Mosquitoes of western Uganda.

The mosquito fauna in many areas of western Uganda has never been studied and is currently unknown. One area, Bwamba County, has been previously studied and documented but the species lists have not been updated for >40 yr. This paucity of data makes it difficult to determine which arthropod-borne viruses pose a risk to human or animal populations. Using CO2 baited-light traps, from 2008 through 2010, 67,731 mosquitoes were captured at five locations in western Uganda including Mweya, Sempaya, Maramagambo, Bwindi (BINP), and Kibale (KNP). Overall, 88 mosquito species, 7 subspecies, and 7 species groups in 10 genera were collected. The largest number of species was collected at Sempaya (65 species), followed by Maramagambo (45), Mweya (34), BINP (33), and KNP (22). However, species diversity was highest in BINP (Simpson's Diversity Index 1-D = 0.85), followed by KNP (0.80), Maramagambo (0.79), Sempaya (0.67), and Mweya (0.56). Only six species Aedes (Aedimorphus) cumminsii (Theobald), Aedes (Neomelaniconion) circumluteolus (Theobald), Culex (Culex) antennatus (Becker), Culex (Culex) decens group, Culex (Lutzia) tigripes De Grandpre and De Charmoy, and Culex (Oculeomyia) annulioris (Theobald), were collected from all five sites suggesting large differences in species composition among sites. Four species (Aedes (Stegomyia) metallicus (Edwards), Anopheles (Cellia) rivulorum Leeson, Uranotaenia (Uranotaenia) chorleyi (Edwards), and Uranotaenia (Uranotaenia) pallidocephala (Theobald) and one subspecies (Aedes (Stegomyia) aegypti formosus (Walker)) were collected in Bwamba County for the first time. This study represents the first description of the mosquito species composition of Mweya, Maramagambo, BINP, and KNP. A number of morphological variations were noted regarding the postspiracular scales, hind tibia, and sternites that make Culex (Culex) neavei (Theobald) challenging to identify. At least 50 species collected in this study have previously been implicated in the transmission of arboviruses of public health importance suggesting a high potential for maintenance and transmission of a wide variety of arboviruses in western Uganda.

Genetically engineered resistance to dengue-2 virus transmission in mosquitoes.

The control of arthropod-borne virus diseases such as dengue may ultimately require the genetic manipulation of mosquito vectors to disrupt virus transmission to human populations. To reduce the ability of mosquitoes to transmit dengue viruses, a recombinant Sindbis virus was used to transduce female Aedes aegypti with a 567-base antisense RNA targeted to the premembrane coding region of dengue type 2 (DEN-2) virus. The transduced mosquitoes were unable to support replication of DEN-2 virus in their salivary glands and therefore were not able to transmit the virus.

Mosquitoes of Northwestern Uganda.

Despite evidence of arbovirus activity in northwestern Uganda (West Nile Sub-region), there is very limited information on the mosquito fauna of this region. The only published study reported 52 mosquito species in northwestern Uganda but this study took place in 1950 and the information has not been updated for more than 60 yr. In January and June 2011, CO2 baited-light traps were used to collect 49,231 mosquitoes from four different locations, Paraa (9,487), Chobe (20,025), Sunguru (759), and Rhino Camp (18,960). Overall, 72 mosquito species representing 11 genera were collected. The largest number of distinct species was collected at Chobe (43 species), followed by Paraa (40), Sunguru (34), and Rhino Camp (25). Only eight of the 72 species (11.1%) were collected from all four sites: Aedes (Stegomyia) aegypti formosus (Walker), Anopheles (Cellia) funestus group, Culex (Culex) decens group, Cx. (Culex) neavei Theobald, Cx. (Culex) univittatus Theobald, Cx. (Culiciomyia) cinereus Theobald, Cx. (Oculeomyia) poicilipes (Theobald), and Mansonia (Mansonoides) uniformis (Theobald). Fifty-four species were detected in northwestern Uganda for the first time; however, these species have been detected elsewhere in Uganda and do not represent new introductions to the country. Thirty-three species collected during this study have previously been implicated in the transmission of arboviruses of public health importance.

Study of viremic profile in febrile specimens of chikungunya in Bandung, Indonesia.

BACKGROUND: Data regarding the viremia profile of chikungunya virus (CHIKV) infected patients especially during the pre-febrile period is limited. OBJECTIVE: To obtain virological kinetic data on CHIKV infections. STUDY DESIGN: A two-week community observation for dengue transmission was conducted in Bandung, Indonesia, from 2005 to 2009. Acute specimens from non-dengue febrile patients were screened by pan-alphavirus conventional RT-PCR. The positives were confirmed for CHIKV RNA by a specific RT-PCR followed by sequencing. Simultaneously these specimens were also cultured in Vero cells and tested for anti-CHIK IgM MAC-ELISA. All the available serial specimens,including the pre-febrile specimens, from confirmed CHIK cases, were tested by virus isolation, RT-PCR, qRT-PCR, and CHIK IgM ELISA. RESULTS: There were five laboratory confirmed CHIK cases identified and studied. Among these, viremia was determined to extend from as early as 6 days prior to until 13 days post fever onset. Quantitative RT-PCR showed viremia peaked at or near onset of illness. CONCLUSION: In this study, individuals were identified with viremia prior to fever onset and extending beyond the febrile phase. This extended viremic phase has the potential to impact transmission dynamics and thus the public health response to CHIK outbreaks.

Intracellular immunization of mosquito cells to LaCrosse virus using a recombinant Sindbis virus vector.

A cDNA of the small RNA genome segment of La Crosse (LAC) virus was inserted, in an antisense orientation, into a double subgenomic Sindbis (dsSIN) virus expression vector generating pTE/3'2J/ANTI-S (15,000bp). In vitro transcription of the pTE/3'2J/ANTI-S template generated genomic RNA that was electrotransfected into BHK-21 cells to produce virus. Northern blot analysis of RNA isolated from infected Aedes albopictus (C6/36) cells showed that the TE/3'2J/ANTI-S virus produced a subgenomic mRNA of the appropriate size, indicating transcription of the LAC cDNA segment. C6/36 cells were infected with either TE/3'2J/ANTI-S, TE/3'2J (a dsSIN virus with no LAC insert), or wild type Sindbis (SIN, strain AR339) viruses and subsequently challenged with LAC virus. LAC virus titers were determined using a capture antibody ELISA. Mosquito cells infected with TE/3'2J/ANTI-S virus yielded at least 4 log10 TCID50/ml less LAC virus than cells infected with either TE/3'2J or AR339 SIN viruses. The use of the infectious SIN virus expression vectors provides a novel approach for high level cytoplasmic expression of genes or sequences of interest in arthropod cells, and for evaluating strategies for intracellular immunization against arboviruses.

Detection of expressed chloramphenicol acetyltransferase in the saliva of Culex pipiens mosquitoes.

Mosquito salivary glands play an important role in the transmission of arthropod-borne pathogens. The ability to express genes in mosquitoes would be a powerful approach to characterize salivary gland genes, and to reveal important vector determinants of pathogen transmission. Here we report the use of a double subgenomic Sindbis (dsSIN) virus, designated TE/3'2J/CAT, and a packaged Sindbis replicon virus, designated rep5/CAT/26S, to express chloramphenicol acetyltransferase (CAT) protein in the salivary glands and saliva of transduced female Culex pipiens pipiens. Indirect immunofluorescence analysis revealed that salivary glands of these mosquitoes infected with either TE/3'2J/CAT or rep5/CAT/26S virus (4 or 6 days post-infection (p.i.)) were positive for both SIN E1 antigen and CAT protein. Saliva collected from mosquitoes transduced with TE/3'2J/CAT virus contained a unique 25 kDa protein that corresponded to the size of CAT protein. Additionally, CAT activity assays revealed that saliva collected from mosquitoes transduced with either TE/3'2J/CAT or rep5/CAT/26S virus could contain greater than 5.0 x 10(-5) units of CAT enzyme (3.0 x 10(6) CAT trimers).

Genetic determinants of Venezuelan equine encephalitis emergence.

Following a period of inactivity from 1973-1991, Venezuelan equine encephalitis (VEE) reemerged during the past decade in South America and Mexico. Experimental studies of VEE virus (VEEV) infection of horses with virus strains isolated during these outbreaks have revealed considerable variation in the ability of equine-virulent, epizootic strains to exploit horses as efficient amplification hosts. Subtype IC strains from recent outbreaks in Venezuela and Colombia amplify efficiently in equines, with a correlation between maximum viremia titers and the extent of the outbreak from which the virus strain was isolated. Studies of enzootic VEEV strains that are believed to represent progenitors of the epizootic subtypes support the hypothesis that adaptation to efficient replication in equines is a major determinant of emergence and the ability of VEEV to spread geographically. Correlations between the ability of enzootic and epizootic VEEV strains to infect abundant, equiphilic mosquitoes, and the location and extent of these outbreaks, also suggest that specific adaptation to Ochlerotatus taeniorhynchus mosquitoes is a determinant of some but not all emergence events. Genetic studies imply that mutations in the E2 envelope glycoprotein gene are major determinants of adaptation to both equines and mosquito vectors.

Isolation and genetic characterization of a hantavirus (Bunyaviridae: Hantavirus) from a rodent, Oligoryzomys microtis (Muridae), collected in northeastern Peru.

This paper describes the isolation and partial genetic characterization of a hantavirus from a pygmy rice rat, Oligoryzomys microtis, collected within the urban area of Iquitos, Loreto Department, Peru. The virus, designated HTN-007, exhibited the highest degree of genetic similarity to Rio Mamore virus, which was originally described from the same rodent species in eastern Bolivia. Comparison of small and medium segment nucleotide sequence data from HTN-007 and Rio Mamore virus revealed 87% and 85% sequence identity, respectively. Based on these analyses, HTN-007 appears to be a variant of Rio Mamore virus. As such, it represents the first successful isolation of Rio Mamore virus and the first evidence for the existence of a hantavirus in Peru. Serologic studies done by immunofluorescence on blood samples of 56 O. microtis trapped at the collection site indicated that 21.4% had antibodies to hantavirus. In view of the proximity of this rodent species to humans and the close phylogenetic relationship of Rio Mamore virus to hantaviruses that have been associated with human disease, Rio Mamore virus may be a hantavirus of some public health importance in tropical South America.

Virulence and viremia characteristics of 1992 epizootic subtype IC Venezuelan equine encephalitis viruses and closely related enzootic subtype ID strains.

Following a 19-year hiatus, Venezuelan equine encephalitis (VEE) reemerged in western Venezuela in December 1992. This outbreak is important in understanding VEE emergence because phylogenetic studies imply that sympatric, enzootic, subtype ID VEE viruses mutated to generate the epizootic/epidemic. Although the 1992-1993 strains belong to subtype IC, a serotype implicated in extensive outbreaks during the 1960s and in 1995, relatively small numbers of human and equine cases occurred in 1992-1993. We, therefore, evaluated the pathogenicity of these Venezuelan enzootic ID and epizootic IC viruses to determine 1) if they exhibit phenotypes like those described previously for more distantly related enzootic and epizootic strains, and 2) if the 1992-1993 outbreak was limited by the inability of these IC viruses to exploit equines as amplification hosts. All strains were virulent in mice and guinea pigs, but were benign for cotton rats, natural hosts of enzootic viruses. However, only the IC strains produced equine disease, with mean peak viremias of 10(5) suckling mouse 50% lethal doses per mL serum, and some titers exceeding 10(7). These viremias approximate those observed previously with VEE strains isolated during more extensive epizootics, suggesting that efficient equine amplification did not limit the scope and duration of the 1992-1993 outbreak. Enzootic ID virus infection protected all horses from challenge with epizootic strain P676, supporting the hypothesis that epizootics bypass regions of enzootic transmission due to natural immunization of equines by enzootic VEE viruses.

Genetic and antigenic diversity among eastern equine encephalitis viruses from North, Central, and South America.

Eastern equine encephalitis virus (EEEV), the sole species in the EEE antigenic complex, is divided into North and South American antigenic varieties based on hemagglutination inhibition tests. Here we describe serologic and phylogenetic analyses of representatives of these varieties, spanning the entire temporal and geographic range available. Nucleotide sequencing and phylogenetic analyses revealed additional genetic diversity within the South American variety; 3 major South/Central American lineages were identified including one represented by a single isolate from eastern Brazil, and 2 lineages with more widespread distributions in Central and South America. All North American isolates comprised a single, highly conserved lineage with strains grouped by the time of isolation and to some extent by location. An EEEV strain isolated during a 1996 equine outbreak in Tamaulipas State, Mexico was closely related to recent Texas isolates, suggesting southward EEEV transportation beyond the presumed enzootic range. Plaque reduction neutralization tests with representatives from the 4 major lineages indicated that each represents a distinct antigenic subtype. A taxonomic revision of the EEE complex is proposed.

Trocara virus: a newly recognized Alphavirus (Togaviridae) isolated from mosquitoes in the Amazon Basin.

This report describes Trocara virus, a newly recognized member of the genus Alphavirus, that has been isolated from Aedes serratus mosquitoes collected at two widely separated sites in the Amazon Basin. Biological, antigenic and genetic characteristics of the new virus are given. Results of these studies indicate that Trocara virus is the first member of a newly discovered antigenic complex within the family Togaviridae genus Alphavirus. The public health and veterinary importance of Trocara virus is still unknown.

Motivations for condom use: do pregnancy prevention goals undermine disease prevention among heterosexual young adults?

Differences in motives for condom use and their implications for understanding frequency of use were investigated in a random, biracial (Black, White) sample of heterosexuals, aged 17 to 25 years (n = 902). Results indicated that sexually active young adults-regardless of race, age, gender, or relationship status-were more likely to use condoms to prevent pregnancy than to prevent disease. Reasons for use mediated the effects of relationship status on condom use per se and moderated the effects of attitudinal and perceptual variables on condom use. Finally, comparisons among condom users motivated by different prevention goals and nonusers (n = 388) revealed that differences among user subgroups were nearly as numerous and, in some cases, more robust than differences between users and nonusers. These findings indicate that condom users comprise distinct subgroups, defined in part by their underlying motives for use, and highlight important conceptual and empirical reasons to distinguish among them.

PIP: This study explores the implications of using condoms for pregnancy prevention versus disease prevention, and examines intervention efforts aimed at understanding and changing condom use behaviors. Differences in motives for condom use and their implications for understanding frequency of use were investigated in a random, biracial (Black, White) sample of heterosexuals. A total of 902 respondents residing in Buffalo, New York, aged 17-25 years were interviewed. Results indicated that sexually active young adults were more likely to use condoms to prevent pregnancy than to prevent disease, regardless of race, age, gender, or relationship status. Reasons for use mediated the effects of relationship status on condom use per se and moderated the effects of attitudinal and perceptual variables on condom use. In addition, comparisons among condom users motivated by different prevention goals and nonusers (388 heterosexuals) revealed that differences among user subgroups were nearly as numerous and, in some cases, more forceful than differences between users and nonusers. Overall, these findings indicate that condom users comprise distinct subgroups, defined in part by their underlying motives for use, and underscore important conceptual and empirical reasons to distinguish among them.

Motivations for sex and risky sexual behavior among adolescents and young adults: a functional perspective.

The implications of a functionalist perspective for understanding sexual risk taking are explored. Key motivational dimensions thought to underlie human behavior (viz., approach vs. avoidance, autonomy vs. relatedness) were used to identify 4 broad domains of sexual motivations and to develop a measure of specific motives within each of these domains. Data from both college student and community samples are used to demonstrate the psychometric adequacy of these scales and to show that having sex for different reasons predicts distinctive patterns of sexual risk taking both cross-sectionally and longitudinally: that selection into specific types of sexual relationships partially mediates these effects; and that these needs may be differentially expressed, or even suppressed, depending on relationship context. Results provide strong support for the functionalist perspective on behavior and indicate that an adequate understanding of sexual risk-taking behavior must take into account the various needs and goals that such behavior can serve.

PIP: Despite knowing how HIV is transmitted, many young people still engage in HIV/AIDS risk behavior. The implications of a functionalist perspective for understanding such sexual risk-taking are explored. Key motivational dimensions thought to underlie human behavior were used to identify 4 broad domains of sexual motivation and develop a measure of specific motives within each domain. Data from both college student and community samples are used to demonstrate the psychometric adequacy of these scales and that having sex for different reasons predicts distinctive patterns of sexual risk-taking both cross-sectionally and longitudinally; that selection into specific types of sexual relationships partially mediates those effects; and that those needs may be differentially expressed, or even suppressed, depending upon relationship context. Strong support is provided for the functionalist perspective on behavior and indicates that an adequate understanding of sexual risk-taking must consider the various needs and goals which such behavior can serve.

Molecular epidemiological studies of veterinary arboviral encephalitides.

Recent studies using molecular genetic approaches have made important contributions to our understanding of the epidemiology of veterinary arboviral encephalitides. Viruses utilizing avian enzootic hosts, such as Western equine encephalitis virus (WEEV) and North American Eastern equine encephalitis virus (EEEV), evolve as relatively few, highly conserved genotypes that extend over wide geographic regions; viruses utilizing mammalian hosts with more limited dispersal evolve within multiple genotypes, each geographically restricted. Similar findings have been reported for Australian alphaviruses. This difference may be related to vertebrate host relationships and the relative mobility of mammals and avians. Whereas EEEV and Venezualan equine encephalitis virus (VEEV) utilize small mammalian hosts in the tropics, most WEEV genotypes probably utilize avian hosts in both North and South America. The ability of mobile, infected avian hosts to disperse alphaviruses may result in continual mixing of virus populations, and thus limit diversification. This high degree of genetic conservation is also exhibited by EEE and Highlands J viruses in North America, where passerine birds serve as amplifying hosts in enzootic transmission foci. Most equine arboviral pathogens, including EEEV, WEEV and Japanese encephalitis virus (JEV), occur in a naturally virulent enzootic state and require only appropriate ecological conditions to cause epizootics and epidemics. However, VEE epizootics apparently require genetic changes to convert avirulent enzootic strains into distinct epizootic serotypes. All of these arboviruses have the potential to cause severe disease of veterinary and human health importance, and further molecular epidemiological studies will undoubtedly improve our ability to understand and control future emergence.

Isolation of chikungunya virus from Aedes aegypti mosquitoes collected in the town of Yawat, Pune District, Maharashtra State, India.

Chikungunya (CHIK) virus is prevalent throughout Southeast Asia and Africa. It has caused numerous large outbreaks in India. No active or passive surveillance has been carried out since the last epidemic occurring in 1971. During a recent outbreak of Dengue (DEN)-like illness in eastern India, Aedes aegypti mosquitoes collected from the affected area were positive for CHIK virus. Evidence of dual infection with CHIK and DEN typel virus was also obtained. A widely circulating low-virulent CHIK virus is a possible explanation for the epidemiological pattern of the CHIK virus disease in this region.

Alphaviruses.

Alphaviruses remain important emerging mosquito-borne, zoonotic pathogens that cause both localized human outbreaks and epizootics (e.g., Venezuelan equine encephalitis) and large human epidemics (e.g., Chikungunya). Alphaviruses are globally dispersed, and each continent has humans at risk from one or more of these arthropod-borne viruses (arboviruses). Symptoms of human alphaviral disease range from frank, severe encephalitis (e.g., eastern and western equine encephalitis) to polyarthritis (e.g., Ross River). Diagnostic techniques to identify human alphaviral infections have changed dramatically with the development and implementation of standardized nucleic acid amplification tests (NAAT). The NAAT is rapidly replacing virus isolation and typing using indirect fluorescent antibody (IFA) assay with monoclonal antibodies (MAbs) as the preferred method of virus identification. The older techniques still have value, however, since alphaviral growth in cell culture is rapid, and IFA with MAbs is inexpensive. This chapter provides detailed, standardized protocols for the identification of alphaviruses from clinical specimens and the serological characterization of human infection-immune sera. Both laboratory approaches are needed to identify and confirm human infections with these agents.

Tracking epidemic Chikungunya virus into the Indian Ocean from East Africa.

The largest documented outbreak of Chikungunya virus (CHIKV) disease occurred in the Indian Ocean islands and India during 2004-2007. The magnitude of this outbreak led to speculation that a new variant of the virus had emerged that was either more virulent or more easily transmitted by mosquito vectors. To study this assertion, it is important to know the origin of the virus and how the particular strain circulating during the outbreak is related to other known strains. This study genetically characterized isolates of CHIKV obtained from Mombasa and Lamu Island, Kenya, during 2004, as well as strains from the 2005 outbreak recorded in Comoros. The results of these analyses demonstrated that the virus responsible for the epidemic that spread through the Indian Ocean originated in coastal Kenya during 2004 and that the closest known ancestors are members of the Central/East African clade. Genetic elements that may be responsible for the scope of the outbreak were also identified.