SAR analysis of a series of acylthiourea derivatives possessing broad-spectrum antiviral activity

A series of acylthiourea derivatives were designed, synthesized, and evaluated for broad-spectrum antiviral activity with selected viruses from Poxviridae (vaccinia virus) and two different genera of the family Bunyaviridae (Rift Valley fever and La Crosse viruses). A compound selected from a library screen, compound 1, displayed submicromolar antiviral activity against both vaccinia virus (EC(50)=0.25 μM) and La Crosse virus (EC(50)=0.27 μM) in cytopathic effect (CPE) assays. SAR analysis was performed to further improve antiviral potency and to optimize drug-like properties of the initial hits. During our analysis, we identified 26, which was found to be nearly fourfold more potent than 1 against both vaccinia and La Crosse viruses. Selected compounds were further tested to more fully characterize the spectrum of antiviral activity. Many of these possessed single digit micromolar and sub-micromolar antiviral activity against a diverse array of targets, including influenza virus (Orthomyxoviridae), Tacaribe virus (Arenaviridae), and dengue virus (Flaviviridae).     

Protection from La Crosse virus encephalitis with recombinant glycoproteins: role of neutralizing anti-G1 antibodies.

La Crosse virus, a member of the California serogroup of bunyaviruses, is an important cause of pediatric encephalitis in the midwestern United States. Like all bunyaviruses, La Crosse virus contains two glycoproteins, G1 and G2, the larger of which, G1, is the target of neutralizing antibodies. To develop an understanding of the role of each of the glycoproteins in the generation of a protective immune response, we immunized 1-week-old mice with three different preparations: a vaccinia virus recombinant (VV.ORF) that expresses both G1 and G2, a vaccinia virus recombinant (VV.G1) that expresses G1 only, and a truncated soluble G1 (sG1) protein prepared in a baculovirus system. Whereas VV.ORF generated a protective response that was mostly directed against G1, VV.G1 was only partially effective at inducing a neutralizing response and at protecting mice from a potentially lethal challenge with La Crosse virus. Nevertheless, a single immunization with the sG1 preparation resulted in a robust immune response and protection against La Crosse virus. These results indicate that (i) the G1 protein by itself can induce an immune response sufficient for protection from a lethal challenge with La Crosse virus, (ii) a neutralizing humoral response correlates with protection, and (iii) the context in which G1 is presented affects its immunogenicity. The key step in the defense against central nervous system infection appeared to be interruption of a transient viremia that occurred just after La Crosse virus inoculation.     

Structural proteins of La Crosse virus.

Preparations of La Crosse virus, a member of the California encephalitis group of bunyaviruses, were found to possess three major virion proteins. Two of the proteins were glycosylated (G1 and G2) and were located on the surface of the virus particles. These two glycoproteins were present in equimolar amounts and possessed apparent molecular weights of 120 X 10(3) and 34 X 10(3). Virion nucleocapsids, isolated by a nonionic detergent and salt treatment, contained another major protein, N (molecular weight = 23 X 10(3)). A large, but minor, protein species L (molecular weight = 180 X 10(3)) was also found in virus preparations. The approximate number of protein molecules per virion has been determined. Electron microscopy of purified La Crosse virus indicated that the virus particle (mean diameter, 91 nm) is enveloped and possesses irregular surface projections (length, 10 nm).     

Prevalence of tick-borne encephalitis virus (TBEV) RNA in Dermacentor reticulatus ticks from natural and urban environment,Poland

Tick-borne encephalitis virus (TBEV) (Flaviviridae, Flavivirus) is an arthropod-borne virus, an etiologic agent of tick-borne encephalitis (TBE), a human infection involving the central nervous system. The disease is endemic in a large region in Eurasia, where it is transmitted mainly by Ixodes ricinus and Ixodes persulcatus ticks. It is known that also Dermacentor reticulatus is involved in circulation of TBEV. However, the current knowledge of D. reticulatus importance in TBE epidemiology is still insufficient. A total of 471 adult D. reticulatus ticks were collected by flagging vegetation in the Bia?owie?a Primeval Forest, Biebrza National Park, Masurian Landscape Park (North-Eastern Poland) and in the city of Warsaw in the years 2007–2010. All collected ticks were examined individually for the presence of RNA of TBEV using nested RT-PCR assay. Positive results were noted in all investigated localities with the infection rate ranging from 0.99 to 12.5 % with a total mean of 2.12 %. The difference in the percentage of infective males and females was not statistically significant.     

Aedes Mosquito Saliva Modulates Rift Valley Fever Virus Pathogenicity

Background

Rift Valley fever (RVF) is a severe mosquito-borne disease affecting humans and domestic ruminants. Mosquito saliva contains compounds that counteract the hemostatic, inflammatory, and immune responses of the host. Modulation of these defensive responses may facilitate virus infection. Indeed, Aedes mosquito saliva played a crucial role in the vector''s capacity to effectively transfer arboviruses such as the Cache Valley and West Nile viruses. The role of mosquito saliva in the transmission of Rift Valley fever virus (RVFV) has not been investigated.

Objective

Using a murine model, we explored the potential for mosquitoes to impact the course of RVF disease by determining whether differences in pathogenesis occurred in the presence or absence of mosquito saliva and salivary gland extract.

Methods

C57BL/6NRJ male mice were infected with the ZH548 strain of RVFV via intraperitoneal or intradermal route, or via bites from RVFV-exposed mosquitoes. The virus titers in mosquitoes and mouse organs were determined by plaque assays.

Findings

After intraperitoneal injection, RVFV infection primarily resulted in liver damage. In contrast, RVFV infection via intradermal injection caused both liver and neurological symptoms and this route best mimicked the natural infection by mosquitoes. Co-injections of RVFV with salivary gland extract or saliva via intradermal route increased the mortality rates of mice, as well as the virus titers measured in several organs and in the blood. Furthermore, the blood cell counts of infected mice were altered compared to those of uninfected mice.

Interpretation

Different routes of infection determine the pattern in which the virus spreads and the organs it targets. Aedes saliva significantly increases the pathogenicity of RVFV.     

Molecular-based identification and phylogeny of genomic and proteomic sequences of mosquito-borne flavivirus

Mosquito-borne flaviviruses (MBFVs) are important cause of emerging and re-emerging human diseases nearly worldwide, transmitted by arthropod vectors (mostly aedes and culex mosquitoes), with particular reference to yellow fever virus, Japanese encephalitis virus, dengue fever virus, St. Louis encephalitis virus, Murray Valley encephalitis virus, etc. In over 100 countries, more than 2.5 billion people are at risk of infection, and approximately 20 million infections are reported annually. Through the analysis of gene sequence data of these virus populations it is possible to infer phylogenetic relationships, which in turn can yield important epidemiological information, including their demographic history. Early attempts to define the evolutionary relationships and origins of viruses in the genus flavivirus are hampered by the lack of genetic information particularly amongst the MBFVs. In this study, complete genome, translated polyprotein, structural and non-structural proteins of MBFVs have been targeted and revealed an extensive series of clades defined by their epidemiology and disease associations. The branching patterns of at the deeper nodes of the resultant trees were different from those reported in the previous study. The significance of these observations is discussed.     

A Multiplex PCR/LDR Assay for the Simultaneous Identification of Category A Infectious Pathogens: Agents of Viral Hemorrhagic Fever and Variola Virus

CDC designated category A infectious agents pose a major risk to national security and require special action for public health preparedness. They include viruses that cause viral hemorrhagic fever (VHF) syndrome as well as variola virus, the agent of smallpox. VHF is characterized by hemorrhage and fever with multi-organ failure leading to high morbidity and mortality. Smallpox, a prior scourge, has been eradicated for decades, making it a particularly serious threat if released nefariously in the essentially non-immune world population. Early detection of the causative agents, and the ability to distinguish them from other pathogens, is essential to contain outbreaks, implement proper control measures, and prevent morbidity and mortality. We have developed a multiplex detection assay that uses several species-specific PCR primers to generate amplicons from multiple pathogens; these are then targeted in a ligase detection reaction (LDR). The resultant fluorescently-labeled ligation products are detected on a universal array enabling simultaneous identification of the pathogens. The assay was evaluated on 32 different isolates associated with VHF (ebolavirus, marburgvirus, Crimean Congo hemorrhagic fever virus, Lassa fever virus, Rift Valley fever virus, Dengue virus, and Yellow fever virus) as well as variola virus and vaccinia virus (the agent of smallpox and its vaccine strain, respectively). The assay was able to detect all viruses tested, including 8 sequences representative of different variola virus strains from the CDC repository. It does not cross react with other emerging zoonoses such as monkeypox virus or cowpox virus, or six flaviviruses tested (St. Louis encephalitis virus, Murray Valley encephalitis virus, Powassan virus, Tick-borne encephalitis virus, West Nile virus and Japanese encephalitis virus).     

An avirulent G1 glycoprotein variant of La Crosse bunyavirus with defective fusion function.

La Crosse virus, a member of the California serogroup of the family Bunyaviridae, causes encephalitis in humans and laboratory rodents. A variant virus (V22) selected with a monoclonal antibody against the large (G1) glycoprotein showed diminished neuroinvasiveness after peripheral inoculation. This variant has an alteration in its fusion function, requiring a lower pH for the activation of fusion and demonstrating reduced efficiency of cell-to-cell fusion of BHK-21 cultures. V22 was studied in detail following the infection by intraperitoneal or intracerebral routes in suckling, weanling, or adult CD-1 mice. It exhibited a marked reduction in its ability to replicate in striated muscle and to produce viremia; however, after intracerebral injection V22 virus replicated almost as rapidly in brain as its parent, La Crosse virus. V22 virus thus represents an example of reduced neuroinvasiveness associated with an alteration at a specific epitope of the G1 glycoprotein. This same epitope also influences the fusion activity of the glycoprotein.     

A Single Immunization with MVA Expressing GnGc Glycoproteins Promotes Epitope-specific CD8+-T Cell Activation and Protects Immune-competent Mice against a Lethal RVFV Infection

Background

Rift Valley fever virus (RVFV) is a mosquito-borne pathogen causing an important disease in ruminants often transmitted to humans after epizootic outbreaks in African and Arabian countries. To help combat the spread of the disease, prophylactic measures need to be developed and/or improved.

Methodology/Principal Findings

In this work, we evaluated the immunogenicity and protective efficacy of recombinant plasmid DNA and modified vaccinia virus Ankara (rMVA) vectored vaccines against Rift Valley fever in mice. These recombinant vaccines encoded either of two components of the Rift Valley fever virus: the viral glycoproteins (Gn/Gc) or the nucleoprotein (N). Following lethal challenge with live RVFV, mice immunized with a single dose of the rMVA-Gn/Gc vaccine showed no viraemia or clinical manifestation of disease, but mounted RVFV neutralizing antibodies and glycoprotein specific CD8+ T-cell responses. Neither DNA-Gn/Gc alone nor a heterologous prime-boost immunization schedule (DNA-Gn/Gc followed by rMVAGn/Gc) was better than the single rMVA-Gn/Gc immunization schedule with regards to protective efficacy. However, the rMVA-Gn/Gc vaccine failed to protect IFNAR^−/− mice upon lethal RVFV challenge suggesting a role for innate responses in protection against RVFV. Despite induction of high titer antibodies against the RVFV nucleoprotein, the rMVA-N vaccine, whether in homologous or heterologous prime-boost schedules with the corresponding recombinant DNA vaccine, only conferred partial protection to RVFV challenge.

Conclusions/Significance

Given the excellent safety profile of rMVA based vaccines in humans and animals, our data supports further development of rMVA-Gn/Gc as a vaccine strategy that can be used for the prevention of Rift Valley fever in both humans and livestock.     

Modeling a single season of Aedes albopictus populations based on host‐seeking data in response to temperature and precipitation in eastern Tennessee

In the southern Appalachia of the U.S., Aedes mosquitoes maintain and transmit La Crosse virus (LACV) which causes La Crosse encephalitis, a neuroinvasive disease of children. In response to mosquito outbreaks, communities organize prevention, detection, and response measures that are dependent on local characteristics of the mosquito population and the community. Knowing Ae. albopictus is an accessory vector of LACV and a nuisance biter, our objective was to build a system of ordinary differential equations to model dynamics in a single season using our data and readily available environmental variables that can reflect the abundance and activity of Ae. albopictus. Consequently, we built an Ae. albopictus single‐season mathematical model for eastern Tennessee to fit our 2013 mosquito collection data in order to understand the population fluctuations. We included precipitation, temperature, and rate of change of temperature in the model because Aedes mosquitoes oviposit desiccant tolerant eggs with peak activity occurring over 26° C and those data are readily available and used frequently as forecast predictors. Our ordinary differential equation model accurately fits the data and facilitates predictions and better understanding of Ae. albopictus populations in southern Appalachia.     

Identification of super-infected Aedes triseriatus mosquitoes collected as eggs from the field and partial characterization of the infecting La Crosse viruses

Background

La Crosse virus (LACV) is a pathogenic arbovirus that is transovarially transmitted by Aedes triseriatus mosquitoes and overwinters in diapausing eggs. However, previous models predicted transovarial transmission (TOT) to be insufficient to maintain LACV in nature.

Results

To investigate this issue, we reared mosquitoes from field-collected eggs and assayed adults individually for LACV antigen, viral RNA by RT-PCR, and infectious virus. The mosquitoes had three distinct infection phenotypes: 1) super infected (SI+) mosquitoes contained infectious virus, large accumulations of viral antigen and RNA and comprised 17 of 17,825 (0.09%) of assayed mosquitoes, 2) infected mosquitoes (I+) contained no detectable infectious virus, lesser amounts of viral antigen and RNA, and comprised 3.7% of mosquitoes, and 3) non-infected mosquitoes (I-) contained no detectable viral antigen, RNA, or infectious virus and comprised 96.21% of mosquitoes. SI+ mosquitoes were recovered in consecutive years at one field site, suggesting that lineages of TOT stably-infected and geographically isolated Ae. triseriatus exist in nature. Analyses of LACV genomes showed that SI+ isolates are not monophyletic nor phylogenetically distinct and that synonymous substitution rates exceed replacement rates in all genes and isolates. Analysis of singleton versus shared mutations (Fu and Li's F*) revealed that the SI+ LACV M segment, with a large and significant excess of intermediate-frequency alleles, evolves through disruptive selection that maintains SI+ alleles at higher frequencies than the average mutation rate. A QTN in the LACV NSm gene was detected in SI+ mosquitoes, but not in I+ mosquitoes. Four amino acid changes were detected in the LACV NSm gene from SI+ but not I+ mosquitoes from one site, and may condition vector super infection. In contrast to NSm, the NSs sequences of LACV from SI+ and I+ mosquitoes were identical.

Conclusions

SI+ mosquitoes may represent stabilized infections of Ae. triseriatus mosquitoes, which could maintain LACV in nature. A gene-for-gene interaction involving the viral NSm gene and a vector innate immune response gene may condition stabilized infection.     

Serendipitous identification of natural Intergenotypic recombinants of hepatitis C in Ireland

Background

Encephalitis caused by flaviviruses, Japanese encephalitis virus (JEV) and West Nile virus (WNV) is responsible for significant morbidity and mortality in many endemic countries. Dengue-2 (Den-2) virus is a recent addition to the list of encephalitogenic viruses, after its Central Nervous System (CNS) invasion capability has been established. There is a wide array of laboratory tools that have helped us not only in the diagnosis of these conditions but also in understanding their pathogenesis and pathology. However, there are no reports of Shell Vial Culture (SVC), a centrifuge enhanced tissue culture assay that has revolutionized viral culturing in terms of rapidity and sensitivity being optimized for these flaviviral encephalitic conditions. The present study is an attempt to standardize and evaluate the usefulness of SVC for the laboratory diagnosis of JE, WN and Den-2 encephalitis cases and to compare it with Indirect Immunofluorescence (IIF) technique that detects cell associated virus antigen. Analysis of the various clinical parameters with respect to viral etiology has also been carried out.

Results

Pediatric patients constituted the major group involved in the study (92%). Etiological diagnosis of viral encephalitis could be established in twenty nine (58%) patients. JE encephalitis was the commonest with 19 (39%) cases being positive followed by, WN (9 cases-18%) and Den-2 (one case). IIF test could detect antigens of JE, WN and Den-2 viruses in 16(32%), 7(14%) and 1 case respectively. Shell vial culture assay picked up all cases that were positive by IIF test. In addition, SVC assay could detect 3 and 2 more cases of JE and WN encephalitis respectively, that were negative by the IIF test.

Conclusion

Shell vial culture is a rapid and efficient tool for the etiological diagnosis of JE, WN and Den-2 encephalitis cases. Early, prompt collection, transport and processing of the CSF samples, would make SVC a better method for the rapid diagnosis of these flaviviral infections.     

Hepatitis C virus resistance to interferon therapy: an alarming situation

Hepatitis C virus is presently a major public health problem across the globe. The main objective in treating hepatitis C virus (HCV) infection is to achieve a sustained virological response (SVR). Interferon-α (IFN-α) and pegylated interferon (PegIFN) in combination with Ribavirin (RBV) are the choice of treatment nowadays against chronic hepatitis C. There are several mechanisms evolved by the hepatitis C virus that facilitate the persistence of virus and further lead the patient’s status as non responder. Various factors involved in patient’s lack ofresponse to the therapy include: (1) viral factors, (2) host factors, (3) molecular mechanisms related to the lack of response and (4) social factors. Herein we have made an attempt to summarize all the related predictors of drug resistance in one article so that the future polices can be planned to overcome this obstacle and potential therapies can be designed by considering these factors.     

Serological evidence of California group and Cache Valley virus infection in Minnesota white-tailed deer

Blood samples were obtained from 138 white-tailed deer (Odocoileus virginianus) harvested at three sites surrounding the greater Minneapolis-St. Paul, Minnesota, metropolitan area (USA) and tested for neutralizing antibody to Cache Valley virus and three California serogroup (Jamestown Canyon, La Crosse, trivittatus) viruses (Bunyaviridae). Deer at each site had neutralizing antibody to one or more California serogroup viruses and/or Cache Valley virus. The majority of adult deer (85%) had antibody to both a California serogroup virus and Cache Valley virus. Antibody prevalence varied significantly with age of the deer. Fawns had a significantly lower prevalence of antibody to either a California serogroup (17%) or Cache Valley virus (39%) than did older (greater than 1-yr-old) deer (89% for a California serogroup virus and 91% for Cache Valley virus). The geometric mean titers of antibody in fawns to California serogroup (1:6) and Cache Valley viruses (1:17) were also less than that seen in older animals (1:11 and 1:28 for California serogroup and Cache Valley viruses, respectively). Of 76 older deer with antibody to the California serogroup, 91% had antibody specific for Jamestown Canyon virus. Jamestown Canyon is the primary California serogroup virus circulating in the suburban/rural Minneapolis-St. Paul area. Transmission occurs in an enzootic pattern similar to that documented in Indiana and Michigan. Cache Valley virus also appears to be enzootically transmitted in this area. However, the impact on domestic or wild animal populations is unknown.     

Coordinated regulation of autophagy and apoptosis determines endothelial cell fate during Dengue virus type 2 infection

Dengue is the most prevalent mosquito-borne viral disease in tropical regions. Severe cases may progress to Dengue hemorrhagic fever, suggesting vascular endothelial dysfunction in disease pathogenesis. In our previous study, we found that Dengue virus type 2 (DENV2) induced apoptosis of vascular endothelial cells via FasL/Fas- and XIAP-associated factor 1 (XAF1)-dependent pathways. In this paper, we demonstrate that DENV2 can induce autophagy in primary human umbilical vein endothelial cells (HUVECs) and the human umbilical vein endothelial cell line EA.hy926. Inhibition of autophagy with 3-methyl adenine promoted apoptosis, while inhibition of apoptosis with Z-VAD-FMK facilitated autophagy in DENV2-infected HUVECs and EA.hy926 cells. Interferon-alpha-inducible protein 6 (IFI6), a putative apoptosis regulator, inhibited DENV2-induced autophagy in EA.hy926 cells, while XAF1, an inhibitor of anti-apoptotic XIAP, facilitated autophagy. Molecular regulators of apoptosis and autophagy interact at multiple levels to determine cell fate. Our data suggest that XAF1 and IFI6 are involved in regulating the balance between autophagy and apoptosis in DENV2-infected endothelial cells.     

A bibliometric investigation of life cycle assessment research in the web of science databases

Purpose

Over the past few decades, life cycle assessment (LCA) methodologies have been developed extensively, and there has been a growing interest in LCA research. However, as attested by scientific literature, few systematic, synthesizing, and visualizing studies have been found on LCA research which show how this field has evolved over time. The goal of this mainly bibliometric, empirical study is to get insight into publication performance of global LCA research, characterize its intellectual structure, and trace its evolution by using the bibliometric method with visual mapping.

Methods

Based on the data from the ISI Web of Science databases Science Citation Index Expanded (SCI-EXPANDED), Social Sciences Citation Index (SSCI), Conference Proceedings Citation Index—Science (CPCI-S) and Conference Proceedings Citation Index —Social Science & Humanities (CPCI-SSH) in the period of 1998–2013, bibliometric methods are used to investigate general development profiles of LCA research, while knowledge domain visualization technologies are employed to conduct a further co-citation analysis.

Results and discussion

The results and discussions of this research mainly shed light on (1) basic statistics of significant publication performances, (2) research focuses and their intellectual base in LCA research, (3) how the streams of research evolved during the whole period of interest.

Conclusions

A new work on systematic and synthesizing study is conducted in this research to evaluate and map LCA research-related context. Some salient scholarly journals and institutions are identified that have shown a significant impact during the exponential growth of LCA research in the past 16 years. Biofuel, process design, solid waste management, and livestock production-related LCA researches are the main areas where interest is surging, confirmed by the active citers in each specialty. Furthermore, from the perspective of science mapping, evolution of LCA research is traced and some pivot publications are identified, which work as structural holes for the LCA-research development in the given time window.     

The properties of a few isolates of the sour cherry necrotic ringspot virus

Results of the investigation of four isolates of the sour cherry necrotic ringspot virus are presented in this paper. The isolates used caused characteristic symptoms on woody indicators “Bing”, “Montmorency”, F 12/1, and on peach seedlings. The virus was transmitted mechanically to some herbaceous species:Antirrhinum majus, Cucumis sativus, Cucurbita maxima, Chenopodium quinoa Crotalaria juncea, Momordica balsamina, Petunia hybrida andLeonorus sibiricus. The attempts to transmit the virus mechanically to further 23 herbaceous species were unsuccessful. The thermal inactivation point of the virus lies between 46 and 58°C and the dilution end-point between 10^?1 and 10^?2. The virus is stable in vitro at room temperature for more than one day. Individual virus isolates gave a positive immunological reaction with the Fulton’s “G” antiserum.     

I. Genetic polymorphisms in «La Jara» region (Toledo,Spain) Blood group systems A1A2BO,Lewis and Secretor character

Saliva and blood samples of 257 unrelated blood donors of both sexes, proceeding from La Jara region, were tested for a total of seven genetic polymorphisms, some of these results having been already published before (Calderón e Ibáñez, 1983). The gene frequencies obtained: (ABO: r=0.62887; p₁ =0.21950; p₂=0.06799; q=0.8365), (Lewis: Le=0.6257; le=0.3743) and (Secretor: Se=0.5910; se=0.4090-which is the lowest frequency found in Spain so far), are compared with those found in some other populations, both from Spain and from abroad.     

Partial nucleotide sequence of the Murray Valley encephalitis virus genome. Comparison of the encoded polypeptides with yellow fever virus structural and non-structural proteins

The sequence of 5400 bases corresponding to the 5'-terminal half of the Murray Valley encephalitis virus genome has been determined. The genome contains a 5' non-coding region of about 97 nucleotides, followed by a single continuous open reading frame that encodes the structural proteins followed by the non-structural proteins. Amino acid sequence homology between the Murray Valley encephalitis and yellow fever (Rice et al., 1985) polyproteins is 42% over the region sequenced. The start points of the various Murray Valley encephalitis virus-coded proteins have been assigned on the basis of this homology and a consistent set of potential proteolytic cleavage sites identified, the sequences of which are similar in Murray Valley encephalitis and yellow fever. The deduced Murray Valley encephalitis gene order is 5'-C-prM (M)-E-NS1-ns2a-ns2b-NS3-3'. The genome organization of Murray Valley encephalitis and yellow fever appears to be identical and the sizes of the predicted virus-coded proteins similar between the two viruses. Both viruses encode a basic capsid protein followed by three glycoproteins; the glycoproteins appear to have the conventional topology of N terminus outside with a C-terminal membrane-spanning domain. There are conserved glycosylation sites in prM, the precursor to the M protein of the virion, and in NS1, a non-structural protein of uncertain function. The glycosylation sites in E, the major envelope protein of the virion, are not conserved as to position. We predict the existence, in flavivirus-infected cells, of two small, hydrophobic peptides, ns2a and ns2b, which show only limited amino acid sequence homology. Finally, about half of the amino acid sequence of NS3 has been obtained; NS3 is a hydrophilic non-structural protein that shows 55% amino acid sequence similarity between Murray Valley encephalitis and yellow fever over the region sequenced and is probably involved in RNA replication.     

Identification of quantitative trait loci for larval morphological traits in interspecific hybrids of Ochlerotatus triseriatus and Ochlerotatus hendersoni (Diptera: Culicidae)

Ochlerotatus triseriatus is the natural vector of La Crosse virus, a common cause of pediatric encephalitis in the United States; the closely related Ochlerotatus hendersoni transmits this virus at low frequency. Adults of these mosquito species are difficult to distinguish morphologically; however, the larval stages show species-specific differences in several characters. We identified genomic regions contributing to the differences between the larvae of these species through interspecific hybridizations. Quantitative trait loci (QTL) were identified by standard interval mapping techniques and by univariate marker association analyses. We examined 159 F₂ progeny from an Oc. hendersoni female by Oc. triseriatus male interspecific cross for variation in the number of saddle and siphon hair branches, attachment of the acus, and morphology of the anal papillae. At least one putative QTL was identified for each of the phenotypes examined. QTL most commonly mapped to linkage group (LG) III, although QTL were identified on LGI and LGII for three phenotypes each. Several of these QTL, and particularly those on LGIII, also map to genome regions controlling adult female body size and ability to orally transmit La Crosse virus. Further studies are required to elucidate the relationships among these traits and the impact they may have had on the ecological specialization and speciation of these mosquitoes.