Enzyme-linked immunosorbent assay for detection of Rift Valley fever virus antigen in mosquitoes.

An enzyme-linked immunosorbent assay (ELISA) was used to measure the quantity of Rift Valley fever (RVF) viral antigen in infected Egyptian Culex pipiens. Infectivity, as measured by plaque assay, was significantly correlated with viral antigen, as measured by the ELISA, in all groups of mosquitoes regardless of the time interval after the infectious blood meal. The proportion of noninfectious viral antigen in these groups increased with time. When individual mosquitoes were assayed the plaque assay and the ELISA techniques had similar sensitivity (100% vs. 93%, respectively) and specificity (94% vs. 94%, respectively) in detecting mosquitoes capable of transmitting virus to susceptible hamsters. The ELISA may be useful in detecting RVF-infected arthropods in the field because it provides a rapid, sensitive, and specific test.

Replication and dissemination of Rift Valley fever virus in Culex pipiens.

Following ingestion of 10(4.2) to 10(7.2) plaque-forming units (PFU) of Rift Valley fever (RVF) virus, 662 of 850 female Culex pipiens (78%) became infected. Those mosquitoes that became infected separated into two distinct groups: 1) those with a nondisseminated infection limited to the gut, and 2) those with a disseminated infection. The former group contained a median of 10(3.2) PFU, while those females with a disseminated infection contained a median of 10(5.5) PFU. Only those females with a disseminated infection were capable of transmitting virus by bite to a susceptible hamster. This is consistent with a mesenteronal escape barrier to viral dissemination. Following intrathoracic inoculation of RVF virus, all females developed a disseminated infection (26/26) and successfully transmitted virus by bite (49/49) if allowed to feed on a susceptible hamster or suckling mouse. Examination of legs and bodies separately provided a rapid and efficient method of determining dissemination status.

Mechanical transmission of Rift Valley fever virus by hematophagous Diptera.

Experimental studies were conducted to determine if hematophagous Diptera were capable of mechanical transmission of Rift Valley fever (RVF) virus to laboratory animals. All species tested (Glossina morsitans, Aedes aegypti, Aedes taeniorhynchus, Culex pipiens, Stomoxys calcitrans, Lutzomyia longipalpis, and Culicoides variipennis) mechanically transmitted the virus to hamsters. Mechanical transmission rates for G. morsitans ranged from 0-100%, with the probability of mechanical transmission positively correlated with initial viremia titer and negatively correlated with the time after virus exposure. Mechanical transmission of RVF virus to lambs was demonstrated with both G. morsitans and Cx. pipiens. These findings demonstrated that mechanical transmission of RVF virus by hematophagous flies may contribute to the natural transmission and dissemination of this virus.

Vector potential of selected North American mosquito species for Rift Valley fever virus.

Selected North American mosquito species were evaluated as potential vectors of Rift Valley fever virus. Field populations of Aedes canadensis, Ae. cantator, Ae. excrucians, Ae. sollicitans, Ae. taeniorhynchus, Ae. triseriatus, Anopheles bradleyi-crucians, Culex salinarius, Cx. tarsalis, and Cx. territans perorally exposed to 10(6.2)-10(7.2) plaque forming units of Rift Valley fever virus readily became infected. Infection rates ranged from 51% (65/127) for Cx. salinarius to 96% (64/67) for Ae. canadensis. Disseminated infection rates were generally greater at 14 days than at 7 days after the infectious bloodmeal, and, with the exception of An. bradleyi-crucians, they were not significantly different than the pooled rate of 59% for each species tested. Only 5/55 (9%) of the An. bradleyi-crucians developed a disseminated infection. For most of the species, about half of the mosquitoes with a disseminated infection transmitted an infectious dose of virus to hamsters. While all species, with the exception of An. bradleyi-crucians, transmitted virus, Ae. canadensis, Ae. taeniorhynchus, and Cx. tarsalis had the highest vector potential of the species tested. Following inoculation of approximately 10(1.6) plaque forming units of virus, 100% of the mosquitoes of each species became infected. For most species, transmission rates were similar for inoculated individuals and those that developed a disseminated infection following peroral infection. Viral titers of transmitting and nontransmitting-disseminated individuals were similar for all species tested. These data suggest that, if Rift Valley fever virus was introduced into North America, several mosquito species would be capable of transmitting it.

The effect of laboratory colonization on the vector-pathogen interactions of Egyptian Culex pipiens and Rift Valley fever virus.

Field and laboratory findings implicated Culex pipiens as a vector of Rift Valley fever (RVF) virus during the 1977-1978 epizootics/epidemics in Egypt. This study evaluated changes in infection and transmission rates, and viral titers in F1 through F16 generation Cx. pipiens mosquitoes orally infected with RVF virus. Infection and transmission rates of RVF virus by this species changed significantly during the colonization process. However, the ultimate viral titers of either the transmitting or the infected nontransmitting mosquitoes were not affected by the colonization process. Following ingestion of virus, Cx. pipiens could be separated into three distinct subpopulations, an uninfected group and two types of infected mosquitoes--transmitters and nontransmitters. Transmitters contained significantly more virus (approximately 100-fold) than nontransmitters. These results demonstrated that not every infected female mosquito should be considered a competent vector, even if the species (population) is known to be a primary vector. Transmission was also accomplished by probing mosquitoes which were unsuccessful in obtaining a blood meal. These data document the long-held suspicion that vector competence studies based upon laboratory-colonized specimens may not represent the field situation.

Serologic evidence of Jamestown Canyon and Keystone virus infection in vertebrates of the DelMarVa Peninsula.

Serological data accumulated during the past decade indicated that a variety of feral and domestic animals of the Delaware-Maryland-Virginia (DelMarVa) Peninsula were infected with Jamestown Canyon (JC) and/or Keystone (KEY) viruses (Bunyaviridae, California serogroup). Neutralizing (N) antibody to JC virus was most prevalent in white-tailed deer, sika deer, cottontail rabbits and horses. KEY virus N antibody was detected most frequently in gray squirrels and domestic goats. N antibody indicative of past infection by one or both viruses also was found in raccoons, horses and humans. JC and/or KEY virus N antibodies were not demonstrable in sera of several other species of small mammals and reptiles. Investigations were extended to evaluate the role of domestic goats as an amplifying host of JC and KEY viruses and to assess their potential as sentinels of virus transmission. Goats maintained in the Pocomoke Cypress Swamp during the summer season of 1978, acquired N antibodies to JC and KEY viruses. Following experimental inoculation with either JC or KEY virus, all goats developed N antibody despite the absence of a demonstrable viremia in most animals. Goats proved to be effective as sentinels for monitoring the transmission of JC and KEY viruses; however, the exceptionally low titers or absence of viremia following inoculation with these viruses would seem to preclude a potential virus-amplifying role for this species. Although findings implicated primarily gray squirrels and white-tailed deer as possible amplifying hosts of KEY and JC virus, respectively, further investigations will be required to clarify their role, particularly since both viruses may be maintained entirely by transovarial transmission.

Winter survival of blood-fed and nonblood-fed Culex pipiens L.

Comparisons were made between groups of Culex pipiens L. with different physiologic histories to test their ability to sucessfully overwinter under field conditions. On 14 December 1978, each group of mosquitoes was marked with a distinctive fluorescent dust and released inside an abandoned ammunition bunker at Fort Washington, Maryland. To insure that dead mosquitoes could be dissected and information obtained on their ovarian development, a sample of females from each group was also released into a plexiglass cage that was attached to the inside wall of the room. The physiologic histories of each group of mosquitoes were as follows: (a) "wild caught", those which had entered the bunker prior to the release date, (b) "lab-reared diapausing nonblood-fed," (c) "lab-reared diapausing blood-fed nongravid, " (d)"lab-reared diapausing blood-fed gravid," (e) "lab-reared nondiapausing nonblood-fed," and (f) "lab-reared nondiapausing blood-fed." By 8 March 1979, all of the lab-reared nondiapausing groups, of mosquitoes released in the room had died, whereas 15.7, 22.4 and 24.7% were recovered from the "lab-reared diapausing nonblood-fed," "lab-reared diapausing blood-fed" (gravid and nongravid) and "wild caught" mosquitoes, respectively. For the mosquitoes in the cage, only 0, 2.1 and 7.0% of the "lab-reared nondiapausing blood-fed," "lab-reared nondiapausing nonblood-fed" and "lab-reared diapausing blood-fed gravid," respectively, survived. This compared to 45.4, 56.8 and 58.0%, respectively, for the "lab-reared diapausing nonblood-fed," "lab-reared diapausing blood-fed nongravid" and the "wild caught" groups. These data provide evidence to support the theory that a significant number of diapausing Cx. pipiens which have taken a prehibernation (possibly viremic) blood meal do not develop eggs and can survive the winter at rates comparable to diapausing nonblood-fed mosquitoes.

Association of a Sindbis-like virus with Ockelbo disease in Sweden.

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.

Characterization of malaria transmission by Anopheles (Diptera: Culicidae) in western Kenya in preparation for malaria vaccine trials.

Malaria transmission was studied for 33 mo in the villages of Kisian and Saradidi in western Kenya in preparation for field trials of malaria vaccines. Abundance estimates of Anopheles gambiae Giles sensu lato and Anopheles funestus Giles, which constituted over 99% of 26,645 anophelines collected, were compared for all-night biting collections inside houses, outdoors, and in tents. The overall numbers of Anopheles per man-night were 2.3 times greater in Kisian than in Saradidi. For the three types of collections, mean sporozoite rates by dissection ranged from 2.2 to 5.4% for 13,072 Anopheles in Kisian and from 9.9 to 13.6% for 7,058 Anopheles in Saradidi; greater than 90% of the infections were Plasmodium falciparum, either alone or mixed with P. malariae or P. ovale. Heaviest transmission from April to July coincided with the end of the long rainy season. Entomological inoculation rates (EIR) averaged 0.82 infective bites per man per night inside houses in Kisian and 0.65 in Saradidi. Outdoors, EIRs averaged 0.09 in Kisian and 0.52 in Saradidi. In tents, which were evaluated to identify methods for exposing nonindigenous volunteers during vaccine efficacy trials, EIRs were 3.3 and 2.5 times less than inside houses for Kisian (EIR = 0.25) and Saradidi (EIR = 0.26), respectively. Exposure in tents averaged one infective bite every 4.0 d in Kisian and every 3.8 d in Saradidi. The use of tents in vaccine efficacy trials should provide adequate exposure for nonindigenous volunteers. Malaria vaccine trials could be conducted efficiently in western Kenya, with timing dependent upon the intensity of transmission required by vaccine trial objectives.

Malaria sporozoite detection by dissection and ELISA to assess infectivity of afrotropical Anopheles (Diptera: Culicidae).

Malaria infection rates determined by dissection and Plasmodium falciparum enzyme-linked immunosorbent assay (ELISA) were compared for 26,935 Anopheles gambiae Giles sensu lato and 17,739 Anopheles funestus Giles collected during 20 mo in western Kenya. ELISA infection rates were about 43% higher than dissection sporozoite rates. In dissection-negative Anopheles, circumsporozoite (CS) protein was detected by ELISA in 5.2% of 10,017 salivary gland samples and in 12.2% of 237 thorax samples. The accuracy of dissection and ELISA techniques was compared by the following tests on a group of 352 field-collected Anopheles (held 10 d to ensure sporogonic development): salivary gland dissection, examination of Giemsa-stained dissection slides, ELISA tests on salivary gland and thorax body parts, and microscopic techniques for determining sporozoite loads. Respective infection rates were 9.9%, 10.8%, and 15.6% for dissection, stained slides, and ELISA. Sporozoite loads were associated significantly with ELISA absorbance values (r = 0.76). Compared with Giemsa-stained dissection slide results, the sensitivity of sporozoite detection was 92.1% for dissection compared with 78.9% for ELISA; specificity was 100.0% for dissection versus 92.0% for ELISA. Immunological detection of CS protein in head-thorax samples of Afrotropical vectors overestimated the proportion of infective Anopheles because the comparison of techniques indicated that 45.4% of the ELISA positive Anopheles did not contain salivary gland sporozoites.

Panveld oviposition sites of floodwater Aedes mosquitoes and attempts to detect transovarial transmission of Rift Valley fever virus in South Africa.

Floodwater aedine mosquito eggs were recovered from soil samples taken from grassland depressions, called pans, in the Orange Free State Province of South Africa. A sedge, Mariscus congestus (Vahl) C.B.Cl., was a useful indicator of Aedes (Ochlerotatus) juppi McIntosh oviposition areas. No transovarial transmission of virus was demonstrated by Ae.juppi females reared from the eggs and allowed to feed shortly after eclosion on hamsters. No virus was recovered from 557 pools of 5425 adult Ae.juppi that were collected as eggs and reared to the adult stage in the laboratory. Rift Valley fever virus replicated to high titres in experimentally infected Ae.juppi females, but horizontal transmission experiments proved inconclusive.

Detection of Rift Valley fever virus antigen by enzyme-linked immunosorbent assay.

A double-antibody (sandwich) enzyme-linked immunosorbent assay (ELISA) was adapted to detect Rift Valley fever virus antigen. Antibodies were purified from hyperimmune mouse and rabbit sera by affinity chromatography, using CNBr-activated Sepharose 4B coupled to a beta-propiolactone-inactivated sucrose-acetone-extracted suckling mouse liver antigen. In the assay, antigen was captured by mouse antibody adsorbed to polystyrene plates and then detected by reacting sequentially with rabbit anti-Rift Valley fever virus antibody and swine anti-rabbit immunoglobulin G conjugated to alkaline phosphatase. ELISA proved to be useful in measuring viral antigen in different animal systems. However, great variation was found in the amount of antigen per PFU encountered in different circumstances. The ELISA system was optimized using supernatant fluids from infected Vero cell cultures and had a sensitivity of 10(5) PFU/ml. Hamsters develop progressive viremia, much as seen in susceptible domestic animals, such as lambs; ELISA could reliably detect 10(6) PFU/ml of viremic hamster serum. Rhesus monkeys with Rift Valley fever infection were positive by ELISA even when viremias were only 5 X 10(3) PFU/ml. ELISA also proved to be useful in measuring viral antigen in infected mosquitoes.