreaks of epidemic polyarthritis, a chronic debilitating disease associated with significant long-term morbidity in Australia and the Pacific region since the 1920s. To address this public health concern, a formalin- and UV-inactivated whole virus vaccine grown in animal protein-free cell culture was developed and tested in preclinical studies to evaluate immunogenicity and efficacy in animal models. After active immunizations, the vaccine dose-dependently induced antibodies and protected adult mice from viremia and interferon α/β receptor knock-out (IFN-α/βR(-/-)) mice from death and disease. In passive transfer studies, administration of human vaccinee sera followed by RRV challenge protected adult mice from viremia and young mice from development of arthritic signs similar to human RRV-induced disease. Based on the good correlation between antibody titers in human sera and protection of animals, a correlate of protection was defined. This is of particular importance for the evaluation of the vaccine because of the comparatively low annual incidence of RRV disease, which renders a classical efficacy trial impractical. Antibody-dependent enhancement of infection, did not occur in mice even at low to undetectable concentrations of vaccine-induced antibodies. Also, RRV vaccine-induced antibodies were partially cross-protective against infection with a related alphavirus, Chikungunya virus, and did not enhance infection. Based on these findings, the inactivated RRV vaccine is expected to be efficacious and protect humans from RRV disease., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Dengue and dengue hemorrhagic fever re-emerged in Bangladesh in 2000 and 2001 and nearly all viruses isolated were dengue type 3. Phylogenetic analyses of the envelope genes of examples of these viruses indicated that they were most closely related to recently emerged dengue type 3 viruses from neighboring Thailand and Myanmar but distinct from those from India and Sri Lanka. Since this strain of dengue virus type 3 had not been associated with unusual patterns of disease in Thailand or Myanmar, it suggested that the outbreak in Bangladesh was due to local factors after the introduction of viruses from countries to the east rather than to the evolution of an unusually virulent strain of virus in Bangladesh.

From September 2000 to June 2003, a community-based program for dengue control using local predacious copepods of the genus Mesocyclops was conducted in three rural communes in the central Vietnam provinces of Quang Nam, Quang Ngai, and Khanh Hoa. Post-project, three subsequent entomologic surveys were conducted until March 2004. The number of households and residents in the communes were 5,913 and 27,167, respectively, and dengue notification rates for these communes from 1996 were as high as 2,418.5 per 100,000 persons. Following knowledge, attitude, and practice evaluations, surveys of water storage containers indicated that Mesocyclops spp. already occurred in 3-17% and that large tanks up to 2,000 liters, 130-300-liter jars, wells, and some 220-liter metal drums were the most productive habitats for Aedes aegypti. With technical support, the programs were driven by communal management committees, health collaborators, schoolteachers, and pupils. From quantitative estimates of the standing crop of third and fourth instars from 100 households, Ae. aegypti were reduced by approximately 90% by year 1, 92.3-98.6% by year 2, and Ae. aegypti immature forms had been eliminated from two of three communes by June 2003. Similarly, from resting adult collections from 100 households, densities were reduced to 0-1 per commune. By March 2004, two communes with no larvae had small numbers but the third was negative; one adult was collected in each of two communes while one became negative. Absolute estimates of third and fourth instars at the three intervention communes and one left untreated had significant correlations (P = 0.009-< 0.001) with numbers of adults aspirated from inside houses on each of 15 survey periods. By year 1, the incidence of dengue disease in the treated communes was reduced by 76.7% compared with non-intervention communes within the same districts, and no dengue was evident in 2002 and 2003, compared with 112.8 and 14.4 cases per 100,000 at district level. Since we had similar success in northern Vietnam from 1998 to 2000, this study demonstrates that this control model is broadly acceptable and achievable at community level but vigilance is required post-project to prevent reinfestation.

Strains of dengue 3 (DEN-3) virus circulating in Thailand prior to 1992 appear to have disappeared from that location and to have been replaced by two new lineages which have evolved locally, rather than being introduced. Similar DEN-3 virus extinctions may have occurred previously in Thailand in 1962 and 1973. Although no causal relationship could be shown, this strain replacement event was accompanied by DEN-3 replacing DEN-2 as the serotype recovered most frequently from patients in Thailand. Although this implies a change in selection pressure, we found no evidence for positive natural selection at the level of either the E protein or the E protein gene. Further, the extinction of the pre-1992 strains and the appearance of the new lineages occurred during an interepidemic period, suggesting that a genetic bottleneck, rather than selection, might have been important in the emergence of these two new strains of virus. The pre-1992 DEN-3 virus lineage could still be found in 1998, to the west, in Myanmar. The ratio of nonsynonymous-to-synonymous nucleotide changes within a DEN-3 virus population from a single patient was less than the ratio among the consensus sequences of DEN-3 viruses from different patients, suggesting that many of the nonsynonymous nucleotide changes which occurred naturally in the E protein were deleterious and removed by purifying selection.

We describe remarkable success in controlling dengue vectors, Aedes aegypti (L.) and Aedes albopictus (Skuse), in 6 communes with 11,675 households and 49,647 people in the northern provinces of Haiphong, Hung Yen, and Nam Dinh in Vietnam. The communes were selected for high-frequency use of large outdoor concrete tanks and wells. These were found to be the source of 49.6-98.4% of Ae. aegypti larvae, which were amenable to treatment with local Mesocyclops, mainly M. woutersi Van der Velde, M. aspericornis (Daday) and M. thermocyclopoides Harada. Knowledge, attitude, and practice surveys were performed to determine whether the communities viewed dengue and dengue hemorrhagic fever as a serious health threat; to determine their knowledge of the etiology, attitudes, and practices regarding control methods including Mesocyclops; and to determine their receptivity to various information methods. On the basis of the knowledge, attitude, and practice data, the community-based dengue control program comprised a system of local leaders, health volunteer teachers, and schoolchildren, supported by health professionals. Recycling of discards for economic gain was enhanced, where appropriate, and this, plus 37 clean-up campaigns, removed small containers unsuitable for Mesocyclops treatment. A previously successful eradication at Phan Boi village (Hung Yen province) was extended to 7 other villages forming Di Su commune (1,750 households) in the current study. Complete control was also achieved in Nghia Hiep (Hung Yen province) and in Xuan Phong (Nam Dinh province); control efficacy was > or = 99.7% in the other 3 communes (Lac Vien in Haiphong, Nghia Dong, and Xuan Kien in Nam Dinh). Although tanks and wells were the key container types of Ae. aegypti productivity, discarded materials were the source of 51% of the standing crop of Ae. albopictus. Aedes albopictus larvae were eliminated from the 3 Nam Dinh communes, and 86-98% control was achieved in the other 3 communes. Variable dengue attack rates made the clinical and serological comparison of control and untreated communes problematic, but these data indicate that clinical surveillance by itself is inadequate to monitor dengue transmission.

Of a panel of forty-six anti-dengue 3 monoclonal antibodies (MAbs) only three neutralised infection of BHK cells by dengue 3 virus. Attempts to select neutralisation escape mutants (n.e.m.) with two of these antibodies failed. The n.e.m. population selected in the presence of the third neutralising antibody, 1H9, had a nucleotide change at position 1157 of the E protein gene resulting in a non-conservative amino acid change at E386 for a Lys to an Asn. A dengue 2 n.e.m. was selected with the flavivirus crossreactive IgG monoclonal antibody 4G2, had deduced amino acid changes at E169 (Ser to Pro) and E275 (Gly to Arg). This dengue 2 n.e.m. population produced smaller plaques in BHK cells than the parental virus, decreased fusion activity (FFWI) and had lost the ability to agglutinate gander erythrocyes at pH 6.0 to 6.7.

Three of 41 IgM monoclonal antibodies derived from dengue 1 virus immunized mice neutralized dengue 1 infection in vitro. All three neutralizing monoclonal antibodies reacted with spatially related epitopes on the E protein of dengue 1 which were also recognized by antibodies in sera from dengue patients. Two neutralization-resistant populations of dengue 1 virus, D1-M10 and D1-M17, were selected by sequential passage of virus in C6/36 cells in the presence of neutralizing IgM monoclonal antibodies M10 and M17, respectively. Single nucleotide changes occurred in the E protein gene of each of these virus populations resulting in single amino acid substitutions at E279 (Phe-Ser) in D1-M10 and at E293 (Thr-Ile) in D1-M17. Both neutralization-resistant populations of virus were more sensitive to elevated temperature than was the wild-type dengue 1 virus and the infectivity and haemagglutinating ability of the neutralization-resistant populations decreased more slowly than that of wild-type virus when exposed to pH in the range 5.8 to 7.0. These are the first epitopes involved in neutralization to have been identified in dengue 1 virus and the first outside domain III of the E protein on any dengue virus.

Proenzyme dipeptidyl peptidase I (DPP I) of Schistosoma japonicum was expressed in a baculovirus expression system utilizing Trichoplusia ni BTI-5B1-4 (High Five) strain host insect cells. The recombinant enzyme was purified from cell culture supernatants by affinity chromatography on nickel-nitriloacetic acid resin, exploiting a polyhistidine tag fused to the COOH-terminus of the recombinant protease. The purified recombinant enzyme resolved in reducing SDS-PAGE gels as three forms, of 55, 39, and 38 kDa, all of which were reactive with antiserum raised against bacterially expressed S. japonicum DPP I. NH(2)-terminal sequence analysis of the 55-kDa polypeptide revealed that it corresponded to residues -180 to -175, NH(2)-SRXKXK, of the proregion peptide of S. japonicum DPP I. The 39- and 38-kDa polypeptides shared the NH(2)-terminal sequence, LDXNQLY, corresponding to residues -73 to -67 of the proregion peptide and thus were generated by removal of 126 residues from the NH(2)-terminus of the proenzyme. Following activation for 24 h at pH 7.0, 37 degrees C under reducing conditions, the recombinant enzyme exhibited exopeptidase activity against synthetic peptidyl substrates diagnostic of DPP I. Specificity constants (k(cat)/K(m)) for the recombinant protease for the substrates H-Gly-Arg-NHMec and H-Gly-Phe-NHMec were found to be 14.4 and 10.7 mM(-)1 s(-1), respectively, at pH 7.0. Approximately 1 mg of affinity-purified schistosome DPP I was obtained per liter of insect cell culture supernatant, representing approximately 2 x 10(9) High Five cells., (Copyright 2000 Academic Press.)

The number of clinical Ross River virus (RRV) infections (epidemic polyarthritis) each year in Australia continues to grow despite extensive vector control programs. There is a need, therefore, for a surveillance program that can give sufficient warning of outbreaks of the disease so that highly focused preventative measures may be undertaken. The ability of a surveillance program, based on voluntary Red Cross blood donations, to predict outbreaks of epidemic polyarthritis was evaluated. Anti-RRV IgM antibody was detected in significant numbers of blood donors from throughout the state of Queensland 6-9 weeks prior to an increase in the number of notified cases of epidemic polyarthritis. At a local level, significant numbers of anti-RRV IgM blood donors were detected in Brisbane in 1996 four weeks prior to an increase in the number of notified cases of epidemic polyarthritis. This system of surveillance is technically simple, rapid (results are obtained in 2-3 days), it samples the human population from throughout the state, and it gives timely warning of outbreaks of epidemic polyarthritis.

In a step towards a tetravalent dengue virus subunit vaccine which is economical to produce, highly immunogenic and stable, a hybrid dengue virus envelope (E) protein molecule has been constructed. It consists of 36 amino acids from the membrane protein, the N-terminal 288 amino acids of the dengue-2 virus E protein plus amino acids 289-424 of the dengue-3 virus E protein. It has been engineered for secretory expression by fusion to a mellitin secretory signal sequence and truncation of the hydrophobic transmembrane segment. Using the baculovirus expression system and serum-free conditions, more than 95% of recombinant dengue-2 virus-dengue-3 virus hybrid E protein (rD2D3E) was secreted into the cell culture supernatant in a stable form with multiple features indicative of preserved conformation. The hybrid molecule reacted with a panel of dengue virus- and flavivirus-specific MAbs which recognize linear or conformational epitopes on dengue virions. Human dengue virus-specific antisera also reacted with the protein. The hybrid rD2D3E protein was able to inhibit the in vitro binding of dengue-2 and dengue-3 viruses to human myelomonocytic cells, suggesting that the receptor-binding epitope(s) was preserved. Adjuvant-free immunization with the hybrid protein induced an antibody response to both dengue-2 and dengue-3 virus in outbred mice, comparable in strength to that of individual rD2E and rD3E proteins. Notably, these antibody responses were primarily of the IgG2a and IgG2b isotype. A strong dengue virus cross-reactive T cell response was also induced in the mice, suggesting that dengue virus hybrid E proteins could form the basis of an efficacious multivalent dengue virus vaccine.

Ross River virus (RRV) is the aetiological agent of epidemic polyarthritis (EPA) a predominantly rheumatic disease afflicting up to 5000 Australians annually. We show here for the first time that macrophages can be productively infected by RRV. Subneutralizing titres of anti-RRV IgG (but not IgM) also showed classical antibody-dependent enhancement (ADE) of RRV infection in macrophage and monocyte cell lines. No correlation between development of EPA and the pre-existence of ADE titres was apparent, nor could sera raised against a related arbovirus, Barmah Forest, enhance RRV infection. Tumour necrosis factor-alpha, implicated in the immunopathogenesis of rheumatoid arthritis, was not secreted by RRV-infected monocytes or macrophages. Macrophage cell lines infected with RRV were, however, capable of producing virus for over 50 days. RRV-induced arthritis may therefore be due to the persistent productive infection of macrophages, perhaps established by a brief period of ADE early in infection.

Forty-two hybridomas secreting IgM antibody against dengue virus were derived from spleen cells of dengue 2 infected mice. Antibody from 27 of these recognised the E protein of this virus. Of the 22 antibodies which neutralised dengue 2, only two cross-reacted with other flaviviruses. These 22 antibodies recognised three discrete domains on dengue virions. Competitive binding studies with IgG monoclonal antibodies suggested that two of the three domains were recognised by both IgG and IgM antibodies and that there were two additional domains which may be recognised exclusively by either IgG or IgM antibodies. IgM antibodies reacting with domains recognised by IgG antibodies that enhanced infection of susceptible cells by dengue 2, had no enhancing properties. None of the IgM antibodies activated the serum complement system after reacting with dengue 2 virions.

Ross River virus is a mosquito-borne alphavirus which causes several thousand cases of arthritis (epidemic polyarthritis) each year. In this study, binary ethylenimine (BEI) was used to destroy the infectivity of this virus without abolishing the antigenicity or immunogenicity of the virion. Mice immunized intramuscularly with BEI-inactivated virus, with or without Alhydrogel adjuvant, produced antibody which neutralized Ross River virus in vitro, and the mice also failed to develop viraemia when challenged intravenously with live virus. Serum neutralization and in vivo protection were greatest when BEI-inactivated virus was administered without adjuvant.

A glycoprotein C-prM of 35,000 M(r) was immuno-precipitated from lysates of Aedes albopictus cells infected with dengue virus type 2 (DEN-2) using antisera directed against the C protein or an amino-terminal fragment of the prM glycoprotein. C-prM was not detected in infected Vero cells. The prM glycoprotein synthesized in infected A. albopictus and Vero cells was cleaved to produce the membrane-associated virion protein (M) and the non-M fragment (pr) immediately preceding or occurring simultaneously with the release of viral particles from cells. The cleavage was less efficient in mosquito cells. The pr fragment was found only in the medium and was not rapidly degraded. To obtain pr-specific and M-specific antisera for these studies, proteins containing fragments of DEN-2 prM fused with staphylococcal Protein A were synthesized in Escherichia coli using the expression vector pRIT2T. The fusion proteins were stable and were used to raise antisera in rabbits for immunoprecipitation of radiolabelled cell extracts and culture medium. This is the first report of the detection of a C-prM protein in flavivirus-infected cells and the identification of the pr component of prM.

Anti-dengue 2 virus core protein monoclonal antibodies (MAbs) reacted with antigens in the cytoplasm and in, or on, the nucleus of dengue 2 and dengue 4, but not dengue 1, dengue 3, Kunjin or Murray Valley encephalitis virus-infected cells. These MAbs also reacted with the core protein from dengue 1, 2 and 4 virions in Western blots. The antigens detected by these MAbs could not be detected in uninfected or heat-shocked cells, but were first detected in infected cells approximately 32 h post-infection. PEPSCAN epitope mapping suggested that all the MAbs react with a region of the dengue 2 virus core protein (9RNTPFNMLKRE19) which is adjacent to a putative nuclear localization sequence (6KKAR9) and spans a possible second site for the initiation of synthesis of core protein (12PFN decreases MLKR18).