Rates of spread of marine pathogens

Epidemics of marine pathogens can spread at extremely rapid rates. For example, herpes virus spread through pilchard populations in Australia at a rate in excess of 10 000 km year(-1), and morbillivirus infections in seals and dolphins have spread at more than 3000 km year(-1). In terrestrial environments, only the epidemics of myxomatosis and calicivirus in Australian rabbits and West Nile Virus in birds in North America have rates of spread in excess of 1000 km year(-1). The rapid rates of spread of these epidemics has been attributed to flying insect vectors, but flying vectors have not been proposed for any marine pathogen. The most likely explanation for the relatively rapid spread of marine pathogens is the lack of barriers to dispersal in some parts of the ocean, and the potential for long-term survival of pathogens outside the host. These findings caution that pathogens may pose a particularly severe problem in the ocean. There is a need to develop epidemic models capable of generating these high rates of spread and obtain more estimates of disease spread rate.

Tetracycline-Inducible packaging cell line for production of Flavivirus replicon particles.

We have previously developed replicon vectors derived from the Australian flavivirus Kunjin that have a unique noncytopathic nature and have been shown to direct prolonged high-level expression of encoded heterologous genes in vitro and in vivo and to induce strong and long-lasting immune responses to encoded immunogens in mice. To facilitate further applications of these vectors in the form of virus-like particles (VLPs), we have now generated a stable BHK packaging cell line, tetKUNCprME, carrying a Kunjin structural gene cassette under the control of a tetracycline-inducible promoter. Withdrawal of tetracycline from the medium resulted in production of Kunjin structural proteins that were capable of packaging transfected and self-amplified Kunjin replicon RNA into the secreted VLPs at titers of up to 1.6 x 10(9) VLPs per ml. Furthermore, secreted KUN replicon VLPs from tetKUNCprME cells could be harvested continuously for as long as 10 days after RNA transfection, producing a total yield of more than 1010 VLPs per 106 transfected cells. Passaging of VLPs on Vero cells or intracerebral injection into 2- to 4-day-old suckling mice illustrated the complete absence of any infectious Kunjin virus. tetKUNCprME cells were also capable of packaging replicon RNA from closely and distantly related flaviviruses, West Nile virus and dengue virus type 2, respectively. The utility of high-titer KUN replicon VLPs was demonstrated by showing increasing CD8(+)-T-cell responses to encoded foreign protein with increasing doses of KUN VLPs. A single dose of 2.5 x 10(7) VLPs carrying the human respiratory syncytial virus M2 gene induced 1,400 CD8 T cells per 10(6) splenocytes in an ex vivo gamma interferon enzyme-linked immunospot assay. The packaging cell line thus represents a significant advance in the development of the noncytopathic Kunjin virus replicon-based gene expression system and may be widely applicable to the basic studies of flavivirus RNA packaging and virus assembly as well as to the development of gene expression systems based on replicons from different flaviviruses.

Entomological investigations in a focus of dengue transmission in Cairns, Queensland, Australia, by using the sticky ovitraps

Sticky ovitraps (patent pending) were used to sample female Aedes aegypti (L.) weekly in a focus of dengue activity in Cairns, Queensland, Australia. In February 2003, transmission of dengue virus serotype 2 began in the suburb of Parramatta Park, peaking in mid-March 2003. This suburb features many older, unscreened houses with high populations of Ae. aegypti. Highest densities (2-3.5 females per trap per week) were obtained during peak dengue transmission (January and February) before mosquito control was initiated. Beginning in late March, female Ae. aegypti collected in sticky ovitraps were tested for dengue viral RNA by using a TaqMan reverse transcription-polymerase chain reaction assay. Dengue viral RNA was detected in six pools of Ae. aegypti collected in late March. The highest minimum infection rate was 116/1000 mosquitoes. After the initiation of larval control (containers treated with S-methoprene or lambda-cyhalothrin) and adult control (interior harborage sites sprayed with lambda-cyhalothrin) in early March, trap collections dropped to

Information technologies for vaccine research

Vaccinology is a combinatorial science which studies the diversity of pathogens and the human immune system, and formulations that can modulate immune responses and prevent or cure disease. Huge amounts of data are produced by genomics and proteomics projects and large-scale screening of pathogen-host and antigen-host interactions. Current developments in computational vaccinology mainly support the analysis of antigen processing and presentation and the characterization of targets of immune response. Future development will also include systemic models of vaccine responses. Immunomics, the large-scale screening of immune processes which includes powerful immunoinformatic tools, offers great promise for future translation of basic immunology research advances into successful vaccines.

Epitope-blocking enzyme-linked immunosorbent assay for detection of antibodies to Ross River virus in vertebrate sera

We describe the development of an epitope-blocking enzyme-linked immunosorbent assay (ELISA) for the sensitive and rapid detection of antibodies to Ross River virus (RRV) in human sera and known vertebrate host species. This ELISA provides an alternative method for the serodiagnosis of RRV infections.

Biological, antigenic and phylogenetic characterization of the flavivirus Alfuy

Alfuy virus (ALFV) is classified as a subtype of the flavivirus Murray Valley encephalitis virus (MVEV); however, despite preliminary reports of antigenic and ecological similarities with MVEV, ALFV has not been associated with human disease. Here, it was shown that ALFV is at least 10(4)-fold less neuroinvasive than MVEV after peripheral inoculation of 3-week-old Swiss outbred mice, but ALFV demonstrates similar neurovirulence. In addition, it was shown that ALFV is partially attenuated in mice that are deficient in alpha/beta interferon responses, in contrast to MVEV which is uniformly lethal in these mice. To assess the antigenic relationship between these viruses, a panel of monoclonal antibodies was tested for the ability to bind to ALFV and MVEV in ELISA. Although the majority of monoclonal antibodies recognized both viruses, confirming their antigenic similarity, several discriminating antibodies were identified. Finally, the entire genome of the prototype strain of ALFV (MRM3929) was sequenced and phylogenetically analysed. Nucleotide (73%) and amino acid sequence (83 %) identity between ALFV and IMVEV confirmed previous reports of their close relationship. Several nucleotide and amino acid deletions and/or substitutions with putative functional significance were identified in ALFV, including the abolition of a conserved glycosylation site in the envelope protein and the deletion of the terminal dinucleotide 5'-CUOH-3' found in all other members of the genus. These findings confirm previous reports that ALFV is closely related to IMVEV, but also highlights significant antigenic, genetic and phenotypic divergence from MVEV. Accordingly, the data suggest that ALFV is a distinct species within the serogroup Japanese encephalitis virus.

Comparative mechanistic studies of de novo RNA synthesis by flavivirus RNA-dependent RNA polymerases

Flavivirus protein NS5 harbors the RNA-dependent RNA polymerase (RdRp) activity. In contrast to the RdRps of hepaci- and pestiviruses, which belong to the same family of Flaviviridae, NS5 carries two activities, a methyltransferase (MTase) and a RdRp. RdRp domains of Dengue virus (DV) and West Nile virus (WNV) NS5 were purified in high yield relative to full-length NS5 and showed full RdRp activity. Steady-state enzymatic parameters were determined on homopolymeric template poly(rC). The presence of the MTase domain does not affect the RdRp activity. Flavivirus RdRp domains might bear more than one GTP binding site displaying positive cooperativity. The kinetics of RNA synthesis by four Flaviviridae RdRps were compared. In comparison to Hepatitis C RdRp, DV and WNV as well as Bovine Viral Diarrhea virus RdRps show less rate limitation by early steps of short-product fort-nation. This suggests that they display a higher conformational flexibility upon the transition from initiation to elongation. (c) 2006 Elsevier Inc. All rights reserved.

Preliminary crystallographic characterization of an RNA helicase from Kunjin virus

Kunjin virus is a member of the Flavivirus genus and is an Australian variant of West Nile virus. The C-terminal domain of the Kunjin virus NS3 protein displays helicase activity. The protein is thought to separate daughter and template RNA strands, assisting the initiation of replication by unwinding RNA secondary structure in the 3' nontranslated region. Expression, purification and preliminary crystallographic characterization of the NS3 helicase domain are reported. It is shown that Kunjin virus helicase may adopt a dimeric assembly in absence of nucleic acids, oligomerization being a means to provide the helicases with multiple nucleic acid-binding capability, facilitating translocation along the RNA strands. Kunjin virus NS3 helicase domain is an attractive model for studying the molecular mechanisms of flavivirus replication, while simultaneously providing a new basis for the rational development of anti-flaviviral compounds.

Translation of the flavivirus Kunjin NS3 gene in cis but not its RNA sequence or secondary structure is essential for efficient RNA packaging

Our previous studies using trans-complementation analysis of Kunjin virus (KUN) full-length cDNA clones harboring in-frame deletions in the NS3 gene demonstrated the inability of these defective complemented RNAs to be packaged into virus particles (W. J. Liu, P. L. Sedlak, N. Kondratieva, and A. A. Khromykh, J. Virol. 76:10766-10775). In this study we aimed to establish whether this requirement for NS3 in RNA packaging is determined by the secondary RNA structure of the NS3 gene or by the essential role of the translated NS3 gene product. Multiple silent mutations of three computer-predicted stable RNA structures in the NS3 coding region of KUN replicon RNA aimed at disrupting RNA secondary structure without affecting amino acid sequence did not affect RNA replication and packaging into virus-like particles in the packaging cell line, thus demonstrating that the predicted conserved RNA structures in the NS3 gene do not play a role in RNA replication and/or packaging. In contrast, double frameshift mutations in the NS3 coding region of full-length KUN RNA, producing scrambled NS3 protein but retaining secondary RNA structure, resulted in the loss of ability of these defective RNAs to be packaged into virus particles in complementation experiments in KUN replicon-expressing cells. Furthermore, the more robust complementation-packaging system based on established stable cell lines producing large amounts of complemented replicating NS3-deficient replicon RNAs and infection with KUN virus to provide structural proteins also failed to detect any secreted virus-like particles containing packaged NS3-deficient replicon RNAs. These results have now firmly established the requirement of KUN NS3 protein translated in cis for genome packaging into virus particles.

Ornamental plants as climatic indicators of arthropod vectors

The importance and risk of vector-borne diseases (eg. leishmaniasis, West Nile Virus, Lyme borreliosis) is going to increase in the European temperate areas due to climate change. Our previous studies have shown that the potential distribution of Leishmania infantum and some Phlebotomus (sand fly) species – a parasite of leishmaniasis, and its vectors – may be expanded even to the southern coastline of the Baltic Sea by the end of the 21st century. The lowland areas of the Carpathian Basin and the main part of Hungary are projected to be suitable for the studied sand fly vectors in the near future. It is important to find some indicator plants to examine whether the sand flies are able to live in a certain climate at a certain time. We studied several Mediterranean and Sub-Mediterranean plant species, and we found that the aggregated distribution of three ligneous species (Juniperus oxycedrus L., Quercus ilex L. and Pinus brutia Ten.) shows high correlation with the union distribution of five sand flies (Phlebotomus ariasi Tonn., Ph. neglectus Tonn., Ph. perfiliewi Parrot, Ph. perniciosus Newst. and Ph. tobbi Adler, Theodor et Lourie). Since these Mediterranean species are highly tolerant of the edaphic characteristics of the planting site, they may prove to be good indicators. The present and upcoming climate of Hungary is seen to be suitable for the selected indicator plant species, and it draws attention to and verifies the potential of the expansion of sand flies, which has been proved by some recent observations of the vectors in Southern Hungary.

Ornamental plants as climatic indicators of arthropod vectors

The importance and risk of vector-borne diseases (e.g., leishmaniasis, West Nile Virus, Lyme borreliosis) is going to increase in the European temperate areas due to climate change. Our previous studies have shown that the potential distribution of Leishmania infantum and some Phlebotomus (sand fly) species – a parasite of leishmaniasis, and its vectors – may be expanded even to the southern coastline of the Baltic Sea by the end of the 21st century. The lowland areas of the Carpathian Basin and the main part of Hungary are projected to be suitable for the studied sand fly vectors in the near future. It is important to find some indicator plants to examine whether the sand flies are able to live in a certain climate at a certain time. We studied several Mediterranean and Sub-Mediterranean plant species, and we found that the aggregated distribution of three ligneous species (Juniperus oxycedrus L., Quercus ilex L. and Pinus brutia Ten.) shows high correlation with the union distribution of five sand flies (Phlebotomus ariasi Tonn., Ph. neglectus Tonn., Ph. perfiliewi Parrot, Ph. perniciosus Newst. and Ph. tobbi Adler, Theodor et Lourie). Since these Mediterranean species are highly tolerant of the edaphic characteristics of the planting site, they may prove to be good indicators. The present and upcoming climate of Hungary is seen to be suitable for the selected indicator plant species, and it draws attention to and verifies the potential of the expansion of sand flies, which has been proved by some recent observations of the vectors in Southern Hungary.

Host cellular regulatory networks in dengue virus-human interactions

Dengue fever is one of the most important mosquito-borne diseases worldwide and is caused by infection with dengue virus (DENV). The disease is endemic in tropical and sub-tropical regions and has increased remarkably in the last few decades. At present, there is no antiviral or approved vaccine against the virus. Treatment of dengue patients is usually supportive, through oral or intravenous rehydration, or by blood transfusion for more severe dengue cases. Infection of DENV in humans and mosquitoes involves a complex interplay between the virus and host factors. This results in regulation of numerous intracellular processes, such as signal transduction and gene transcription which leads to progression of disease. To understand the mechanisms underlying the disease, the study of virus and host factors is therefore essential and could lead to the identification of human proteins modulating an essential step in the virus life cycle. Knowledge of these human proteins could lead to the discovery of potential new drug targets and disease control strategies in the future. Recent advances of high throughput screening technologies have provided researchers with molecular tools to carry out investigations on a large scale. Several studies have focused on determination of the host factors during DENV infection in human and mosquito cells. For instance, a genome-wide RNA interference (RNAi) screen has identified host factors that potentially play an important role in both DENV and West Nile virus replication (Krishnan et al. 2008). In the present study, a high-throughput yeast two-hybrid screen has been utilised in order to identify human factors interacting with DENV non-structural proteins. From the screen, 94 potential human interactors were identified. These include proteins involved in immune signalling regulation, potassium voltage-gated channels, transcriptional regulators, protein transporters and endoplasmic reticulum-associated proteins. Validation of fifteen of these human interactions revealed twelve of them strongly interacted with DENV proteins. Two proteins of particular interest were selected for further investigations of functional biological systems at the molecular level. These proteins, including a nuclear-associated protein BANP and a voltage-gated potassium channel Kv1.3, both have been identified through interaction with the DENV NS2A. BANP is known to be involved in NF-kB immune signalling pathway, whereas, Kv1.3 is known to play an important role in regulating passive flow of potassium ions upon changes in the cell transmembrane potential. This study also initiated a construction of an Aedes aegypti cDNA library for use with DENV proteins in Y2H screen. However, several issues were encountered during the study which made the library unsuitable for protein interaction analysis. In parallel, innate immune signalling was also optimised for downstream analysis. Overall, the work presented in this thesis, in particular the Y2H screen provides a number of human factors potentially targeted by DENV during infection. Nonetheless, more work is required to be done in order to validate these proteins and determine their functional properties, as well as testing them with infectious DENV to establish a biological significance. In the long term, data from this study will be useful for investigating potential human factors for development of antiviral strategies against dengue.

2016 Flanders Virus Cases

Flanders virus was discovered in 1961 in the town of Flanders on Long Island, New York. The virus is in the virus family Rhabdoviridae, and it is widely distributed in Canada, the United States, and Mexico. Flanders virus does not cause disease. The virus is frequently found in birds, such as Red-winged blackbirds, House sparrows, and starlings. It is also found in bird-feeding mosquitoes, such as the black-tailed mosquito (Culiseta melanura) and the northern and southern house mosquitoes (Culex pipiens and Culex quinquefasciatus, respectively). Presence of the virus in an area serves as a sentinel or warning for West Nile virus and as a trigger for public health control and prevention interventions targeting West Nile virus.

Clínica médica e cirúrgica em espécies pecuárias e equinos

O presente trabalho teve como objetivo a descrição das atividades desenvolvidas no âmbito do estágio curricular do Mestrado Integrado em Medicina Veterinária pela Universidade de Évora. Numa primeira parte é apresentada a casuística acompanhada ao longo do estágio, com referência mais pormenorizada a alguns casos clínicos acompanhados nas diversas áreas de intervenção: clínica médica e cirúrgica de espécies pecuárias e equinos. A segunda parte deste relatório é composta por uma revisão bibliográfica sobre os temas “exame neurológico em equinos”, “ síndrome do poldro lavanda” e “vírus do Nilo ocidental”. Na última parte serão relatados de três casos clínicos acompanhados neste âmbito; Clinical medical and surgery in livestock species and horses Abstract: The current report describes the activities developed during the Curricular externship as parts of the Veterinary Medicine Integrated Masters from University of Evora. The first part includes the casuistry that took place along the externship, giving enphasis to some of the clinical cases assessed in different livestock species and equine clinic areas. The second part presents a literature review approaching “neurologic examination in horses”, “lavender foal syndrome” and “west Nile vírus” as its main themes. For the ending part there are described three clinical cases followed during the externship.