West Nile virus vaccines

West Nile virus (WNV) is a mosquito-borne flavivirus that is emerging as a global pathogen. In the last decade, virulent strains of the virus have been associated with significant outbreaks of human and animal disease in Europe, the Middle East and North America. Efforts to develop human and veterinary vaccines have taken both traditional and novel approaches. A formalin-inactivated whole virus vaccine has been approved for use in horses. DNA vaccines coding for the structural WNV proteins have also been assessed for veterinary use and have been found to be protective in mice, horses and birds. Live attenuated yellow fever WNV chimeric vaccines have also been successful in animals and are currently undergoing human trials. Additional studies have shown that immunisation with a relatively benign Australian variant of WNV, the Kunjin virus, also provides protective immunity against the virulent North American strain. Levels of efficacy and safety, as well as logistical, economic and environmental issues, must all be carefully considered before vaccine candidates are approved and selected for large-scale manufacture and distribution.

Flavivirus isolations from mosquitoes collected from Saibai Island in the Torres Strait, Australia, during an incursion of Japanese encephalitis virus

Adult mosquitoes (Diptera: Culicidae) were collected in January and February 2000 from Saibai Island in the Torres Strait of northern Australia, and processed for arbovirus isolation during a period of Japanese encephalitis (JE) virus activity on nearby Badu Island. A total of 84 2 10 mosquitoes were processed for virus isolation, yielding six flavivirus isolates. Viruses obtained were single isolates of JE and Kokobera (KOK) and four of Kunjin (KUN). All virus isolates were from members of the Culex sitiens Weidemann subgroup, which comprised 53.1 % of mosquitoes processed. Nucleotide sequencing and phylogenetic analysis of the pre-membrane region of the genome of JE isolate TS5313 indicated that it was closely related to other isolates from a sentinel pig and a pool of Cx. gelidus Theobald from Badu Island during the same period. Also molecular analyses of part of the envelope gene of KUN virus isolates showed that they were closely related to other KUN virus strains from Cape York Peninsula. The results indicate that flaviviruses are dynamic in the area, and suggest patterns of movement south from New Guinea and north from the Australian mainland.

Ross River virus disease in Australia, 1886-1998, with analysis of risk factors associated with outbreaks

Ross River virus (RE) is a mosquito-borne arbovirus responsible for outbreaks of polyarthritic disease throughout Australia. To better understand human and environmental factors driving such events, 57 historical reports oil RR Outbreaks between 1896 and 1998 were examined collectively. The magnitude, regularity, seasonality, and locality of outbreaks were found to be wide ranging; however, analysis of climatic and tidal data highlighted that environmental conditions let differently ill tropical, arid, and temperate regions. Overall, rainfall seems to be the single most important risk factor, with over 90% of major outbreak locations receiving higher than average rainfall in preceding mouths. Many temperatures were close to average, particularly in tropical populations; however, in arid regions, below average maximum temperatures predominated, and ill southeast temperate regions, above average minimum temperatures predominated. High spring tides preceded coastal Outbreaks, both in the presence and absence of rainfall, and the relationship between rainfall and the Southern Oscillation Index and Lit Nina episodes suggest they may be useful predictive tools, but only ill southeast temperate regions. Such heterogeneity predisposing outbreaks supports the notion that there are different RE epidemiologies throughout Australia but also Suggests that generic parameters for the prediction and control of outbreaks are of limited use at a local level.

Differences in climatic factors between Ross River virus disease outbreak and nonoutbreak years

Ross River virus is a common mosquito-borne arbovirus responsible for outbreaks of polyarthritic disease throughout Australia. To better understand climatic factors preceding outbreaks, we compared seasonal and monthly rainfall and temperature trends in outbreak and nonoutbreak years at four epidemic-prone locations. Our analyses showed that rainfall in outbreak years tended to be above average and higher than rainfall in nonoutbreak years. Overall temperatures were warmer during outbreak years. However, there were a number of distinct deviations in temperature, which seem to play a role in either promoting or inhibiting outbreaks. These preliminary findings show that climatic differences occur between outbreak and nonoutbreak years; however, seasonal and monthly trends differed across geo-climatic regions of the country. More detailed research is imperative if we are to optimize the surveillance and control of epidemic polyarthritic disease in Australia.

Reflecting on ethical and legal issues in wildlife disease

Disease in wildlife raises a number of issues that have not been widely considered in the bioethical literature. However, wildlife disease has major implications for human welfare. The majority of emerging human infectious diseases are zoonotic: that is, they occur in humans by cross-species transmission from animal hosts. Managing these diseases often involves balancing concerns with human health against animal welfare and conservation concerns. Many infectious diseases of domestic animals are shared with wild animals, although it is often unclear whether the infection spills over from wild animals to domestic animals or vice versa. Culling is the standard means of managing such diseases, bringing economic considerations, animal welfare and conservation into conflict. Infectious diseases are also major threatening processes in conservation biology and their appropriate management by culling, vaccination or treatment raises substantial animal ethics issues. One particular issue of great significance in Australia is an ongoing research program to develop genetically modified pathogens to control vertebrate pests including rabbits, foxes and house mice. Release of any self-replicating GMO vertebrate pathogen gives rise to a whole series of ethical questions. We briefly review current Australian legal responses to these problems. Finally, we present two unresolved problems of general importance that are exemplified by wildlife disease. First, to what extent can or should 'bioethics' be broadened beyond direct concerns with human welfare to animal welfare and environmental welfare? Second, how should the irreducible uncertainty of ecological systems be accounted for in ethical decision making?

NS1 protein secretion during the acute phase of West Nile virus infection

The West Nile virus (WNV) nonstructural protein NS1 is a protein of unknown function that is found within, associated with, and secreted from infected cells. We systematically investigated the kinetics of NS1 secretion in vitro and in vivo to determine the potential use of this protein as a diagnostic marker and to analyze NS1 secretion in relation to the infection cycle. A sensitive antigen capture enzyme-linked immunosorbent assay (ELISA) for detection of WNW NS1 (polyclonal-ACE) was developed, as well as a capture ELISA for the specific detection of NS1 multimers (4G4-ACE). The 4G4-ACE detected native NS1 antigens at high sensitivity, whereas the polyclonal-ACE had a higher specificity for recombinant forms of the protein. Applying these assays we found that only a small fraction of intracellular NS1 is secreted and that secretion of NS1 in tissue culture is delayed compared to the release of virus particles. In experimentally infected hamsters, NS1 was detected in the serum between days 3 and 8 postinfection, peaking on day 5, the day prior to the onset of clinical disease; immunoglobulin M (IgM) antibodies were detected at low levels on day 5 postinfection. Although real-time PCR gave the earliest indication of infection (day 1), the diagnostic performance of the 4G4-ACE was comparable to that of real-time PCR during the time period when NS1 was secreted. Moreover, the 4G4-ACE was found to be superior in performance to both the IgM and plaque assays during this time period, suggesting that NS1 is a viable early diagnostic marker of WNV infection.

Real-time reverse transcriptase PCR for the endogenous koala retrovirus reveals an association between plasma viral load and neoplastic disease in koalas

Koala retrovirus (KoRV) is a newly described endogenous retrovirus and is unusual in that inserts comprise a full-length replication competent genome. As koalas are known to suffer from an extremely high incidence of leukaemia/lymphoma, the association between this retrovirus and disease in koalas was examined. Using quantitative real-time reverse transcriptase PCR it was demonstrated that KoRV RNA levels in plasma are significantly increased in animals suffering from leukaemia or lymphoma when compared with healthy animals. Increased levels of KoRV were also seen for animals with clinical chlamydiosis. A significant positive association between viral RNA levels and age was also demonstrated. Real-time PCR demonstrated as much as 5 log variation in KoRV proviral DNA levels in genomic DNA extracted from whole blood from different animals. Taken together these data indicate that KoRV is an active endogenous retrovirus and suggests that it may be causally linked to neoplastic disease in koalas.

Risks for ross river virus disease in tropical Australia

Background There are no analytical studies of individual risks for Ross River virus (RRV) disease. Therefore, we set out to determine individual risk and protective factors for RRV disease in a high incidence area and to assess the utility of the case-control design applied for this purpose to an arbovirus disease. Methods We used a prospective matched case-control study of new community cases of RRV disease in the local government areas of Cairns, Mareeba, Douglas, and Atherton, in tropical Queensland, from January I to May 31, 1998. Results Protective measures against mosquitoes reduced the risk for disease. Mosquito coils, repellents, and citronella candles each decreased risk by at least 2-fold, with a dose-response for the number of protective measures used. Light-coloured clothing decreased risk 3-fold. Camping increased the risk 8-fold. Conclusions These risks were substantial and statistically significant, and provide a basis for educational programs on individual protection against RRV disease in Australia. Our study demonstrates the utility of the case-control method for investigating arbovirus risks. Such a risk analysis has not been done before for RRV infection, and is infrequently reported for other arbovirus infections.

Hepatitis B surface antigen vector delivers protective cytotoxic T-lymphocyte responses to disease-relevant foreign epitopes

Although hepatitis B surface antigen (HBsAg) per se is highly immunogenic, its use as a vector for the delivery of foreign cytotoxic T-lymphocyte (CTL) epitopes has met with little success because of constraints on HBsAg stability and secretion imposed by the insertion of foreign sequence into critical hydrophobic/amphipathic regions. Using a strategy entailing deletion of DNA encoding HBsAg-specific CTL epitopes and replacement with DNA encoding foreign CTL epitopes, we have derived chimeric HBsAg DNA immunogens which elicited effector and memory CTL responses in vitro, and pathogen- and tumor-protective responses in vivo, when the chimeric HBsAg DNAs were used to immunize mice. We further show that HBsAg DNA recombinant for both respiratory syncytial virus and human papillomavirus CTL epitopes elicited simultaneous responses to both pathogens. These data demonstrate the efficacy of HBsAg DNA as a vector for the delivery of disease-relevant protective CTL responses. They also suggest the applicability of the approach of deriving chimeric HBsAg DNA immunogens simultaneously encoding protective CTL epitopes for multiple diseases. The DNAs we tested formed chimeric HBsAg virus-like particles (VLPs). Thus, our results have implications for the development of vaccination strategies using either chimeric HBsAg DNA or VLP vaccines. HBsAg is the globally administered vaccine for hepatitis B virus infection, inviting its usage as a vector for the delivery of immunogens from other diseases.

Cytotoxic T-lymphocyte epitope vaccination protects against human metapneumovirus infection and disease in mice

Human metapneumovirus (hMPV) has emerged as an important human respiratory pathogen causing upper and lower respiratory tract infections in young children and older adults. In addition, hMPV infection is associated with asthma exacerbation in young children. Recent epidemiological evidence indicates that hMPV may cocircullate with human respiratory syncytial virus (hRSV) and mediate clinical disease similar to that seen with hRSV. Therefore, a vaccine for hMPV is highly desirable. In the present study, we used predictive bioinformatics, peptide immunization, and functional T-cell assays to define hMPV cytotoxic T-lymphocyte (CTL) epitopes recognized by mouse T cells restricted through several major histocompatibility complex class I alleles, including HILA-A*0201. We demonstrate that peptide immunization with hMPV CTL epitopes reduces viral load and immunopathollogy in the lungs of hMPV-challenged mice and enhances the expression of Th1-type cytokines (gamma interferon and interleukin-12 [IL-12]) in lungs and regional lymph nodes. In addition, we show that levels of Th2-type cytolkines (IL-10 and IL-4) are significantly lower in hMPV CTL epitope-vaccinated mice challenged with hMPV. These results demonstrate for the first time the efficacy of an hMPV CTL epitope vaccine in the control of hMPV infection in a murine model.

Ross River virus disease clusters and spatial relationship with mosquito biting exposure in Redland Shire, southern Queensland, Australia

The spatial heterogeneity in the risk of Ross River virus (family Togaviridae, genus Alphavirus, RRV) disease, the most common mosquito-borne disease in Australia, was examined in Redland Shire in southern Queensland, Australia. Disease cases, complaints from residents of intense mosquito biting exposure, and human population data were mapped using a geographic information system. Surface maps of RRV disease age-sex standardized morbidity ratios and mosquito biting complaint morbidity ratios were created. To determine whether there was significant spatial variation in disease and complaint patterns, a spatial scan analysis method was used to test whether the number of cases and complaints was distributed according to underlying population at risk. Several noncontiguous areas in proximity to productive saline water habitats of Aedes vigilax (Skuse), a recognized vector of RRV, had higher than expected numbers of RRV disease cases and complaints. Disease rates in human populations in areas which had high numbers of adult Ae. vigilax in carbon dioxide- and octenol-baited light traps were up to 2.9 times those in areas that rarely had high numbers of mosquitoes. It was estimated that targeted control of adult Ae. vigilax in these high-risk areas could potentially reduce the RRV disease incidence by an average of 13.6%. Spatial correlation was found between RRV disease risk and complaints from residents of mosquito biting. Based on historical patterns of RRV transmission throughout Redland Shire and estimated future human population growth in areas with higher than average RRV disease incidence, it was estimated that RRV incidence rates will increase by 8% between 2001 and 2021. The use of arbitrary administrative areas that ranged in size from 4.6 to 318.3 km2, has the potential to mask any small scale heterogeneity in disease patterns. With the availability of georeferenced data sets and high-resolution imagery, it is becoming more feasible to undertake spatial analyses at relatively small scales.

Viruses: agents of coral disease?

The potential role of viruses in coral disease has only recently begun to receive attention. Here we describe our attempts to determine whether viruses are present in thermally stressed corals Pavona danai, Acropora formosa and Stylophora pistillata and zoanthids Zoanthus sp., and their zooxanthellae. Heat-shocked P. danai, A. formosa and Zoanthus sp. all produced numerous virus-like particles (VLPs) that were evident in the animal tissue, zooxanthellae and the surrounding seawater; VLPs were also seen around heat-shocked freshly isolated zooxanthellae (FIZ) from P. danai and S. pistillata. The most commonly seen VLPs were tail-less, hexagonal and about 40 to 50 nm in diameter, though a diverse range of other VLP morphotypes (e.g. rounded, rod-shaped, droplet-shaped, filamentous) were also present around corals. When VLPs around heat-shocked FIZ from S. pistillata were added to non-stressed FIZ from this coral, they resulted in cell lysis, suggesting that an infectious agent was present; however, analysis with transmission electron microscopy provided no clear evidence of viral infection. The release of diverse VLPs was again apparent when flow cytometry was used to enumerate release by heat-stressed A. formosa nubbins. Our data support the infection of reef corals by viruses, though we cannot yet determine the precise origin (i.e. coral, zooxanthellae and/or surface microbes) of the VLPs seen. Furthermore, genome sequence data are required to establish the presence of viruses unequivocally.

Perspective on the host response to human metapneumovirus infection: what can we learn from respiratory syncytial virus infections?

Human metapneumovirus (HMPV) is a recently discovered pathogen first identified in respiratory specimens from young children suffering from clinical respiratory syndromes ranging from mild to severe lower respiratory tract illness. HMPV has worldwide prevalence, and is a leading cause of respiratory tract infection in the first years of life, with a spectrum of disease similar to respiratory syncytial virus (RSV). The disease burden associated with HMPV infection has not been fully elucidated; however, studies indicate that HMPV may cause upper or lower respiratory tract illness in patients between ages 2 months and 87 years, may co-circulate with RSV, and HMPV infection may be associated with asthma exacerbation. The mechanisms and effector pathways contributing to immunity or disease pathogenesis following infection are not fully understood; however, given the clinical significance of HMPV, there is a need for a fundamental understanding of the immune and pathophysiological processes that occur following infection to provide the foundation necessary for the development of effective vaccine or therapeutic intervention strategies. This review provides a current perspective on the processes associated with HMPV infection, immunity, and disease pathogenesis. (c) 2005 Elsevier SAS. All rights reserved.