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This paper characterizes Guanarito virus, the etiologic agent of Venezuelan hemorrhagic fever. Based on its morphology and antigenic properties, Guanarito virus appears to be a new member of the Tacaribe complex of the genus Arenavirus, family Arenaviridae. Complement fixation and indirect fluorescent antibody tests showed that Guanarito virus and its antiserum are broadly cross-reactive with other members of the Tacaribe complex, but it can be differentiated from other members of the complex by neutralization test. Guanarito virus causes mortality in suckling mice and adult guinea pigs, but not in adult mice. Inoculated rhesus monkeys developed viremia and became ill; however, they subsequently recovered and responded with production of antibody. To date, all isolates of Guanarito virus have come from sick persons or wild rodents living within a single geographic focus in the central plains of Venezuela.

Inherited rickettsial symbionts of the genus Wolbachia occur commonly in arthropods and have been implicated in the expression of parthenogenesis, feminization and cytoplasmic incompatibility Wolbachia from the Asian tiger mosquito, Aedes albopictus, to replace the natural infection of Drosophila simulans by means of embryonic microinjection techniques. The transferred Wolbachia infection behaves like a natural Drosophila infection with regard to its inheritance, cytoskeleton interactions and ability to induce incompatibility when crossed with uninfected flies. The transinfected flies are bidirectionally incompatible with all other naturally infected strains of Drosophila simulans, however, and as such represent a unique crossing type. The successful transfer of this symbiont between distantly related hosts suggests that it may be possible to introduce this agent experimentally into arthropod species of medical and agricultural importance in order to manipulate natural populations genetically.

Here in is described the clinical and laboratorial findings of a laboratory-acquired infection caused by the virus SP H 114202 (Arenavirus, family Arenaviridae) a recently discovered agent responsible for a viral hemorrhagic fever. The patient was sick for 13 days. The disease had an abrupt onset characterized by high fever (39 degree C.), headache, chills and myalgias for 8 days. In addition, on the 3rd day, the patient developed nausea and vomiting, and in the 10th, epigastralgia, diarrhea and gengivorrhagia. Leucopenia was seen within the 1st week of onset, with counts as low as 2,500 white cells per mm3. Counts performed after the 23rd day of the onset were within normal limits. With the exception of moderate lymphocytosis, no changes were observed in differential counts. An increase in the titer of antibodies by complement fixation, neutralization and ELISA (IgM) was detected. Suckling mice and baby hamsters were inoculated intracerebrally with 0.02 ml of blood samples collected in the 2nd and 7th days of disease. Attempts to isolate the virus were also made in Vero cells. No virus was isolated. This virus was isolated before in a single occasion in São Paulo State, in 1990, from the blood of a patient with hemorrhagic fever with a fatal outcome. The manipulation of the virus under study, must be done carefully, since the transmission can occur through aerosols.

During February 1992, field studies on the epidemiology of Venezuelan hemorrhagic fever (VHF) were carried out in a rural area of Portuguesa State in central Venezuela. The objective of this work was to determine the prevalence of infection with Guanarito virus, the etiologic agent of VHF, among wild rodents and humans living within an endemic focus of the disease. A total of 234 rodents, representing nine different species, were collected and their spleens were cultured for virus. Thirty-one Guanarito virus isolates were made from two rodent species: 19 from 40 Sigmodon alstoni and 12 from 106 Zygodontomys brevicauda. Guanarito virus antibody rates among these two species were 5.1% and 15.0%, respectively. Nine of the 12 Z. brevicauda that yielded virus from their spleens also had Guanarito virus antibodies in their sera. In contrast, none of the 19 Guanarito virus-positive S. alstoni had antibodies to the virus. These data suggest that S. alstoni usually develops a persistent nonimmunizing infection with Guanarito virus, while Z. brevicauda develops an immunizing infection. Based on knowledge of the behavior of other human pathogenic arenaviruses, these results imply that S. alstoni is the principal rodent reservoir of Guanarito virus in nature. To determine the prevalence of Guanarito virus infection among humans in the same region, 195 people living near one of the rodent collecting sites were bled and their sera were tested for antibodies to the virus. Five individuals (2.6%) had Guanarito virus antibodies; all were adults, and two had been diagnosed previously as having VHF.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood meals from 579 Lutzomyia longipalpis (Diptera:Psychodidae), collected in an endemic focus of American visceral leishmaniasis in Colombia, were identified by precipitin test. Sand fly collections were made during a 16-month period from the inside walls of two houses, a pigpen, and rock crevices in a small community (El Callejon) within the endemic area. Feeding patterns of the sand flies varied with locality and date of collection. Overall, bovine feedings predominated, but feedings were also recorded on pigs, equines, humans, dogs, opossums, birds, and reptiles. Calculation of the forage ratios for each host species indicated that cows and pigs were the preferred hosts of Lu. longipalpis in El Callejon. Results of this study suggest that Lu. longipalpis is an opportunistic feeder and is not highly anthropophilic nor strongly attracted to dogs.

The development of Leishmania major Yakimoff & Schokhor in the New World sand fly Lutzomyia longipalpis (Lutz & Neiva) was examined by light and electron microscopy. In this unnatural host, parasites differentiated into 10 typical morphological forms, multiplied at three sites, migrated anteriorly and established in the foregut, and attached to gut surfaces. In the blood meal, amastigotes divided and transformed into two successive dividing, stumpy promastigote stages. Elongate nectomonad promastigotes developed from stumpy forms and subsequently rounded up in some flies into paramastigotes and opisthomastigotes. Differentiation into round opisthomastigotes and the apparent fusion of paramastigotes in the blood meal were novel observations in this study. Three nectomonad promastigotes--elongate, short, and metacyclic--were free-swimming in the midgut lumen. Elongate nectomonad promastigotes were highly oriented in the midgut, with their flagella embedded between the epithelial microvilli. Short haptomonad promastigotes were the predominant form attached to the intima of the stomodeal valve, whereas pear-shaped haptomonad promastigotes and paramastigotes colonized surfaces of the esophagus and pharynx. Peripylarian attachment of promastigotes and paramastigotes in the pylorus, ileum, and colon was noted in 21% of flies, suggesting that suprapylarian leishmanias have not lost the ability to colonize the hindgut. L. longipalpis was a successful biological host for L. major, allowing complete development of the parasite.

Some of the world's most devastating diseases are transmitted by arthropod vectors. Attempts to control these arthropods are currently being challenged by the widespread appearance of insecticide resistance. It is therefore desirable to develop alternative strategies to complement existing methods of vector control. In this review, Charles Beard, Scott O'Neill, Robert Tesh, Frank Richards and Serap Aksoy present an approach for introducing foreign genes into insects in order to confer refractoriness to vector populations, ie. the inability to transmit disease-causing agents. This approach aims to express foreign anti-parasitic or anti-viral gene products in symbiotic bacteria harbored by insects. The potential use of naturally occurring symbiont-based mechanisms in the spread of such refractory phenotypes is also discussed.

Mark-release-recapture studies were carried out during 1990-1991 in El Callejón, Colombia, an endemic focus of American visceral leishmaniasis, to study the longevity, dispersal, and flight range of the principal vector, Lutzomyia longipalpis Lutz & Neiva. Several groups of wild-caught (n = 1,539) and laboratory-reared F1 (n = 2,208) sand flies were marked with fluorescent dusts and released. Recaptures at daytime resting sites, on animal bait, and in CDC light traps were made for 20 d following each release. From 2 to 9% of L. longipalpis were recaptured, a rate which differed between the sexes (7.7% male, 1.5% female). Overall, 49% of the L. longipalpis were recaptured between 0 and 50 m, 48% within 100 and 300 m, and nearly 3% at > or = 0.5 km from the release site. Sex differences in recapture site, distance flown, and direction were observed. Our results indicate that the dispersal behavior of peridomestic populations of L. longipalpis compares more closely with that of Old World sand fly species from similar habitats than to the sylvan Neotropical phlebotomines.

The secretion, morphology, and chemical composition of the peritrophic membrane was studied in the sand fly, Phlebotomus perniciosus Newstead. The membrane was secreted from the entire midgut epithelium. An electron-dense fine granular secretion, possibly chitin, was present along the length of the microvilli immediately until 24 h after feeding. From 12-48 h, an electron-lucid coarse granular component, possibly protein, was also secreted from the microvillar surface. By light microscopy, the mature 36-h membrane characteristically consisted of a dark anterior cap and posterior open ring, with a transparent intervening membrane and anterior plug. Ultrastructure of the fully formed membrane at 24-48 h was highly variable. Undifferentiated membranes appeared as a single electron-lucid layer; differentiated membranes were more complex, sometimes two-layered, containing electron-lucid and -dense fibers and granules. Results of binding to succinylated wheat germ agglutinin, histochemistry, and amino acid analysis indicated that the membrane was composed of chitin, glycoprotein, and protein. Eighteen amino acids were identified in membrane proteins; aspartic-glutamic acids (and amides), serine, glycine, and lysine (45% by weight) may be important in cross-linking membrane components.

A series of experiments were done to study the effect of simulated summer and winter temperatures on the development of Phlebotomus perniciosus (Diptera:Psychodidae) and on the survival of Toscana virus in transovarially infected insects. Sand flies maintained at 28 degrees C developed relatively fast, with adults emerging from 40 to 55 days after initial oviposition. Similar results were obtained with insects reared at 25 degrees C. In contrast, sand flies maintained at 15 degrees C developed slowly up to the fourth larval instar; at that point, further development ceased and the insects entered diapause. Diapause could be terminated by increasing the ambient temperature to 25 degrees C. The ambient temperatures at which the immature forms were reared (15 degrees C, 25 degrees C, and 28 degrees C) had no effect on the subsequent F1 adult filial infection rates with Toscana virus (49.1%, 47.5%, and 46.5%, respectively). The results of these experiments provide a model of how Toscana virus survives the winter in endemic areas by maintenance in diapausing P. perniciosus larvae. In another experiment, venereal transmission of Toscana virus was shown from transovarially infected males to non-infected virgin females. This is the first demonstration of sexual transmission of a phlebovirus by sand flies. If venereal transmission occurs in nature, it would provide an alternative method of virus amplification in the vector population, in the absence of viremic vertebrates.

Three viruses isolated from anopheline mosquitoes in Indonesia have been identified as bluetongue and epizootic hemorrhagic disease viruses. Another virus isolate showed no relationship to other orbiviruses tested and should be regarded as a new virus; the name Golok is proposed for it. The mosquitoes were collected in 1980 and 1981 in a program designed to isolate flaviviruses infecting humans. It is apparent that such collections of arthropods which feed on large mammals could be screened for other viruses which may infect domestic livestock.

Primer-extension sequencing of the RNA template of polio, dengue, Rift Valley fever, and Japanese encephalitis (JE) viruses has provided new information on their geographic distribution, origin, and evolution. In a previous study of 46 diverse JE virus strains, we demonstrated the existence of three distinct JE genotypes in Asia. We now report the occurrence of a fourth genotype. In the present study, 19 JE virus isolates, representing various geographic regions of Asia and a 50-year time span, were compared with each other and with Murray Valley encephalitis, West Nile, and Kunjin viruses. Twelve of the JE strains from the Indonesian Archipelago and the Philippines had not been previously examined; the remainder were representatives of the three previously identified genotypes. Two hundred forty nucleotides from the pre-M gene region of the virus were used in these comparisons. Using 12% divergence as a cut-off point, the 19 JE strains fell into four distinct genotypic groups; maximum divergence across the comparison region was 21%. The newly recognized fourth genotype was comprised of five Indonesian isolates that were 7% divergent from the rest of the JE viruses.

The phlebotomine sand fly fauna of two coffee plantations in a Leishmania-endemic area of Norte de Santander, Colombia was studied. Regular insect collections using a variety of methods were made for three and a half years. Information was obtained on diurnal resting sites, host range and seasonal abundance for 17 species, of which five (Lutzomyia spinicrassa, Lu. serrana, Lu. shannoni, Lu. ovallesi and Lu. gomezi) were far more numerous than the others, anthropophilic and present throughout the year. The behaviour of these and the remaining 12 species is discussed in relation to their potential role in transmission of Leishmania (Viannia) braziliensis in the area.

Characterization is given of a new parasite, Leishmania equatorensis sp. n., which was isolated from the viscera of a sloth (Choloepus hoffmanni) and a squirrel (Sciurus granatensis), captured in humid tropical forest on the Pacific Coast of Ecuador. Data based on biological and molecular criteria, as well as numerical zymotaxonomical analysis, indicate that this parasite is a new species of the L. braziliensis complex. L. equatorensis is clearly distinguishable from all other known species within this complex, using the following molecular criteria: reactivity patterns with specific monoclonal antibodies, isoenzyme electrophoresis, and restriction-endonuclease fragment patterns of kinetoplast DNA (k-DNA).

The aphid alarm pheromone, trans-beta-farnesene (TBF), was found to stimulate feeding in both male and female Lutzomyia longipalpis Lutz & Neiva. Four other structurally related compounds (farnesol; 808 farnesene; trans, trans-farnesyl acetate; farnesyl methyl ether) were slightly less stimulating to these insects. The effect of TBF varied with sand fly age and the concentration of the chemical used. In contrast, TBF did not stimulate feeding in either sex of four other sand fly species (L. shannoni Dyar, Phlebotomus papatasi (Scopoli), P. argentipes Annandale & Brunetti, P. perniciosus Newstead). TBF might be useful in enhancing L. longipalpis field collections or in developing a poison bait for the control of this species.

The life cycle of Leishmania panamensis in Phlebotomus papatasi was studied to characterize barriers limiting parasite colonization, differentiation, migration, and attachment in an unnatural sand fly host. The insects were fed a suspension of L. panamensis-infected macrophages and human erythrocytes, and were examined up to 16 days post-infection by light and electron microscopy. Histologic examination of 401 flies showed the peritrophic membrane to be the first important barrier to parasite establishment in the gut lumen. In most flies, parasites were unable to escape from the closed peritrophic sac, which was either excreted or retained intact in the midgut. After five days, only 31% of the flies were infected; attached parasites colonized the pylorus-ileum and/or colon regions of the hindgut. Anterior migration into the cardia region of the midgut occurred in less than 1% of infected flies; no parasites colonized the foregut. In the bloodmeal and residual bloodmeal, five morphologic forms developed from ingested amastigotes: stumpy, spatulate, elongate, short nectomonad promastigotes, and paramastigotes. Abnormal retention of amastigotes in macrophages and delayed development of promastigote stages was observed. The primary form attached in the hindgut was a pear-shaped haptomonad promastigote. Differentiation of L. panamensis in Ph. papatasi appeared to be similar to that described in natural hosts, except that metacyclic infective forms were not observed, and some forms developed in unusual locations. Phlebotomus papatasi was a partly refractory biological host for L. panamensis. The peritrophic membrane adversely affected the infection rate; rare anterior migration and a lack of metacyclic promastigotes may preclude transmission by bite.

A shuttle plasmid was developed that is capable of replicating both in Escherichia coli and in Rhodococcus rhodnii, a bacterial symbiont of the Chagas' disease vector Rhodnius prolixus. We have been able to transform R. rhodnii with this plasmid, infect aposymbiotic R. prolixus with the transformed symbionts, select with the antibiotic thiostrepton, and re-isolate genetically altered symbionts from the insects following successive molts. Symbiotic bacteria are potentially valuable as vehicles for the stable introduction of foreign genes into insects with the goal of eventually altering the ability of the insect to transmit a pathogenic agent.

Transmission electron microscopy was used to examine replication of the New Jersey serotype of vesicular stomatitis virus (VSNJ) (Rhabdoviridae: Vesiculovirus) in Lutzomyia shannoni (Diptera: Psychodidae), a recently implicated sand fly vector. Following ingestion of an infectious blood meal, female sand flies were fixed and examined at approximately 12-hr intervals for six days. The New Jersey serotype of vesicular stomatitis virus was first detected in the abdominal midgut after 34 hr of incubation. Virus next appeared in fat body and the thoracic midgut at 48 hr, while salivary glands first contained visible virus in apical cavities 5-6 days after infection. Flight muscles and nervous tissue occasionally contained small numbers of VSNJ virions, while virus was never detected in the ovaries or malphigian tubules. The midgut and fat body appeared to be major sites of VSNJ virus replication. In all tissues examined, virus matured primarily by budding from the plasma membrane. Virions were occasionally observed within vacuoles, along with nucleocapsids. In the midgut, budding occurred exclusively from the basolateral plasma membrane, while maturation in salivary gland cells involved apical budding. Accumulation of virions adjacent to basal laminae surrounding several tissues suggested that this structure physically impedes virus dissemination within the sandfly. The paucity of virus budding 120-144 hr after infection suggested that the VSNJ virus infection was modulated in Lu. shannoni.

Phlebotomine sand flies appear to be involved in the epizootiology of 10 of the 16 currently recognized vesiculoviruses. Evidence in support of this includes isolation of viruses from wild-caught male and female sand flies; demonstration of oral infection, replication, and bite transmission of viruses by sand flies; the temporal and/or spatial association between infected sand flies and infected vertebrates; and demonstration of transovarial virus transmission. The present review summarizes the known sand fly-vesiculovirus relationships.

An outbreak of severe haemorrhagic illness began in the municipality of Guanarito, Portuguesa State, Venezuela, in September, 1989. Subsequent detailed study of 15 cases confirmed the presence of a new viral disease, designated Venezuelan haemorrhagic fever. Characteristic features are fever, toxicity, headache, arthralgia, diarrhoea, conjunctivitis, pharyngitis, leucopenia, thrombocytopenia, and haemorrhagic manifestations. Other features include facial oedema, cervical lymphadenopathy, nausea/vomiting, cough, chest or abdominal pain, and convulsions. The patients ranged in age from 6 to 54 years; all were residents of rural areas in central Venezuela, and 9 died. Infection with Guanarito virus, a newly recognised arenavirus, was shown by direct culture or by serological confirmation in all cases. Epidemiological studies suggest that the disease is endemic in some rural areas of central Venezuela and that it is rodent-borne. Venezuelan haemorrhagic fever has many similarities to Lassa fever and to the arenavirus haemorrhagic fevers that occur in Argentina and Bolivia.

The effect of ivermectin (0.1 microgram/ml) on blood digestion, ovarian development, and ovipositional attributes of Aedes aegypti was studied using standard morphological and histological techniques. Uncoordinated movements and paralysis were observed in most ivermectin-treated females within 1 h after ingestion of blood containing the chemical. Eight days after the blood meal, 23.5% of the treated females had died, whereas no mortality occurred in controls. Formation of the peritrophic membrane and digestion of the blood meal were delayed in the surviving treated mosquitoes. The most striking effect of ivermectin on Ae. aegypti at this dosage was on ovarian development. Changes observed among ivermectin-treated mosquitoes included: blood digestion without development of ovarian follicles; degeneration of primary follicles and formation of ovarian dilatations within 24 h after ingestion of the chemical; significant reduction in the rate of vitellogenesis and follicle development; decreased egg production; reduced egg hatching; abnormal egg size and shape; and increased percentages of unhatched embryonated and sterile eggs. Although the precise action of ivermectin on Ae. aegypti is unknown, our studies indicate that the chemical directly or indirectly affects at least three major organ systems (nervous, digestive, and reproductive).

Characterization of Leishmania colombiensis sp.n. is presented, which on the basis of biological and molecular criteria, appears to be a new member of the L. braziliensis complex. A total of nine isolates of the new parasite were made in Colombia and Panama between 1980 and 1986: two from human cases of cutaneous leishmaniasis, six from phlebotomine sand flies, and one from a sloth. Although most closely related to L. lainsoni, L. colombiensis sp.n. is clearly distinguishable from other members of the genus by its reactivity with monoclonal antibodies, isoenzyme electrophoresis, and restriction endonuclease fragment patterns of kinetoplast DNA (k-DNA).

We tested if genetic exchange was observable between two strains of Leishmania major (Trypanosomatidae) during mixed infection of the sand fly Phlebotomus papatasi. Previous studies suggested that genetic exchange may occur in natural populations of Leishmania at a low frequency, but experimental crosses examining small numbers of progeny (less than 60) did not reveal hybrid parasites. Accordingly, a strategy was devised to increase the number of progeny that could be screened by 100-fold. Clonal derivatives from two strains that were infective to flies and contained numerous restriction fragment length polymorphisms were characterized and selected for resistance to methotrexate or tunicamycin by gene amplification. A successfully mixed infection of P. papatasi was obtained, and a method was developed for directly plating promastigotes from the gut contents of infected flies onto selective media. Twenty-five hundred independent progeny were scored for the presence of both drug resistance markers. No hybrid parasites were observed, indicating that the frequency of genetic exchange in this cross must be less than 4 x 10(-4). The lines and methods established in this work may prove useful in future studies of the mechanism and frequency of gene exchange in Leishmania.

Mosquitoes of 3 species (Aedes aegypti, Ae. albopictus, and Culex quinquefasciatus) were fed on human blood containing various concentrations of ivermectin. Three effects (death, decreased egg production, and reduced egg hatching) were observed in the insects, depending upon the concentration of ivermectin ingested. The LD50 of ivermectin in human blood for the 3 mosquito species was estimated to be 126, 208, and 698 ng/ml, respectively. Mosquitoes dying after ingestion of ivermectin developed signs of acute toxicity including paralysis, lethargy, incoordination, and difficulty in movement. Death usually occurred within 48-72 hr. With sublethal blood concentrations of the chemical, mosquitoes survived, but there was a marked reduction in both the number and viability of their eggs. This infertility was only temporary, however, as subsequent refeeding of the insects on uncontaminated blood resulted in the production of normal numbers of fertile eggs. Blood levels of ivermectin which made 50% of the eggs infertile in Ae. aegypti and Ae. albopictus were calculated 3.4 and 4.3 ng/ml, respectively. These latter concentrations of the chemical are within the range found in blood of humans and domestic animals receiving ivermectin for treatment of parasitic infections. This finding suggests that the widespread use of ivermectin in veterinary and human medicine may have an unrecognized effect on mosquito populations.

Laboratory-reared female sand flies (Lutzomyia shannoni) were experimentally infected, orally and by intrathoracic inoculation, with the New Jersey serotype of vesicular stomatitis (VSNJ) virus. Virus replication occurred in the insects following infection by both routes. Virus titers greater than 10(4) plaque forming units of VSNJ virus were present in heads of orally infected sand flies 12 days after virus ingestion, confirming that a persistent disseminated infection had occurred. Both orally and parenterally infected Lu. shannoni transmitted VSNJ virus by bite to susceptible rodents and by transovarial transmission to a small percentage of their F1 progeny. The significance of these findings in the epizootiology of VSNJ virus on Ossabaw Island, Georgia, an enzootic focus of this virus, is discussed.

Variation in susceptibility to infection with cultured promastigotes of Leishmania major was detected among 3 different geographic strains (ISRAEL, EGYPT, and INDIA) of the sand fly Phlebotomus papatasi. The ISRAEL strain showed the greatest susceptibility and was chosen for subsequent genetic selection experiments. After 13 generations of genetic selection in this strain, a stable refractory line was obtained in which only 7.5% of the insects could be infected. A highly susceptible line was also obtained in 5 generations of selection. Infection rates in the susceptible line were stabilized at about 94%. A description of the selection process and characteristics of the 2 lines are given.

Studies of the genetic factors controlling Leishmania major infection in Phlebotomus papatasi were carried out using 2 different sand fly lines: one highly susceptible and the other refractory to the parasite. L. major infection rates in both F1 and F2 generations from reciprocal crosses and in backcrosses between the parent lines showed that susceptibility and refractoriness of Ph. papatasi to infection with L. major are controlled by less than 1 gene. Neither susceptibility nor refractoriness is dominant over the other.

A total of 340 Leishmania strains, isolated from humans, animals, and sand flies from various regions of Colombia, were examined by isozyme electrophoresis. Seven different Leishmania species were identified. Leishmania panamensis and L. braziliensis were the most common, representing 53.8% and 30.3% of the total, respectively. Isolation rates of the other species were as follows: L. chagasi, 9.4%; L. guyanensis, 2.6%; L. amazonensis, 1.8%; L. mexicana, 0.8%; and a new species requiring additional study, 1.2%. Statistical analyses of representative L. panamensis and L. braziliensis isolates indicated that the populations of these 2 species are genetically very similar. L. panamensis may have a continuous distribution in Colombia west of the eastern Andes Mountains and L. braziliensis may have a continuous distribution east of the western Andes Mountains. Information is given on disease manifestations of the parasites in human hosts and on isolation records from sand flies and animals.

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.

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.

Selected human sera from 59 different localities in Africa, the Mediterranean littoral, eastern Europe and Asia were examined by plaque reduction neutralization test against eight sandfly (Phlebotomus) fever virus serotypes (Sicilian, Naples, Arumowot, SudAn 754-61, Karimabad, Salehabad, Gordil and Saint Floris) known to occur in the Old World. Results of these studies provide new information on the geographic distribution and prevalence of human infection with each of the viruses. Specific neutralizing antibodies were detected against all of the agents except Salehabad. Naples and Sicilian antibodies were encountered most frequently and had the widest geographic range; moreover they were found only in areas where Phlebotomus papatasi occurs. Age-specific antibody rates for several of the viruses are presented. These data and the epidemiology of sandfly fever are discussed.

This paper describes a series of experiments which were done to determine the behavior of 14 different phleboviruses in laboratory-reared sand flies (Phlebotomus papatasi, P. perniciosus and Lutzomyia longipalpis) after oral and parenteral infection. Most of the viruses replicated in the sand flies after intrathoracic inoculation; however, the insects were quite refractory to oral infection. Six of 11 phleboviruses tested were transovarially transmitted in one or more sand fly species. The percentage of infected F1 offspring produced by parenterally infected female parents ranged from 1.5-60%, depending on the virus type used. These data support the hypothesis that some of the phleboviruses are maintained in sand flies by transovarial transmission.

Six isolates of a new phlebotomus fever serogroup virus, designated Arboledas virus, were obtained from sand flies (Lutzomyia spp.) collected in northeastern Colombia. One of the isolates was made from a pool of male sand flies. By immunofluorescence, Arboledas virus is related to Caimito and Pacui viruses; by neutralization test, it is distinct. Arboledas virus neutralizing antibodies were found in the sera of opossums (Didelphis marsupialis) and humans living in the study area. D. marsupialis inoculated with the virus developed a viremia of four days' duration, and sand flies (Lutzomyia gomezi) feeding on a viremic opossum were readily infected. Transovarial transmission of Arboledas virus was also demonstrated in experimentally infected Lu. gomezi. Results of the above laboratory studies suggest that Arboledas virus is maintained in nature by two mechanisms: vertical (transovarial) transmission in the insect vector, and an alternating marsupial-sand fly cycle. The implications of this complex maintenance cycle for other phleboviruses are discussed.

Transovarial transmission of all four dengue serotypes was demonstrated in Aedes albopictus mosquitoes. The rates of such transmission varied with the serotype and strain of virus. In general, the highest rates were observed with strains of dengue type 1 and the lowest with dengue type 3. Surprisingly, despite the use of viral strains of the four dengue serotypes which gave the highest rates with Ae. albopictus, transovarial transmission was observed in Aedes aegypti only with dengue type 1, and then only at a relatively low rate. Five different strains of Ae. aegypti were employed, including one that was known to be relatively susceptible to oral infection with dengue viruses. The findings support the view that Ae. aegypti, while of major importance from the point of view of transmission of dengue to man, may be relatively unimportant in the overall natural history of dengue viruses.

Four strains of vesicular stomatitis virus--New Jersey (Hazelhurst, Guatemala, Panama, and Concan) were compared by cross-neutralization and complement-fixation tests. They were indistinguishable by complement-fixation test; however, by plaque-reduction neutralization method, slight antigenic differences were observed between the Hazelhurst and the three other strains. It is concluded that these antigenic differences are insufficient to warrant reclassification of vesicular stomatitis virus--New Jersey into two distinct subtypes, as has been recently proposed.

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.

The levels of albumin, IgG, IgM, and complement (C3) were determined by enzyme-linked immunosorbent assay in Aedes albopictus and Phlebotomus papatasi at regular intervals after feeding on human blood. Albumin disappeared most rapidly; by 48 hr, detectable levels of albumin had decreased 100-fold. In contrast, IgG and IgM survived longer and were still detectable at low levels several days after ingestion. C3 was intermediate in its rate of degradation. In general, serum protein disappearance occurred more rapidly in Ae. albopictus than in Ph. papatasi, despite a larger volume of blood ingested by the former.

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.

Four groups of hamsters were infected sequentially with various combinations of Arumowot, Chagres, and Gabek Forest viruses. Following each infection, the survival, level of viremia, and immune response of the animals were monitored. All of the agents produced viremia in the hamsters, regardless of the order of their administration. The antibody response, as measured by plaque reduction neutralization test, was monotypic even after two consecutive phlebovirus infections. Arumowot and Chagres viruses produced nonfatal infections in adult hamsters, which were characterized by viremia of several days duration and subsequent antibody formation. In contrast, Gabek Forest virus produced a fulminating and rapidly fatal disease in phlebovirus nonimmune animals. In hamsters previously infected with Chagres and/or Arumowot viruses, Gabek Forest infection was less severe, indicating some degree of cross-protection. The degree of cross-protection was in part related to the sequence of previous phlebovirus infections. No evidence of immune enhancement or other immunopathologic events were observed in the animals.

Transovarial transmission of two strains of yellow fever virus was demonstrated in three colonized geographical strains of Aedes aegypti following infection by intrathoracic inoculation. Infected progeny were detected in F1 offspring from only the first three ovarian cycles (OVC). The overall minimum filial infection rate for the first three OVC was 1:596; rates were loser in the second and third OVC. Virus survived in eggs for longer than 4 months and was recovered from progeny of three immersions of first OVC eggs. Infected progeny averaged a longer time to pupation than noninfected siblings. Transovarial transmission of virus was also demonstrated in first OVC progeny of Aedes mascarensis.

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.

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.

An alphavirus isolated from Culiseta mosquitoes has been associated with Ockelbo disease, an exanthema arthralgia syndrome occurring in Sweden. The isolate was made from mosquitoes collected in Edsbyn (central Sweden), an area with considerable Ockelbo disease morbidity. This isolate proved to be indistinguishable from Sindbis virus by complement-fixation and hemagglutination-inhibition tests, and was antigenically related to Sindbis in plaque reduction neutralization tests. Patients with Ockelbo disease developed neutralizing antibodies to the virus in their convalescent sera, suggesting that it is the etiologic agent of the disease.