Genome-scale phylogeny of the alphavirus genus suggests a marine origin.

The genus Alphavirus comprises a diverse group of viruses, including some that cause severe disease. Using full-length sequences of all known alphaviruses, we produced a robust and comprehensive phylogeny of the Alphavirus genus, presenting a more complete evolutionary history of these viruses compared to previous studies based on partial sequences. Our phylogeny suggests the origin of the alphaviruses occurred in the southern oceans and spread equally through the Old and New World. Since lice appear to be involved in aquatic alphavirus transmission, it is possible that we are missing a louse-borne branch of the alphaviruses. Complete genome sequencing of all members of the genus also revealed conserved residues forming the structural basis of the E1 and E2 protein dimers.

Silent circulation of arboviruses in Cameroon.

OBJECTIVES: To investigate the silent circulation and transmission of arthropod-borne viruses (arboviruses) in the Fako Division of Cameroon. DESIGN: This survey was conducted based on clinical observations and laboratory diagnosis; field collections of mosquitoes. SETTING: This study was conducted in the Fako Division of South West Cameroon. SUBJECTS: One hundred and two sera were obtained from febrile patients (with negative laboratory findings for malaria and typhoid fever) at clinics in the Fako Division, and diurnal anthropophilic mosquitoes (4,764) collected. INTERVENTIONS: Virus isolation was attempted from these, and sera were screened for antibodies against 18 African arboviruses by haemagglutination inhibition (HI) and complement fixation (CF) tests. RESULTS: No virus was isolated. Fifty three of 79 (67.1%) sera reacted with one or more viral antigens. Twenty nine sera (36.7%) reacted with members of the genus Alphavirus, with Chikungunya (CHIKV) and O'nyong-nyong (ONNV) viruses as the most frequent (34.2%). Forty six sera (58.2%) reacted with members of the genus Flavivirus: 24 (30.4%) were cross-reactive, but 11.4% reacted monotypically with Zika, 5.1% with yellow fever virus (YFV), 5.1% with dengue virus-2 (DENV-2), 2.5% with DENV-1 and 1.3% with Wesselsbron virus, respectively. The plaque reduction neutralisation test used to specify the agent that elicited the response could not resolve 33.3% of the cross reactions between CHIKV and ONNV. Neutralising antibody titres against ONNV and CHIKV were very high indicating probable re-infection. CONCLUSION: Our results indicate previously undetected circulation of arboviruses in Cameroon, and suggest that they are important, overlooked public health problems.

Vesicular stomatitis virus (Indiana serotype): transovarial transmission by phlebotomine sandlies.

Transovarial transmission of vesicular stomatitis virus (Indiana serotype) by experimentally infected Lutzomyia trapidoi and Lutzomyia ylephiletrix to their progeny was demonstrated. Virus was recovered from all developmental stages; mean virus titers from egg to first generation adult showed a four-log increase, indicating that virus multiplication occurred during development of the sandflies. Virus titers in first generation adult females were comparable to those found in their parents. These infected female sandflies transmitted vesicular stomatitus virus Indiana by bite to susceptible animals and transmitted the virus transovarially to their offspring (second generation). Results demonstrate a possible mechanism for transmission and maintenance of this virus in nature without a vertebrate (heat) host reservoir.

A novel vasodilatory peptide from the salivary glands of the sand fly Lutzomyia longipalpis.

Salivary gland lysates of the sand fly Lutzomyia longipalpis contain a potent vasodilator that aids the fly to feed on the blood of its vertebrate hosts. Chromatographic analysis, antibody reactivity, and data obtained from bioassays of the salivary erythema-inducing factor indicate striking similarity with human calcitonin gene-related peptide. The erythema-inducing factor is, however, at least one order of magnitude more potent than calcitonin gene-related peptide.

Transovarial transmission of Japanese encephalitis virus by mosquitoes.

Female Aedes albopictus and Aedes togoi mosquitoes infected with Japanese encephalitis virus either by intrathoracic inoculation or by ingestion of a virus-sucrose-erythrocyte mixture transmitted the virus to a small percentage of their F1 progeny. Adult F1 female Aedes albopictus thus infected transmitted the virus in turn to newly hatched chickens by feeding on them.

Seroepidemiology of selected alphaviruses and flaviviruses in bats in Trinidad.

A serosurvey of antibodies against selected flaviviruses and alphaviruses in 384 bats (representing 10 genera and 14 species) was conducted in the Caribbean island of Trinidad. Sera were analysed using epitope-blocking enzyme-linked immunosorbent assays (ELISAs) specific for antibodies against West Nile virus (WNV), Venezuelan equine encephalitis virus (VEEV) and eastern equine encephalitis virus (EEEV), all of which are zoonotic viruses of public health significance in the region. Overall, the ELISAs resulted in the detection of VEEV-specific antibodies in 11 (2.9%) of 384 bats. Antibodies to WNV and EEEV were not detected in any sera. Of the 384 sera, 308 were also screened using hemagglutination inhibition assay (HIA) for antibodies to the aforementioned viruses as well as St. Louis encephalitis virus (SLEV; which also causes epidemic disease in humans), Rio Bravo virus (RBV), Tamana bat virus (TABV) and western equine encephalitis virus (WEEV). Using this approach, antibodies to TABV and RBV were detected in 47 (15.3%) and 3 (1.0%) bats, respectively. HIA results also suggest the presence of antibodies to an undetermined flavivirus(es) in 8 (2.6%) bats. Seropositivity for TABV was significantly (P<0.05; χ2) associated with bat species, location and feeding preference, and for VEEV with roost type and location. Differences in prevalence rates between urban and rural locations were statistically significant (P<0.05; χ2) for TABV only. None of the aforementioned factors was significantly associated with RBV seropositivity rates.

Size-conserved chromosomes and stability of molecular karyotype in cloned stocks of Leishmania major.

Molecular karyotypes for 5 stocks of Leishmania major were derived by pulsed-field gradient gel electrophoresis and transverse alternating field electrophoresis. Chromosome sizes obtained by the two methods agreed within less than or equal to 50kb. A set of 10 size-concordant chromosome bands between approx. 350-1000 kb was found in all stocks, plus a variable number of polymorphic chromosomes. Cloned gene probes, and DNA purified from individual chromosomes, hybridized to individual size-concordant chromosomes in different stocks, indicating a high degree of sequence homology among bands of similar size. Since the stocks were isolated over a 25-year period in a wide geographic area, we interpret these size-conserved chromosomes to be characteristic of the L. major karyotype. We were unable to identify irreversible genomic rearrangements in Leishmania cloned from the midgut of sandflies or cultivated from the skins of infected mice, which might have explained the origin of the size-variable chromosomes. For stocks that are maintained in the laboratory, the molecular karyotype appears to be a stable characteristic of a cloned population of Leishmania.

RNA genome stability of Toscana virus during serial transovarial transmission in the sandfly Phlebotomus perniciosus.

We have carried out a T1 ribonuclease fingerprinting analysis of the RNA genomes of Toscana virus isolates from successive generations of an experimentally virus-infected laboratory colony of Phlebotomus perniciosus sandflies. This analysis detected no virus RNA genome changes during transovarial transmission of the virus over 12 sandfly generations (a period of almost 2 years). These results demonstrate that although RNA viruses can exhibit high rates of mutational change under a variety of conditions, Toscana virus RNA genomes can be maintained in a stable manner during repeated transovarial virus transmission in the natural insect host. The implications of these results for insect RNA virus evolution are discussed.

Jatobal virus is a reassortant containing the small RNA of Oropouche virus.

Jatobal (JAT) virus was isolated in 1985 from a carnivore (Nasua nasua) in Tucuruí, Pará state, Brazil and was classified as a distinct member of the Simbu serogroup of the Bunyavirus genus, family Bunyaviridae on the basis of neutralization tests. On the basis of nucleotide sequencing, we have found that the small (S) RNA of JAT virus is very similar (>95% identity) to that of Oropouche (ORO) virus, in particular, the Peruvian genotype of ORO virus. In comparison, limited nucleotide sequencing of the G2 protein gene, encoded by the middle (M) RNA, of JAT and ORO viruses, revealed relatively little identity (<66%) between these two viruses. Neutralization tests confirmed the lack of cross-reactivity between the viruses. These results suggest that JAT virus is a reassortant containing the S RNA of ORO virus. JAT virus was attenuated in hamsters compared to ORO virus suggesting that the S RNA of ORO virus is not directly involved in hamster virulence.

Isolation, characterization and geographic distribution of Caño Delgadito virus, a newly discovered South American hantavirus (family Bunyaviridae).

Rodents collected from the Venezuelan llanos (plains) during field studies of viral hemorrhagic fever were tested for evidence of hantavirus infection. Hantavirus antibody was found in one (7.7%) of 13 Oryzomys bicolor, one (3.4%) of 29 Rattus rattus, 10 (6.0%) of 166 Sigmodon alstoni and one (2.2%) of 45 Zygodontomys brevicauda. Hantavirus-specific RNA was detected in lung tissues from four antibody-positive rodents: two S. alstoni from Portuguesa State and one S. alstoni each from Cojedes and Barinas States. A hantavirus isolate (herein identified as VHV-574) was recovered from lung tissue from a hantavirus RNA-positive S. alstoni collected from Portuguesa State. The results of serological tests and analyses of small and medium RNA segment nucleotide sequence data indicated that VHV-574 represents a novel hantavirus (proposed name 'Caño Delgadito') that is distinct from all previously characterized hantaviruses. The results of analyses of nucleotide sequence data from the four hantavirus RNA-positive S. alstoni suggested that Caño Delgadito virus is widely distributed in the Venezuelan llanos.

A method for the isolation and identification of dengue viruses, using mosquito cell cultures.

An improved method for the isolation and identification of dengue viruses is described. Viruses were isolated in mosquito cell cultures (C6/36 or AP-61), identified by indirect fluorescent antibody technique, and typed by complement-fixation test, using the cell culture fluid as antigen. The sensitivity of this method was compared with mosquito inoculation in comparative titrations of 16 low passage dengue virus strains. Although lower virus titers were obtained by the mosquito cell culture technique, its decreased sensitivity was compensated for by the much larger volume (588X) which could be assayed. By incubating the mosquito cells at 32 degrees C, dengue viruses can be identified and typed within 6 days after inoculation.

The prevalence of encephalomyocarditis virus neutralizing antibodies among various human populations.

The prevalence of encephalomyocarditis virus (EMC) antibodies among selected human populations in various regions of the world was determined by the plaque reduction neutralization method. Antibody rates among children ranged from 1.0 to 33.9%, while those among adults varied from 3.2 to 50.6%. No differences between sexes were found in the frequency of EMC infection. The pattern of age-specific antibody rates observed among the study populations suggests that EMC infection occurs primarily during childhood. There appeared to be no association between the presence of EMC antibodies and potential exposure to rats. Sera from diabetic, suspected encephalitis, and myocarditis patients were also examined for EMC neutralizing antibodies. The prevalence of antibodies among these groups was not significantly different from that of control populations in the same geographic regions. No association was demonstrated between EMC infection and these three diseases. The results of this study indicate that EMC infection in man is fairly common but that most human cases are probably asymptomatic and/or unrecognized.

Control of zoonotic visceral leishmaniasis: is it time to change strategies?

Zoonotic visceral leishmaniasis (ZVL) is an important emerging parasitic disease. This article reviews the recommended control methods for the disease and concludes that they have only been partially effective. The continued endemicity of ZVL, its recent appearance in urban areas of Latin America, and its increasing importance as an opportunistic infection among persons infected with human immunodeficiency virus indicate that present control methods for the disease are ineffective and that new control strategies are needed. Prevention of the disease in dogs appears to be the best approach for interrupting the domestic cycle of ZVL. The most feasible approach would seem to be a canine vaccine that protects dogs from developing parasitemia and from becoming peridomestic reservoirs of the parasite.

A simple method for experimental infection of phlebotomine sand flies with Leishmania.

Mouse macrophages, grown in continuous cell culture at 37 degrees C, were inoculated with the promastigote stage of various human pathogenic Leishmania species. Under these culture conditions, the parasites rapidly entered the cells and transformed into amastigotes. Two or 3 days after inoculation, the infected macrophages were mixed with washed human erythrocytes and were fed to female sand flies (Phlebotomus papatasi and Lutzomyia longipalpis) through a chick skin membrane. Within 7-10 days after feeding, large numbers of promastigotes were observed in the anterior portion of the insects' guts, indicating that the infected sand flies were capable of transmitting the parasites by bite. This relatively simple and rapid technique should facilitate studies on the biology of Leishmania in their insect vectors. It also eliminates the need for animals as a source of amastigotes.

Clinical and serologic responses of volunteers infected with phlebotomus fever virus (Sicilian type).

Twenty volunteers were inoculated with various doses of human serum containing Phlebotomus fever virus (Sicilian type) to determine their clinical and serologic responses as well as the human infectious dose50 of the virus. All infected subjects developed fever which varied in duration from 6 to 74 hours. The most common symptoms during sandfly fever were headache, anorexia, myalgia, photophobia, low back and retro-orbital pain. Infected individuals developed a marked leukopenia characterized by an initial lymphopenia followed by protracted neutropenia. Little complement fixing antibody was detected in convalescent sera but most subjects developed significant rises in hemagglutination inhibiting antibodies. All infected subjects developed specific neutralizing antibodies with titers ranging from 1:40 to 1:2,560. Of the three serologic tests performed, the plaque reduction neutralization method appears to be the most sensitive test for detecting antibodies to Phlebotomus fever viruses.

Effect of insecticide spraying for malaria control on the incidence of sandfly fever in Athens, Greece.

Sera from 637 Athens residents of various age groups were examined by plaque reduction neutralization test for antibodies against Naples and Sicilian Phlebotomus fever viruses. A marked change in the prevalence of antibodies to both agents was observed in persons born after 1946, when residual insecticide spraying for malaria control was initiated in Greece. The prevalence of Naples and Sicilian neutralizing antibodies among residents greater than or equal to 30 years of age was 36% and 13%, respectively. In contrast, only 4% of persons less than or equal to 29 years of age had Naples antibodies and all were negative to Sicilian. These serologic data confirm previous clinical observations that sandfly fever becam uncommon in Athens after initiation of the insecticide spraying program. Presumedly the spraying program was effective in reducing the Phlebotomus population to levels where virus transmission was minimal. New information on the specificity and duration of Phlebotomus fever neutralizing antibodies is also presented.

Laboratory studies of transovarial transmission of La Crosse and other arboviruses by Aedes albopictus and Culex fatigans.

Transovarial transmission of La Crosse virus by experimentally infected Aedes albopictus females to 2.7% of their F1 generation offspring was demonstrated. Progeny of both sexes were infected. Mean virus titers in parent mosquitoes and infected F1 generation adults were 10(4.6) and 10(3.4) plaque forming units/insect, respectively. The La Crosse-infected offspring were randomly distributed among the female parents. After two serial passages in A. albopictus, a marked change occurred in the plaque morphology of the virus but this had no apparent effect on the subsequent vertical transmission rate. In contrast, transovarial transmission did not occur in La Crosse-infected Culex fatigans or in A. albopictus and C. fatigans infected with vesicular stomatitis-Indiana, Cache Valley, Batai, Arumowot, and Itaporanga viruses. Results of this experiment suggest that the La Crosse model might be useful in studying the mechanism of transovarial transmission in additional mosquito species.

The mechanism of arbovirus transovarial transmission in mosquitoes: San Angelo virus in Aedes albopictùs.

The mechanism of transovarial transmission of San Angelo (SA) virus in Aedes albopictus was investigated.A mosquito line with SA virus filial infection rates of almost 100% was developed by selection. Results of crosses and back-crosses between this transovarial transmission-efficient line and noninfected mosquitoes indicated that SA virus was transmitted in Ae. albopictus by maternal inheritance. The infection status of the male parent was of no consequence; the virus was passed from generation to generation through the females. Transovarially infected mosquitoes contained less virus than insects infected by inoculation. The behavior of SA virus in Ae. albopictus was similar to that of sigma virus in Drosophila melanogaster, suggesting that some females in the transovarial transmission-efficient line had developed a chronic infection of their germinal cells (oogonia). Serial transovarial passage of SA virus in Ae. albopictus did not alter its plaque morphology, infectivity for mosquitoes, or pathogenicity for vertebrates. Transovarially infected mosquitoes transmitted the virus by bite to mice.

The location of San Angelo virus in developing ovaries of transovarially infected Aedes albopictus mosquitoes as revealed by fluorescent antibody technique.

Aedes albopictus adult female mosquitoes, transovarially infected with San Angelo (SA) virus, were examined by fluorescent antibody technique during various stages of ovarian development to determine how the virus enters the egg. Upon emergence from the day of adulthood, it was visible in the follicular epithelium, oocytes and nurse cells of the primary follicles. In the 72-hour period between the ingestion of blood and oviposition, there was a marked increase in the amount of viral antigen in the oocyte, indicating rapid virus accumulation. After oviposition, SA viral antigen was also seen in the secondary ovarian follicles. The observed sequence of infection of the mosquito ovariole with SA virus is analogous to that described with certain endosymbionts of insects.

Maintenance of Toscana virus in Phlebotomus perniciosus by vertical transmission.

Toscana virus was maintained in a laboratory colony of Phlebotomus perniciosus by vertical (transovarial) transmission for 13 consecutive generations over a 23-month period. No significant biological changes were noted in the virus after prolonged vertical passage in the sand flies, and transovarially infected females were able to transmit the agent by bite to susceptible animals. Chronic infection of Ph. perniciosus with Toscana virus had no apparent effect on the insects' rate of eclosion. In the absence of selection and with random matings, the virus infection rates in each subsequent generation of the colony decreased, suggesting that Toscana virus cannot be maintained in Ph. perniciosus by transovarial transmission alone. Alternative mechanisms for virus maintenance are discussed.