Rift Valley fever virus in the cardia of Culex pipiens: an immunocytochemical and ultrastructural study.

In this paper, we consider the movement of Rift Valley fever (RVF) virus from infected mosquito midgut epithelial cells into the hemocoel as an important factor in the ultimate ability of the insect to transmit the virus. Our results are therefore significant in the context of vector competence. The mosquito Culex pipiens was identified as the primary vector of RVF in an epidemic that occurred in Egypt in the 1970s. On this basis, we have carried out several studies of RVF virus in this mosquito species. In the research reported here, we used immunocytochemical and transmission electron microscopic techniques to study the occurrence of RVF virus in the mosquito cardia and aspects of the histology and ultrastructure of this organ. The cardia is a complex organ consisting of both foregut and midgut tissue and is the location of the foregut-midgut junction. The cardia is of interest because it appears to provide routes of RVF virus egress from the midgut lumen and it is consistently infected in mosquitoes with disseminated infections, making it a potentially important site of viral amplification and an ideal site for studying RVF viral morphogenesis. In orally infected mosquitoes, large numbers of RVF virions were observed budding into the basal labyrinth associated with the outer cardial epithelial cells and into the noncellular matrix associated with the inner cardial epithelial cells and the cells of the intussuscepted foregut. In mosquitoes infected by injection of virus into the hemocoel and then held for different incubation periods, viral antigen was first detected in the cells of the intussuscepted foregut in the cardia and later in the cardial epithelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Blister beetle dermatosis in Hawaii caused by Thelyphassa apicata (Fairmaire).

Members of the family Oedemeridae are known as false blister beetles. Although they have a worldwide distribution, oedemerid blister beetle dermatosis has been described only in the Pacific basin and the Caribbean. We report a case of the disorder in the Hawaiian Islands caused by Thelyphassa apicata (Fairmaire). To our knowledge this is the first case report described in that locale, and the first caused by this species.

An immunocytochemical study of the distribution of Rift Valley fever virus in the mosquito Culex pipiens.

The dissemination of Rift Valley fever (RVF) virus in females of the epidemic vector Culex pipiens was studied immunocytochemically. Among orally infected specimens, viral antigen was detected in all major midgut regions, although individuals varied with respect to which regions were infected. Among specimens with disseminated infections (infections beyond the midgut epithelium), antigen was detected in most tissues, including those of the nervous and endocrine systems. However, no antigen was found in the ovarian follicular epithelia, oocytes/nurse cells, or female accessory gland. A viral dissemination index (DI) based on several tissues was determined for each orally infected specimen and used to estimate the extent of viral dissemination in the hemocoel. Plots of DI values as a function of time after ingestion of an infectious blood meal indicated that dissemination was sporadic, and that once virus escapes from the midgut, its spread to other tissues is rapid. The most common sequence of tissue/organ infection following escape of virus from the midgut epithelium appears to be as follows: intussuscepted foregut, fat body, salivary glands and thoracic ganglia, epidermis, and ommatidia of the compound eyes. Reduced fecundity and survival identified in other studies of RVF virus-infected Cx. pipiens are probably explainable on the basis of extensive tissue and organ infection resulting in an overall energy drain. Infection of regulatory tissues and organs such as the ganglia of the central nervous system, neurosecretory cells, and the corpora allata may also have detrimental effects on the overall functioning of a mosquito, even in the absence of widespread infection.

The distribution of Rift Valley fever virus in the mosquito Culex pipiens as revealed by viral titration of dissected organs and tissues.

Distribution of Rift Valley fever virus (RVFV) was studied in the mosquito Culex pipiens. Mosquitoes were dissected on days 1-7 after an infectious bloodmeal, and RVFV plaque assays were performed on the legs, posterior midgut, ovaries, salivary glands, thoracic ganglia, and remaining organs and tissues (remnants). On days 7-12 and 14 following an infectious bloodmeal, mosquitoes were tested for their ability to transmit virus and then dissected. Dissemination (systemic infection) rates averaged 22% on days 1-14 and transmission rates 33% on days 7-14. There were no significant differences in the viral titers of midgut samples among the nondisseminated infected (virus limited to alimentary canal), disseminated infected nontransmitting, and transmitting groups of mosquitoes. The sequence of infection of the organs and tissues studied appeared to be as follows: midgut, hemolymph, remnants: salivary glands, ovaries, and thoracic ganglia. Some individuals were found to have disseminated infections as early as 12 hr following an infectious bloodmeal. Trauma, simulated by vigorous shaking immediately following the viremic bloodmeal, did not affect either infection or dissemination rates.

Reduced survival of adult Culex pipiens infected with Rift Valley fever virus.

The effect of Rift Valley fever (RVF) viral infection on the survival of female Culex pipiens was examined. In 3 experiments in which mosquitoes ingested RVF virus, there was a 44% decrease in survival to days 14-16 for transmitting vs. nontransmitting mosquitoes, and a 48% decrease in survival for individuals with disseminated vs. nondisseminated infections. These results were corroborated by other experiments in which survival of mosquitoes intrathoracically inoculated with RVF virus was compared with that of those inoculated with diluent. In both the per os and inoculation tests, uninfected mosquitoes survived significantly longer than infected mosquitoes. Even though mosquitoes with disseminated infections had a lower survival rate than did uninfected mosquitoes, dissemination and transmission rates were similar at days 7 and 14-18 after the infectious bloodmeal. This suggests that nondisseminated individuals were developing disseminated infections and becoming capable of transmitting virus between days 7 and 14-18 at approximately the same rate older transmitters were dying. The decreased survival associated with RVF viral infection should be considered in predictive models of this disease.

Use of the avidin-biotin-peroxidase complex immunocytochemical procedure for detection of Rift Valley fever virus in paraffin sections of mosquitoes.

The avidin-biotin-peroxidase complex (ABC) immunocytochemical procedure was used to locate Rift Valley fever (RVF) virus antigen in infected Aedes albopictus C6/36 cultured cells and in serial paraffin sections of intrathoracically-injected Egyptian Culex pipiens. Fixation of the cultured cells in formaldehyde or periodate-lysine-paraformaldehyde, and fixation of whole mosquitoes with formaldehyde resulted in good preservation of morphology and excellent differential staining between uninfected and infected specimens. Two primary antibodies against RVF virus were tested on the cultured cells: a polyclonal rabbit antiserum and a mixture of mouse monoclonal antibodies. Only the mouse monoclonal antibody mixture was tested on the Cx. pipiens. Specific staining of tissues in paraffin sections occurred at dilutions as high as 1:5,000. In both cultured Ae. albopictus cells and sections of Cx. pipiens, only the cytoplasm was positive for antigen. A preliminary list of tissues in Cx. pipiens that exhibited specific or nonspecific staining is given.

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.