M. G. R. Varma

Cell lines from larvae of Aedes (Stegomyia) malayensis Colless and Aedes (S) pseudoscutellaris (Theobald) and their infection with some arboviruses.

Abstract The establishment of 2 cell lines from first stage larvae of the mosquitoes, Aedes malayensis and Aedes pseudoscutellaris is described. The cells of the A. malayensis line are diploid while those of A. pseudoscutellaris are predominantly polyploid. West Nile and Japanese B encephalitis viruses produced a cytopathic effect (CPE) in the A. malayensis cells. Both these viruses and Dengue-2 virus also produced a cytopathic effect in the A. pseudoscutellaris cells. There was some evidence that the CPE was influenced by the nature of the container. A. malayensis cells of early subcultures grown in Falcon plastic flasks formed large syncytia while those of later subcultures infected under identical conditions produced only small syncytia. Cells of the A. malayensis line grown on glass did not show any cytopathic response in early subcultures, but in cells of higher subcultures grown on glass, the viruses produced a definite CPE presenting as degenerate clumps of cells and floating single cells. It is suggested that the different result is due to continual adaptation of the cells to the culture conditions in the early stages. Cells of the A. pseudoscutellaris line, following infection with viruses formed syncytia, clearly visible on plastic surfaces, but less obvious on glass. An unadapted strain of Dengue-2 in infected human serum produced syncytia in the cells, but did not produce any plaques in a stable line in pig kidney cells. The infected cultures recovered after some days and formed healthy monolayers. Such cells were subcultured without showing any cytopathic response, but continued to produce virus.

Cytopathic effect and plaque formation by arboviruses in a continuous cell line (XTC-2) from the toad Xenopus laevis.

Forty-six arboviruses were tested for c.p.e. and/or plaque formation in an amphibian cell line. C.p.e. was observed with a high proportion of the viruses tested. Comparative plaque assay, in the XTC-2 cells at 28 degrees C and Vero cells at 37 degrees C, suggests that these systems are comparable in sensitivity and susceptibility to infection. Practical uses of this cell line are discussed.

Resistance and cross‐resistance in guinea‐pigs and rabbits to immature stages of ixodid ticks

ABSTRACT. Infestation of guinea‐pigs and rabbits with larvae of any one of five species of ticks, Rhipicephalus appendiculatus Neumann, Rhipicephalus evertsi evertsi Neumann, Amblyomma hebrauem Koch, Amblyomma variegatum Fabricius and Ixodes ricinus L., conferred resistance in the animals when exposed to subsequent infestations with the same tick species. Resistance to infestations by other tick species was not observed.

Isolations in a Mosquito (Aedes pseudoscutellaris) Cell Line (Mos. 61) of Yellow Fever Virus Strains from Original Field Material

A simple, rapid and inexpensive method of isolating yellow fever (YF) virus from naturally infected mosquitoes, human liver and the serum of a sentinel monkey by inoculation of a continuous line of mosquito cells is described. The mosquito cells were more sensitive than suckling mice and marginally better than Vero cells for primary isolation. This is the first time that mosquito cells have been successfully used for primary isolation of YF virus from field material.

Resistance and cross-resistance in rabbits to adults of three species of African ticks (Acari: Ixodidae)

Resistance to Rhipicephalus appendiculatus, Amblyomma variegatum and Amblyomma hebraeum was investigated in the laboratory by infesting rabbits with adults of each of the three species followed by homospecific or heterospecific secondary infestations. Significantly lower female engorged weights and egg mass weights were taken as evidence of protective immunity. Following a single infestation with adults, rabbits developed homospecific protective immunity (resistance) to only R. appendiculatus and A. hebraeum; primary infestation with A variegatum did not protect against secondary infestation with the same species. There was no cross-resistance (heterospecific protective immunity) between the species except for one-way protection between R. appendiculatus and A. variegatum; primary infestation with R. appendiculatus protected against secondary infestation with A. variegatum, but not vice versa. The results from ELISA did not indicate any correlation between serum antibodies to soluble antigens from salivary gland extracts and protective immunity. Post-infestation sera from rabbits infested with each of the three species reacted strongly to their respective salivary gland extracts. Despite the high reactivity of A. variegatum serum with salivary gland antigens from all three species, A. variegatum-infested rabbits did not show any homospecific or heterospecific immunity; on the other hand, although R. appendiculatus serum did not react positively to A. variegatum antigens, infestation with R. appendiculatus protected against a subsequent A. variegatum infestation.

Infection of the mosquito Aedes aegypti with infectious west Nile virus-antibody complexes

Aedes aegypti fed through chick skin membranes on West Nile virus-homologous antiserum mixtures shown by an anti-globulin neutralization test to be highly infectious complexes (in terms of plaque formation in tissue culture) failed to become infected. Control mosquitoes fed on West Nile virus--normal rabbit serum mixtures containing similar or smaller amounts of infectious virus were shown to become infected. Mosquitoes ingesting suspensions of West Nile virus previously incubated with Murray Valley encephalitis or Ntaya antiserum became infected at significantly lower rates (P = less than 0-05) than controls ingesting West Nile virus-normal rabbit serum mixtures. West Nile virus--17D yellow fever or dengue--2 antiserum mixtures did not produce significantly reduced infection rates in Ae. aegypti when compared to controls.

Infection of an Aedes aegypti cell line with infectious arbovirus-antibody complexes

Aedes aegypti cells exposed to infectious complexes of WN or YF virus and homologous antiserum produced lower yields of virus over a 10-day observation period than were produced by Aedes aegypti cells treated with a comparable dose of virus mixed with non-immune serum. When Ae. aegypti cells were infected with WN virus mixed with MVE, NTA, DEN-2 or YF antisera the virus yield over 10 days was lower than in cell cultures infected at similar titres with mixtures of WN virus with non-immune serum. If Ae. aegypti cell cultures were infected with mixtures of YF virus and WN or DEN-2 antiserum the resulting production of virus was lower over 10 days than in virus mixed with non-immune serum. Human serum samples from the field were tested for the presence of antibody by preincubation of the serum with WN virus prior to inoculation on to mosquito cell cultures. The results indicated that this method is as sensitive in detecting antibody as a mouse neutralization test using regression analysis of average survival time.

The role ofRhipicephalus appendiculatus andR. evertsi evertsi males in inducing resistance in laboratory animals: preliminary studies

Guinea-pigs infested with male ticks of the speciesRhipicephalus appendiculatus, and rabbits infested withR. evertsi evertsi, acquired immunity to conspecific female ticks. The hosts were first infested with male ticks and thereafter were challenged with males and females of the same species. The mean weight of the engorged females ofR. appendiculatus fed on guinea pigs previously infested with male ticks was 509.0 (±41.4) mg compared with that of females fed on control guinea pigs (651.2±31.8 mg). Similar weight differences were observed forR.e. evertsi females which fed on rabbits previously infested three times with male ticks. The mean weight of the female ticks which fed on these rabbits was 520.1 (±29.8) mg compared with 640.7 (±30.2) mg ofR.e. evertsi females which fed on control hosts. The concentration of gammaglobulins in the sera of rabbits was monitored at various intervals after the first infestation. It was found, for the first time, that infestation of laboratory animals with male ticks conferred immunity, but to a lesser degree than infestation with both sexes. It was also shown that the level of gammaglobulins increased from 3.4±0.28 g l−1 to 7.3±0.24 g l−1 in sera of rabbits hosts as a result of the feeding activity of males, but to a lesser extent than in sera of rabbits on which both sexes had fed (10.8±2.4 g l−1).