Climatic and geographic influences on arboviral infections and vectors.

Those components of climate that are likely to have major effects upon the geographical distribution, seasonal incidence and prevalence of vector-borne diseases are described. On the basis of existing and predicted climatic variations, examples are given of the types of changes that are to be expected, using several internationally important human and animal arboviral diseases including Rift Valley fever and African horse sickness.

The barriers to bluetongue virus infection, dissemination and transmission in the vector, Culicoides variipennis (Diptera: Ceratopogonidae).

Transmission of bluetongue virus (BTV) by a vector species of Culicoides was studied using immunohistochemistry, virus titration and in vitro transmission tests. Adult female C. variipennis were used from two colonies that are either "transmission competent" or "transmission refractory" after oral infection with BTV. Intrathoracic (i.t.) injection of BTV into the haemocoel always resulted in a fully disseminated infection and transmission of virus in saliva. However, after ingestion of an infectious blood meal, only 30% (approximately) of midges from either colony became persistently infected. Although none of the orally infected insects from the "refractory" colony were able to transmit virus, 12% of those from the "competent" colony (containing > or = 10(3.0)TCID50 of virus/midge) did transmit BTV in their saliva. The most important barriers to BTV transmission in Culicoides vector species appeared to be a mesenteron infection barrier (MIB), which controls initial establishment of persistent infection, a mesenteron escape barrier (MEB) which can restrict virus to gut cells and a dissemination barrier (DB) which can prevent virus which enters the haemocoel from infecting secondary target organs. Culicoides variipennis do not appear to present either a salivary gland infection barrier (SGIB), or a salivary gland escape barrier (SGEB) to BTV.

Determination of dengue virus serotypes in individual Aedes aegypti mosquitoes in Colombia.

Adult Aedes aegypti mosquitoes were collected in Puerto Triunfo, central Colombia, where dengue is endemic, during a six month period. Viral infection within the head of each individual mosquito was identified by an immunofluorescent assay (IFA) using a flavivirus-specific monoclonal antibody. The dengue virus serotype, present in each flavivirus-positive specimen, was then determined in portions of the remaining thorax using IFAs with serotype-specific monoclonal antibodies. Among 2065 female Aedes aegypti collected and tested, twenty-four flavivirus-positive individuals were found (minimum infection rate 11.6%), three identified as dengue type-1 and twenty-one as dengue type-2 virus. This was consistent with the isolation of only these two serotypes of dengue virus from dengue fever patients within this town. No vertical transmission of dengue virus could be detected in 1552 male Aedes aegypti collected. This method is inexpensive, simple, rapid to perform and suitable for use in developing countries to identify and distinguish different serotypes of dengue virus in their vectors during eco-epidemiological investigations.

Post-inoculation changes in enzyme activity of Aedes aegypti infected with Chikungunya virus.

Levels of acetylcholinesterase, non-specific esterases, glutathione-S-transferase and glucose-6-phosphate dehydrogenase in Aedes aegypti (L.) mosquitoes inoculated intrathoracally with Chikungunya virus were elevated, as compared to uninoculated control insects. A number of these enzymes are important in the insects defence mechanism against xenobiotics, such as pesticides. Malathion bioassays indicated a reduction in the susceptibility of experimentally injected insects with virus or virus-free inoculum, compared to non-inoculated controls. However, insects which were mock-inoculated (injected with no inoculum) showed a similar reduction in susceptibility suggesting that the observed effect was due to the mobilization of a defence reaction in the mosquitoes in response to injury during inoculation.

Changes in enzyme titres with age in four geographical strains of Aedes aegypti and their association with insecticide resistance.

The enzymes acetylcholinesterase, glutathione S-transferase (GST), glucose 6-phosphate dehydrogenase (G6PD), and general esterases were assayed in four strains of Aedes aegypti mosquitoes aged between 1 and 30 days. Microtitre plate methods were used to assay activity in the homogenates of individual mosquitoes. The levels of GST and G6PD declined with the age of the mosquitoes, while the activity for the other enzymes remained constant. Soluble protein content was also found to decline with mosquito age in all the strains. Insecticide bioassays showed that two strains (Trinidad and Virtudes) of Ae. aegypti were resistant to DDT, deltamethrin and malathion, whereas two other strains (Bangkok and Indian) were susceptible to all four classes of insecticides tested. Higher esterase activity levels in the resistant compared to the susceptible strains were assumed to be the cause of organophosphate resistance. The combination of DDT and deltamethrin resistance in two strains with normal GST and G6PD characteristics suggests that a kdr-type nerve insensitivity mechanism may be involved.

Arbovirus-mosquito interactions and vector specificity.

Mosquito-borne arboviruses cause important and expanding disease problems. In this article, Colin Leake reviews the increasing knowledge of the complex interaction of arboviruses with their mosquito vectors and mosquito cells, in vitro, and considers the factors influencing vector specificity and vector competence.

The pathogenesis of Japanese encephalitis virus in Culex tritaeniorhynchus mosquitoes.

Culex tritaeniorhynchus were inoculated intrathoracically with mosquito and human strains of Japanese encephalitis virus and maintained at 26 degrees C or 32 degrees C. Virus titration and localization of viral antigen by avidin-biotin immunoperoxidase staining were done at intervals up to 21 days. Marked differences were noted in the tempo of organ involvement at the 2 temperatures; at both there was initial infection of fat body cells followed by selective infection of the central nervous system (CNS), with consistent infection of cells of the compound eye, patchy involvement of cephalic, thoracic and abdominal ganglia and no infection of Johnston's organ. CNS infection was always present 4 days after infection, when salivary gland involvement was first seen at 32 degrees C; at 26 degrees C CNS infection preceded salivary gland infection by 2 weeks. Late involvement of gut cells, pericardial cells and oviducts was also found, with no involvement of muscle.

Isolation of Japanese encephalitis virus from clinical specimens using a continuous mosquito cell line.

During the 1983 Japanese encephalitis (JE) epidemic in northern Thailand, we systematically attempted to isolate JE virus (JEV) from clinical specimens collected from 49 consecutive JE patients at 1 provincial hospital. Fresh acute plasma and cerebrospinal fluid (CSF) samples and postmortem brain samples were immediately inoculated onto cultured monolayers of Aedes pseudoscutellaris (LSTM-AP-61) cells which had been shipped to the epidemic site. None of 49 plasma samples yielded virus. None of 30 fresh CSF samples from nonfatal cases yielded virus, but 5 of 15 (33%) CSF samples from fatal cases did. Inoculation of fresh brain specimens obtained at autopsy yielded virus in every case attempted (7 of 7), whereas postmortem needle biopsy specimens of brain yielded virus in only 1 of 4 cases. Isolates were most frequently successful using thalamic tissue (6 of 7 cases), but isolates were also commonly obtained from frontal cortex (4/7), occipital cortex (4/7), cerebellum (4/7), medulla (4/7) and pons (2/7).

Virus isolations from mosquitoes collected during the 1982 Japanese encephalitis epidemic in northern Thailand.

From 16 June to 15 August, 1982 CDC light traps were used to collect mosquitoes in the province of Kamphaengphet, N. Thailand. 353,042 mosquitoes comprising 59 species were collected and identified, and 345,173 were placed in pools for attempted virus isolation by inoculation of C6/36 Aedes albopictus mosquito cell cultures. Viruses were isolated from 63 mosquito pools. These comprised 56 flaviviruses, identified as 35 isolates of Japanese encephalitis (JE) virus strains, 18 strains of Tembusu (TEM) virus and three untyped flaviviruses (FLA); three alphaviruses, identified as the first isolates of Getah (GET) virus to have been made in Thailand; and four viruses which are still unidentified. Most virus isolates were from Culex tritaeniorhynchus mosquitoes collected in carbon dioxide baited light traps. JE virus was isolated only over a ten-day period and the last isolate was obtained one week before the peak of admission of human encephalitis cases at Kamphaengphet Provincial Hospital. Rapid screening of isolates grown on Ae. pseudoscutellaris (LSTM-AP-61) mosquito cells by indirect immunofluorescence using flavivirus group-specific and JE-specific monoclonal antibodies showed a high degree of correlation with plaque reduction neutralization tests. An antigen capture enzyme immunoassay (EIA) test successfully identified about 50% of the JE virus positive pools, but the method saved considerable processing time.

Fatal outcome in Japanese encephalitis.

Forty-nine consecutive patients with laboratory-confirmed acute Japanese encephalitis were studied to identify risk factors present at hospital admission which were associated with a fatal outcome. Sixteen patients (33%) died. The following constellation of findings correlated with a fatal outcome: infectious virus in cerebrospinal fluid (CSF), low levels of Japanese encephalitis virus-specific IgG and IgM in both CSF and serum, and a severely depressed sensorium. Age, sex, days ill before admission, distance from home to the hospital, past medical history, CSF protein content, and CSF leukocyte count were not significant risk factors. Among patients hospitalized for acute Japanese encephalitis, a vigorous virus-specific immunoglobulin response, both systemically and locally within the central nervous system, is a good marker for survival, and may be an inherently important factor in recovery from illness.

Japanese encephalitis: immunocytochemical studies of viral antigen and inflammatory cells in fatal cases.

The distribution of virus and the composition of the mononuclear inflammatory response were studied in the brains of 7 children who died with Japanese encephalitis. Viral antigen was localized to neurons, with greatest involvement in the thalamus and brainstem. Quantitation of perivascular inflammatory responses showed a preponderance of T cells, but only 7 to 30% of these cells were T suppressor/cytotoxic cells. Inflammatory cells invading the parenchyma were predominantly macrophages with small numbers of T cells. B cells remained localized to perivascular cuffs. Viral antigen was progressively cleared in patients with survival of 6 days or more.

Intense transmission of Japanese encephalitis virus to pigs in a region free of epidemic encephalitis.

Epidemic Japanese encephalitis recurs annually in the northern provinces of Thailand, but in the southern provinces cases of human encephalitis are rare. We investigated transmission of Japanese encephalitis virus (JEV) to pigs in southern Thailand. Blood specimens from one hundred young pigs at abattoirs in three southern provinces were tested for JEV hemagglutination inhibiting (HAI) antibodies. Seventy-four percent were positive. Ten seronegative sentinel pigs were placed at five locations in one southern province. Seven of the ten pigs developed JEV HAI and JEV IgM ELISA antibodies within two weeks of placement. JEV was isolated from all seven seroconverting sentinel pigs from blood specimens collected 3 to 11 days after placement. Fifteen light-trap mosquito collections at the five locations all included known JEV vectors, some in large numbers. We conclude that there is intense transmission of JEV to pigs in southern Thailand despite the rare occurrence of human encephalitis in the same region.

The vector competence of colonized Aedes (Stegomyia) katherinensis for dengue-2 virus.

Colonized Aedes (Stegomyia) katherinensis mosquitoes from Australia were infected with the PR-159 strain of dengue-2 virus using a membrane feeding technique and by intrathoracic inoculation. Virus replication to low levels was detected when mosquitoes infected by both routes were assayed using the virus-sensitive Ae. pseudoscutellaris (LSTM-AP-61) mosquito cell line in a microculture system. Analysis by indirect immunofluorescence revealed the expected 100% infection rates in inoculated mosquitoes compared with only 45% in orally infected mosquitoes. Few of the orally infected mosquitoes showed any viral antigen associated with the head and no virus transmission was detected. Preliminary studies also demonstrated that Ae. (S.) katherinesis was refractory to oral infection with Japanese encephalitis virus but was readily infected by intrathoracic inoculation. On the basis of this data, it is concluded that there is a high threshold of infection in this mosquito and that it is unlikely that Ae. (S.) katherinensis could be important as a vector of dengue-2 virus in Australia.

A kinetic and ultrastructural comparison of alphavirus infection of cultured mosquito and vertebrate cells.

Vero cells and Aedes pseudoscutellaris cells showed rapid production of Semliki Forest virus (SFV) whereas in Aedes aegypti and Anopheles stephensi cells no rapid production of SFV was observed. Ultrastructurally the only virally induced cell inclusion in early infection was the cytopathic vacuole type 1. Later in infection, in mosquito cells, electron-dense bodies appear and budding of new virions appears to be very efficient. In Vero cells large accumulations of envelope proteins and nucleocapsids saturate the plasma membrane suggesting an inefficient budding process. After this time cytopathic vacuoles type 2 appear in Vero cells and a mechanism for their formation is proposed. Subsequent death of Vero cells appears to centre on progressive build-up of envelope protein on the rough endoplasmic reticulum (which is never seen in mosquito cells) and subsequent vacuolation of the rough endoplasmic reticulum followed by cell lysis.

An outbreak of type 2 dengue fever in the Seychelles, probably transmitted by Aedes albopictus (Skuse).

Between December 1976 and September 1977 the Seychelles group of islands in the Indian Ocean was struck by an extensive epidemic of dengue fever. The peak of the epidemic was in the last week of February. Type 2 dengue virus was isolated from patients and mosquitos. Aedes albopictus was the sole vector. The clinical picture was that of classical dengue. Haemorrhagic fever and the shock syndrome were not observed.Absenteeism from schools and offices, anamnestic questioning, and prevalence of antibodies in sera collected after the epidemic was over, indicated that approximately 75% of the population had been infected. Serological evidence was obtained of an epidemic of dengue in the islands more than 40 years earlier. This was confirmed by archival records.