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Isolation of arboviruses in Kenya, 1966–1971
114 TRANSACTIONSOF THE ROYAL SOCIETYOF TROPICALMEDICINEAND HYGIENE.VoL 68. No. 2. 1974.
ISOLATION OF ARBOVIRUSES IN KENYA, 1966-1971 D. METSELAAR
(Medical Research Centre, Nairobi, Department of the Royal Tropical Institute of Amsterdam) B. E. H E N D E R S O N
Department of Pathology, USC School of Medicine, Los Angeles, California, G. B. KIRYA, P. M. T U K E I
East African Virus Research Institute, Entebbe A. DE GEUS
Introduction In 1966 of more than 200 arboviruses described from all over the world, 48 had been isolated in Africa, but only 7 of them were from Kenya. Outbreaks of disease in domestic animals had led to the isolation of Rift Valley Fever, African Horse Sickness, Bluetongue and Nairobi Sheep disease viruses. Continued interest in diseases and disease vectors of domestic animals by veterinarians in Kenya resulted in the isolation of Thogoto virus from ticks. In addition O'nyong-nyong virus was isolated from febrile patients and mosquitoes and Nyando virus from mosquitoes (quoted in Taylor's catalogue, 1967). More recently the isolation of Congo virus from a cow and Mount Elgon Bat virus from the salivary glands of a bat were reported (SIMPSON et al., 1967; /~£ETSELAARet al., 1969). That other arboviruses were also being transmitted within Kenya was suggested by the results obtained from serological surveys carried out in 1936 (SAWYER and WHITMAN) and 1952 (HAvDOW) when humans with yellow fever antibodies were observed. More evidence was obtained when antibodies against Wesselsbron and West Nile (HENDERSON et al., 1970a) and Sindbis and Bunyamwera viruses (G~.S~.Ret al., 1970) were detected in human sera. Although these past investigations have yielded valuable information, systematic surveillance of arbovirus activity in Kenya was not started until recently. The present work was initiated in 1966 to provide distributional information on viruses being transmitted by biting arthropods; an attempt was made to isolate viruses from mosquitoes and from the serum of patients suffering from fever. The present communication is a brief review of this work.
Mosquito breeding, climate and collection areas In most of Kenya, there are 2 rainy seasons. The main season is from April to June, and a second season is from October to November. In much of the coastal plain, mosquito collections can be organized the whole year round because of permanent marshes and occasional showers in the dryer periods. Part of the marshes are tidal and may produce large numbers of Aedes (Skusea) pembaensis Theobald, which breeds in association with the crab Sesarma meinerti de Man ( G o n ~ et al., 1957). On the plateau of Kenya, peri-annual mosquito breeding occurs in the marshy borders of the great lakes. Away from these lakes, mosquito collections are more directly dependent on the rainy season. 5 coastal areas and 2 plateau areas were chosen for the mosquito catches; in one of the coastal areas human blood was also collected (Map). Port Reitz is located at sea level near Mombasa; the region contains extensive tidal marshes. Collections were made between 29 June and 5 August, 1966. Mtwapa and Kikambala are located along the coast 16-24 km. north of Mombasa. Mtwapa is surrounded by extensive marshes, partly tidal; near Kikambala, there is a patch of broken forest. Collections were made between 4 October and 10 December 1967. Thanks are due to the Senior Parasitologist and staff members of the Division of Vector-Borne Diseases of the National Public Health Laboratory Services of the Kenya Government, to the Senior Research Officer and staff members of the Veterinary Department and to the Medical Officers at Msambweni Hospital for hospitality received in field laboratories and on premises. The assistance of Mrs. E. C. C. van Someren, who verified the identifications of the mosquitoes and corrected some, is gratefully acknowledged.
D. METSELAAR, B. E. HENDERSON, G. B. KIRYA, P. M. TUKEI AND A. DE GEUS
115
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Malindi is located 120 kin. north of Mombasa. The area is surrounded by many marshes. Marshes are present also in Sokoke Forest, an extensive broken forest west and south of the township. Collections were made during 15 November-19 December 1966; 9 March-23 April 1968; 7 December 1968-5 February 1969 and 7 November-19 December 1969. Garsen is located on the TaRa River, 110 kin. north of Malindi and 45 km. inland from the Indian Ocean. Downstream from Garsen the river flows through extensive permanent marshes. Collections were made between 2 and 8 March 1968. Kwale District is a coastal district south of Mombasa. The landscape is like that at Mtwapa, but with only a few marshes. Until 1969 human sera were collected in Kwale District as part of a comprehensive study of patients with febrile disease of unknown origin (DE GEUS et al., 1969). After termination of this study the area was visited during 7-30 July 1970; 8 November-2 December 1970; 19 February-11 March 1971 and 17 June-30 July 1971. During the latter 4 visits mosquitoes were collected over a wide area, and human blood was collected in one of the hospitals. Lake Naivasha is a fresh water lake with a marshy border zone located in the Rift Valley, 2,000 m. above sea level. The lake has an abundant bird population, including migrant species. Collections were made between 1 August and 20 September 1967. Langata and Ngong Forests, 2 adjacent forests, are part of an extensive forested area located near Nairobi at an altitude oI 1,800 m. Collections were made periodically during the wet seasons of 1967 and 1968. The coUections of mosquitoes in Sokoke Forest were initiated because of the previous finding of yellow fever immunity in non-human primates in this forest (SMITHBURN, 1949). The collections at Ngong and Langata Forest were organized because of strong indications that in 1943 a yellow fever patient had contracted infection in this forest (H~DoW, 1964). Materials and methods
Collection and storage of mosquitoes The majority of mosquitoes were collected from man at ground level. Catches were conducted in the
116
ISOLATION OF ARBOVIRUSES IN KENYA, 1966--1971
afternoon, in the evening, or occasionally throughout the night. Additional collections were made from man on tree platforms. Mosquitoes were identified in the field, killed with chloroform and pooled according to species for attempted virus isolation. Those species in which the females could not be differentiated were combined in groups. The mosquitoes pooled during 1966-1967 were kept frozen at --20°C. until inoculated into infant mice brought to the field; from 1968 onwards, however, the pools were sealed in plastic bags, stored in liquid nitrogen (WOLFF and CROON, 1967) and sent to Nairobi for inoculation.
Virus isolationsfrom mosquito pools and human sera Mosquito pools were triturated with a cooled mortar and pestle and suspended in cooled diluent (0.75% bovine plasma albumin--Armour Fraction V--in phosphate-buffered saline, pH 7.4). The pool sizes varied with the availability of mouse litters and the numbers of mosquitoes collected of each species. The mosquito suspensions were clarified by centrifugation at 4°C. and inoculated into litters of 8 newborn mice by 3 routes: intracerebrally (IC) (0.01 ml.), intraperitoneally (0.02 ml.) and subcutaneously (0.03 ml.). The remaining suspension was stored at --20°C. or --70°C. for reisolation attempts. The mice were observed daily for 3 weeks. Brains of sick mice were diluted 1 : 500 and passaged by the IC route into newborn mice. With one exception, established virus strains were sent to the East African Virus Research Institute, Entebbe, Uganda for identification. 5 virus strains that could not be identified in Entebbe have been fowarded to the Yale Arbovirus Research Unit (YARU) at New Haven, U.S.A. If possible, blood was taken by venepuncture. Otherwise fmgerprick blood was collected in small tubes containing phosphate buffered saline. After centrifugation, sera were stored in liquid nitrogen and sent to Nairobi for inoculation into litters of newborn mice, after which they were kept for reisolation attempts. Sera were inoculated into mice irrespective ot the presence of malaria parasites in Giemsa stained thick films. Haemagglutination-inhibition (HI) and/or complement fixation (CF) tests were performed for screening isolates against antisera of viruses representative of East Africa and, if necessary, sera of viruses from other areas were also used. Final identification was done by means of cross-neutralization tests CHIT). Antigens were prepared by either fluorocarbon (Arcton 113) or sucrose acetone extraction of infected suckling mouse brain or liver. Hyperimmune mouse antiserum was prepared by 4 sequential intraperitoneal inoculations of a 10% suspension of infected suckling mouse brain on days 7, 14, 21 and 28. HI tests were performed according to the method of CLARKEand CASALS(1958) and CF tests according to CASEY (1965); both tests were performed in microtitre plates. N T were done by the constant serum-varying virus dilution technique. Equal volumes of serum and virus dilution were mixed, incubated at 37°C for 1 hour and inoculated by the IC route into litters of 5 newborn or into 6 adult mice. Results Approximately 146,300 mosquitoes were collected in the 7 areas of Kenya studied, comprising 2 subfamilies, 5 genera and 50 species or groups of species and were processed in 1,420 pools and inoculated into suckling mice. The results of the mosquito collections, the number of isolations and the identifications that have been made so far, are summarized in Tables I and II. From 14 species or groups of species, 1 or more virus strains were isolated, giving a total of 34 strains. Moreover, viruses were isolated from 4 of the 419 sera from patients with febrile diseases. Of the 38 strains isolated, 12 remain, so far, unidentified of which several probably are hitherto undescribed viruses. The remaining 26 isolates include strains of arbovirus group A (Middelburg and Semliki Forest), group B (Banzi and Wesselsbron), the Bunyamwera group (Btmyamwera and Biliefe), Simbu group (Sango), Bwamba group (Pongola), California group (Tahyna) and 2 tmgrouped viruses (AR 1169/64 and Rift Valley Fever). Apart from yellow fever vaccine virus, no arboviruses are kept in our laboratory other than from those isolated by us in Kenya. First and single isolates are, therefore, genuine even when no re-isolation was achieved. Of the others, the possibility of laboratory pick-up was considered by studying the protocols. 2 isolates that could be pick-ups were omitted from the descriptions.
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I S O L A T I O N OF ARBOVIRUSES I N K E N Y A ,
1966-1971
TABLEII. Summaryof isolations. Locality of isolation
Strain PRMP -MMP
Port Reitz --
Aedes pembaensis
Maiindi
Mansonia
---
30 37 47 158 179
--
249
--
---------
312 346 379 433 483 498 525 607 626 636 637 714 766 824 52 54
-----
-----
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63 117 131 160 26 49 67 68 71 4 36 449 462 62 335
--
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Culex antennatus Anopheles coustani Mansonia africana Culex zombaensis Mansonia
uniformis
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Culex thalassius-trit-group Anopheles coustani Culex
antennatus
Culex sitiens Anopheles coustani Mansonia uniformis Anopheles coustani Eretmapodites subsimplicipes Aedes pembaensis ,, ,,
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Aedes cummmstz Anopheles eoustani Culex nakuruensis ,,
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Culex rubinotus Mansonia africana Aedes dentatus ,
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H u m a n serum ,,
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Date collected
180 425 450 100 100 20
26- 7-66 28- 7-66 5-12-66 30- 3-68 27- 3-68 2 0 - 3--68 4-- 4 - 6 8 11-12-68 10-12--68 29- 1-69 19- 1-69 30- 1-69 17- 1-69 11-11-69 12-11-69 12-11-69 27-11-69 6-12-69 7-12-69 16-12-69 19-10--67 21-10--67 30-10-67 9-11-67 21-11--67 30-11-67 13-12-67 6- 3-68 23-- 8 - 6 7 8- 9-67 5- 9-67 8- 9-67 22- 7-68 28-11-68 13-11-68 9- 1-69 10-11-70 1 9 - 6--71
100 100
Mansonia africana
--
Garsen Naivasha
umformis
Pool size
,,
15 75 90 95 40 65 100 85 5 100 100 95 5 140 140 200 110 150 40 60 145 290 230 385 100 65
Results of identification
Re-isolation No
Yes No No No No
Yes No Yes No No
Yes No N.D. No No
Yes N.D. N.D. N.D. No
Yes Yes
Yes No No No No No No No
Tahyna Tahyna Sango AR
1169/64
Btmyamwera Pongola new?* Bunyamwera new? Shokwe? new? Bunyamwera Biliefe Middelburg Middelburg
Middelburg in process new? AR
1169/64
new?* Btmyamwera Bunyamwera n e w ?* Bunyamwera new?*
Bunyamwera Bunyamwera A A A
Ungrouped Bunyamwera Bunyamwera Bunyamwera A
Pongola
Bwamba
Banzi Banzi Banzi
B B B B A B
Banzi Sendiki Forest Wesselsbron i n process
Yes
Bunyamwera
Middelburg
No N.D. Yes
California California Simbu Ungrouped Bunyamwera Bwamba
new?*
Yes Yes Yes
Virus group
Biliefe process Rift Valley Fever
Bunyamwera
in
Ungrouped
not done. to Yale Arbovirus Research U n i t .
Discussion
Middelburg virus was repeatedly isolated in South Africa from Aedes (Ochlerotatus) caballas Theobald during an epizootic amongst sheep in which the virus and Wesselsbron virus were involved (KoKERNOT et al., 1957a). On the same occasion and later the virus was isolated several times from a number of other Aedes species. In West and Central Africa pools of Mansonia (Mansonioides) africana (Theobald), Mansonia (Mansonioides) uniformis Theobald, several Aedes species and mixed pools of mosquitoes yielded the virus. We isolated Middelburg virus from Aedes cumminsii group mosquitoes, Anopheles (Anopheles) coustani group, Culex (Culex) antennatus (Becket) and Gulex (Culex) sitiens Wiedemann (Table II). We succeeded in biological transmission of this virus between suckling mice by means of Aedes (Stegomyia) aegypti (Linnaeus) mosquitoes. Most of the mosquitoes from which the virus was isolated are mosquitoes usually breeding and biting in open country, not in forests. This is in accordance with the finding that in Tongaland, South Africa, up to 24% of sera of cattle and sheep had neutralizing antibodies (NA) (KOKEmqOTet al., 1961). Biological transmission between Ae. caballas and sheep was achieved by KOKEmqOT et al. (1957a). Sheep, cattle and Ae. caballas may, therefore, play a r61e in the dissemination of the virus. So far, the virus was not isolated from other vertebrate hosts, including humans. Of hundreds of human sera from countries in the southern part of Africa only a few showed antibodies (quoted by McIntosh in Taylor's catalogue, 1967). Semliki Forest Virus (SFV), first isolated in Uganda from Aedes (Aedimorphus) abnormalis Theobald collected in a woodland belt (SMIT~mORN and HADDOW, 1944) was further isolated from Aedes (Aedimorphus) argemeopunctatus Theobald in Mozambique (McINToSH et al., 1961); from males of Eretmapodites grahami Edwards (STO~qES, 1960) in Cameroon; from Anopheles (Cellia) funestus Giles in Kenya (HENDERSON et al., 1970a) and from Aedes (Neomelaniconeon) palpalis in Central African Republic (Institute Pasteur, Bangui, 1970). Most ff not all of these species are associated with forest or the mosquitoes that yielded the virus had been collected at the forest edge. The isolation reported in this paper is from Ae. (Aedimorphus) dentatus group collected in Ngong Forest. Elsewhere in Africa the virus was isolated once from a hedgehog and twice from birds of different species one of which is associated with woodland. In Africa the occurrence of human sera positive for NA is generally low. However, in Zalre higher figures were found in inhabitants of bush savannah and river border forests. In these environments high values for NA
D. METSELAAR, B. E. HENDERSON, G. B. KIRYA, P. M. TUKEI AND A. DE GEUS
119
were also found in chimpanzees (quoted by Woodall in Taylor's catalogue, 1967.) NA have also been found in domestic quadrupeds in South Africa (KOKERNOT et al., 1961). SFV has not been associated with naturally occurring human disease. It is frequently used as a laboratory agent but only 1--subclinical-laboratory infection has been reported (CLARKE,1961). The variety of mosquitoes capable of carrying the virus in nature and the various genera which are capable of transmitting it in the laboratory (quoted by WoodaU in Taylor's catalogue, 1967) may provide an explanation for its wide geographical distribution. From the isolation in nature and serology it may be concluded that Semliki Forest virus is more associated with woodland and forests than with open grassland. Banzi virus was originally isolated from blood of a patient with wdld fever and from a pool of Culex (Neoculex) rubinotus Theobald in South Africa (SMITHBURNet al., 1959b) subsequently from the blood of a feverish child in Tanzania (WoODALLet al., 1962). The mosquitoes from which Banzi virus was isolated in South Africa and Kenya (Table II) may indicate that the risk of infection is more associated with open country than with forests. So far only mild disease has been observed. Wesselsbron virus is the cause of epizo6tics in sheep and was first isolated in South Africa by WEISZ et al. (1956) from a lamb. Subsequently it was isolated from mosquitoes belonging to the Anopheles subfamily and Aedes, Mansonia and Culex genera. Isolations from mosquitoes were made in South and West Africa and in Thailand. More isolations were made in Africa from a camel and a gerbil (Desmodillus) species. Several natural and laboratory infections in man have been reported. Relatively mild illness was observed. Serological surveys in South Africa, Mozambique and Angola showed that 507 out of 4,293 (11.8%) human sera and 254 out of 762 (33.3%) sera of sheep, cattle and goats showed NA (quoted by Mclntosh in Taylor's catalogue, 1967; Institute Pasteur Dakar, 1971; MClNTOSH et al., 1972). MAURICE and PROVOST(1969) found high rates of haemagglutlnation inhibiting antibodies (HIA) in sheep, goats and antelopes in Chad and Cameroon, ROOHAINet al. (1972) found a kori bustard (Audeatis kori) with these antibodies in South Ethiopia. The sources of the isolations and serology indicate that the virus may be more associated with open country than with forest. The isolation in Kenya from Ae. dentatus in Ngong Forest should then be an exception. Ae. dentatus however, takes, an intermediate position and frequently rests and bites at the forest edge (METSELAARet al., 1973). Serological tests in Kenya indicated that the virus may circulate in the northern and eastern (Coastal) parts of Kenya, to a lesser extent in the western parts (HENDERSONet al., 1970a; GESERet al., 1970). Because in northern Kenya Wesselsbron immunity seemed to parallel that of West Nile virus--probably a bird virus--HENDERSON et al. (1970a) suggested that Wesselsbron virus is also disseminated by birds. GESERet al. (1970) expressed the opinion, supported by experiments with Wesselsbrou virus (HENDERSONet al., 1970b), that at the Kenya Coast immunity to arboviruses of group B could play a r61e in the protection of the population against manifest infections of yellow fever. Bunyamwera virus was first isolated in 1943 ill Uganda from a pool of 14 Aedes species (SMITHBURN et al., 1946). Since then it has been recovered from Aedes (Neomelaniconeon) circumluteolus Theobald (KOKERNOT et al., 1958; MClNTOSH et al., 1972) and Ae. pembaensis (quoted by Woodall in Taylor's catalogue, 1967) in South Africa, M. africana in Nigeria (DooRMANand DRAPER, 1968) from Culex species in the Central African Republic (Institute Pasteur Bangui, 1971) and South Africa (MclNTOSH et al., 1972) and from Aedes (Finlaya) ingrami Edwards in Cameroon (Institute Pasteur Dakar, 1971). Apart from some of these mosquitoes we isolated the virus from M. uniformis and Eretmapodites subsimplicipes Edwards (Table II). From man it has been isolated in South Africa (KOKERNOTet al., 1958); Nigeria (BEARCROFT et al., 1963) and Uganda (WEINBRENet al., 1959). Mild disease, once with neck stiffness, once with blurring of vision was reported. The natural vertebrate host(s) of Bunyamwera virus are still unknown. Several related viruses, grouped together in the Bunyamwera group (CASALSand WHITMAN,1960) have been found in and outside Africa and because of serological overlapping, results of serological surveys should be interpreted with caution. Taking this into account NA have been found in substantial proportions (sometimes more than 30%) of human sera from various parts of Africa, from Borneo and South America. Antibodies have also been found in chimpanzee sera from Zaire (Congo) artd sera of domestic animals from South Africa (quoted by Woodall in Taylor's catalogue, 1967). In West Kenya, BOWEN et al. (1973) found neutralizing antibodies in 18 out of 161 sera (11%) from people of 10 years of age and older. GESER et al. (1970) found HIA in 52 out of 822 (6%) of human sera of all ages from West Kenya, in 58 out of 1,042 (5-5%) from Central Kenya and in 221 out of 834 (26.5%) from a coastal area of Kenya. In and near the latter area we isolated 6 strains of Bunyamwera virus. The various mosquito species from which the virus was isolated point to a wide variety of habitats of the virus. Probably man frequently penetrates these habitats and risks infection with Bunyamwera virus. In view of the substantial proportion of human sera
120
ISOLATIONOF ARBOVlRUSESIN KENYA, 1966-1971
positive with Bunyamwera (or related) antibodies and the infrequent observation of disease, most infections are presumably mild or subclinical. Beliefe virus was isolated in Uganda from a pool of Aedes abnormalis/tarsalis. HENDERSONet al. (1968) are of the opinion that it is different from other members of the Bunyamwera group and intermediate between Bunyamwera and Shokwe viruses. We isolated the virus in the coastal area of Kenya, once from mosquitoes, once from a patient. The patient, an adult male, suffered a fever for 3 days with rigors and a temperature peak of 103.6°F. He complained of some joint pains 2 weeks after discharge from the hospital. The natural vertebrate host(s) of Beliefe virus are unknown. Sango virus was repeatedly isalated from cattle in Nigeria, the first time was from serum of a cow at the slaughter house of Ibadan (CAusw¢ et al., 1972). The same authors isolated the virus from a pool of Culicoides. Our strain was isolated from M. uniformis collected in an area where herds of cattle frequently pass when driven to the slaughter-house of Mombasa. Nothing is known about the possible r61e of Sango virus Jn human disease. It is likely that cattle play a r61e in its dissemination. CAUSEYet al. did not isolate the virus from thousands of other vertebrate specimens.
Pongola virus was first isolated from Ae. circumluteolus in South Africa (Kor~RNOT et al., 1957b). Further isolations in that country were made from the same mosquito, a pool of M. uniformis and M. africana, from a pool of M. uniformis and a mixed pool of mosquitoes. (WORTH et al., 1960; MCINTOSH et al., 1972). In Uganda, Nigeria and the Central African Republic the virus was isolated from M. africana (WooDALL et al., 1962; BOORMANand D~PER, 1968; Institute Pasteur Bangui, 1971). In North Kenya it was isolated from M. africana, Culex (Culex) zombaensis Theobald and Aedes dentatus (HENDERSON et al., 1970a). 2 more vectors were found in the coastal plain of Kenya (Table II). The large number of vector species may indicate that Pongola virus favours a variety of habitats. Virus isolation from man or possible vertebrate hosts have not been reported. Serological surveys showed NA in 55 out of 427 (13%) sera of domestic animals and in 470 out of 2,813 (16.7%) human sera from South Africa, Angola and Mozambique. In South Africa the residents with antibodies were predominantly found in tropical and lowland areas, less in highland areas. (SMITHBURNet al., 1959a; MCINTOSHet al., 1962a, b; KOKERNOTet al., 1965a, b). It is likely therefore, that Pongola virus is widespread in Africa and has a wide range of vectors. Nothing is known about the natural vertebrate host(s) of Pongola virus. The vector species and the place of 15 isolates in Kenya do not suggest association with forest habitats of the virus. Domestic animals may play a r61e in its dissemination. Notwithstanding serological evidence of human infections little is known of disease in man. The name Tahyna has recently been given priority over Lumbo for a member of the California group of viruses isolated in Africa. Tahyna virus from Europe and Lumbo virus from Africa had at first been regarded as different viruses, but subsequent examinations made it clear that differentiation is not justified. In Europe Tahyna virus was isolated from Aedes species (quoted by Bardos in Taylor's catalogue, 1967). In Africa the virus was first isolated from Ae. pembaensis at Lumbo in a coastal plain of Mozambique, later by us from the same species at the coast near Mombasa. In Europe hares and rabbits are probably vertebrate hosts of the virus. A high proportion of hares in Czechoslovakia have antibodies (SIMKOVA, 1966) and HANNOUN(1968) isolated the virus from a rabbit in south France. The virus was isolated twice from human sera during a survey of healthy people ia Central Europe. In that part of the world Tahyna virus has been associated with febrile illness and an influenza-like syndrome. Under these circumstances the diagnosis was made because of serum conversion (B~DoS and SLUI~, 1963). Serological surveys in Europe have shown that man and his domestic animals are frequently infected (quoted by Bardos in Taylor's catalogue, 1967). In Africa BARDOSand SEFCOVICOVA(1961 ) found NA in 6% of 107 human sera from Uganda; KOKERNOT et al. (1962) in 16 out of" 128 (12.5%) from Mozambique and Natal Province of South Africa, but only in 1 out of 736 human sera from other parts of South Africa and Angola; KuNz et al. (1964) found NA in some of an unstated number of sera from West Africa; RODHAINet al. (1972) detected HIA in 7 sera from adults out of 300 sera from all age groups of people living near the Omo River in South Ethiopia. In Africa virus isolation was not reported from vertebrates. From the above it may be concluded that Tahyna or a related virus probably occurs in areas wide apart in Africa, but has a somewhat patchy distribution. Ae. pembaensis may be an efficient vector and because of the peculiar breeding habits of this mosquito, the virus may be most easily found in coastal plains near tidal marshes. Disease in man is probably infrequent and mild.
D. METSELAAR, B. E. HENDERSON~ G. B. KIRYA~ P. h4. TUKEI AND A. DE GEUS
121
AR 1169/64 virus was first isolated from a pool of M. uniformis in the Sudan (SCHMIDT et al., 1965). We isolated the agent from the same species at Malindi. OKIA and TUKEI (1972) isolated 2 strains from organs of the rodent Praomys jacksoni in Uganda. Other particulars of the virus or its r61e in human disease are not known to us. Rift Valley fever (R VF) virus was first isolated from sheep during a epizo6tic in domestic animals in the Rift Valley in Kenya (DAUBNEYet al., 1931). 4 investigators were infected when handling animals and virus. Later experiences have shown that infections in non-immune veterinarians and laboratory workers are quite common. Natural infections in humans have been reported frequently also, especially from South Africa at times of epizo6tics (GEAR et al., 1951, 1955). Typically, infected patients feel very ill, fever and joint pains are severe, blurring of vision and delirium not infrequent, The disease is, however, seldom if ever fatal. Epizo6tics in sheep and cattle are not uncommon in Africa. SCOTT et al. (1963) found a high level of antibodies in camels in North Kenya, MAURICE and PROVOST(1969) in wild antelopes and buffalo in Chad. These domestic and wild animals therefore, probably play a r61e in the dissemination of the virus. Other vertebrates may be reservoir hosts but these are still unknown. Rodents have been suspected to play a r61e in the natural cycle but SCOTTand HEISH (1959) neither isolated the virus nor detected antibodies in the sera of 285 rodents trapped in the Rift Valley of Kenya. During an epizo6tic in South Africa GEAR et al. (1955) did not find antibodies in 82 rodents either. Only a polecat was positive with NA. Sera of 13 birds and 27 wild animals of 6 species were all negative. PELLISlERand ROUSSELET(1954) examined 122 monkeys of 11 species in 2 institutions of Brazzaville but do not state whether these had been recently captured. 12 sera from 7 species of monkeys of which 113 individuals had been examined showed CF antibodies. On the other hand SMITIIBORNet al. (1948) did not find any NA in sera of 72 wild monkeys of 9 species in an area of Uganda where RVF virus was circulating. At least 10 of the animals had been taken from a forest where shortly before the virus had been isolated from mosquitoes. In Kenya DAVIES et al. (1972) tested sera from 333 baboons, 218 of which from an area where the year before an epizo6tic had been observed. All sera were negative for RVF antibodies. The virus was isolated from pools of Eretmapodites species and Aedes (Aedimorphus) tarsalis Newstead in Uganda (SMITHBURNet al., 1948); from Aedes (Ochlerotatus) caballus Theobald, Culex (Culex) theileri Theobald and Ae. circumluteolus in South Africa (GEAR et al., 1955). Isolations have, therefore, been made in association with forest and grassland country. Our isolate was from a male adult out-patient with mild illness and a temperature of 39.5°C. It was identified by Dr. F. G. Davies at the Veterinary Research Laboratory at Kabete. A serum sample taken a few months later had a neutralizing index of 4.0 logs. Examination of domestic animals in the area where the patient was living did not reveal an epizo6tic (F. G. DAVIES,pers. comm.). The isolate appears, therefore, to have been made from a sporadic case. Yellow fever virus was not isolated in the 2 areas of Kenya where it may have circulated in the past, nor did Ae. aegypti or Aedes (Stegomyia) simpsoni (Theobald), collected at the coast, yield yellow fever or any other virus. We have attempted to differentiate the viruses according to association with forest or more open country. With the information available, more precise definition of the ecology of the viruses was not possible.
Summary The isolation in Kenya of arboviruses from mosquitoes and patients is reported. Middelburg and Semliki Forest virus (group A), Banzi virus (group B), Bunyamwera and Beliefe virus (Bunyamwera group), Sango virus (Simbu group), Pongola virus (Bwamba group), Tahyna virus (California group), ungrouped AR 1169/64 and a number of viruses still to be identified were isolated from mosquitoes. Of the latter some may be hitherto undescribed viruses. Bunyamwera, Rift Valley Fever and 2 unidentified viruses were isolated from patients. The isolates are discussed against the background of information from isolations and serology performed elsewhere. Attempts are made to define their r61e in human disease. Some supertidal suggestions about the ecology are made. REFERENCES BARDOS, V. & SEFCOVlCOVA,L. (1961). J. Hyg. Epidem., Microbiol. Immun., 5, 501. & SLUKA,T. (1963). Cas. Ldk. cesk., 102, 394. BEARCROFT,W. G. C., PORTERFIELD,J. S. • SUTTON,R. N. P. (1963). Trans. R. Soc. trop. Med. Hyg, 57, 308.
122
ISOLATIONOF ARBOVIRUSESIN KENYA,1966--1971
BOORMAN,J. P. T. & DRAPER, C. C. (1968). Ibid., 62, 269. BOWEN, E. T. W., SIMPSON,D. I. H., PLATT,G. S., WAY,H., BRIGHT,W. F., DAY, J., ACHAPA,S. & ROBERTS, J. M. D. (1973). Ibid., 67, 702. CASALS,J. & WHITMAN,L. M. (1960). Am. ft. trop. filed. Hyg., 9, 73. CASEY, H. L. (1965). Publ. Hlth Monogr., No. 74, pp. 31-34. Washington D.C. : U.S. Government Printing Office. CAUSEY,O. R., KEMP, G. E., CAIJSEY,C. E. & LEE, V. H. (1972). Ann. trop. A4ed. Parasit., 66, 357. CLARKE,D. H. (1961). Am. -7. trop. IVied. Hyg., 10, 67. CLARKE,P. H. & CASALS,J. (1958). Ibid., 7, 561. DAImNEY, R., HUDSON,J. R. & GARNI-IAM,P. C. C. (1931). -7. Path. Bact., 34, 545. DAVIES, F. G., CLAUSEN,B. & LUND, L. J. (1972). Trans. R. Soc. trop. Med. Hyg., 66, 363. GEAR, J., DE MEILLON,B., MEASROCH,V. & DAVIES,D. H. (1951). S. AfT. med. -7, 25, 908. ---, - - , LE ROUX, A. F., KOFSKY, R., INES, R. R., STEYN, J. Y., OLEFF,W. D. & SCHULTZ,K. H. (1955). Ibid., 29, 514. GESER, A., HENDERSON,B. E. & CHRISTENSEN,S. (1970). Bull. Wld Hlth Org., 43, 539. GEUS, A. DE, KRANENDONK,O. & BOHLANDER,H. J. (1969). E. Afr. reed. -7, 46, 491. GOINY, H., VAN SOMEREN,E. C. C. & HEISH, R. H. (1957). Ibid., 34, 1. HADDOW,A. J. (1952). Ann. trop. 214ed. Parasit., 46, 135. (1964). Trans. R. Soc. trop. 2tffed. Hyg., 59, 436. HANNOUN, CL. (1968). 8th Int. Congr. trop. ivied. 2~Ialar., Teheran, p. 674. HENDERSON,B. E., KIRYA,G. B., LULE, M. & SEKYALO,E. (1968). E.A. Virus Research Inst., Annual Report, 1968, p. 29. , METSELAAR,n., KIRYA, G. B. & TIMMS, G. L. (1970a). Bull. IlTld Hlth Org., 42, 787. , CHESHIRE,P. P., KIRYA, G. B. & LULE, M. (1970b). Am. -7. trop. Med. Hyg., 19, 110. INSTITUTEPASTEURBANGUI.Annual Reports for the years 1970 and 1971. INSTITUTEPASTEURDAKAR.Annual Report for the year 1971. KOKERNOT,R. H., DE MEILLON,B., PATERSON,H. E., HEYMAN,C. S. & SMITImlmN, K. C. (1957a). S. Aft. J. med. ScL, 22, 145. , SMITI-mURN,K. C., WEINBREN,M. P. & DE MEILLON,B. (1957b). Ibid., 22,~1. - - , --, DE M~ILLON, B. & PATERSON,H. E. (1958). Am. J. trop. Med. Hyg., 7, 579. , - & KLIIOE,E. (1961). Ann. trop. Med. Parasit., 55, 73. •, MCINToSH, B. M., WORTH,C. B., DE MORALS,T. & WEINBREN,M. P. (1962). A m . J . trop. Med. Hyg., 11, 678. ~., SZLAMP,E. L., LEVlTH, J. & MCINTOSH, B. M. (1965a). Trans. R. Soc. trop. Med. Hyg., 59, 553. ~ , CASACA,V. M. R., WEINBREN,M. P. & MClNTOSH, B. M. (1965b). Ibid., 59, 563. KrJNZ, CH., BUCKLEY,S. M. & CASALS,J. (1964). Am. -7. trop. Med. Hyg., 13, 738. MCINTOSH, B. M., WORTH, C. & KOKERNOT,R. H. (1961). Trans. R. Soc. trop. Med. Hyg., 55, 192. , SERAFINI,E. T., DICKINSON,D. B. & WEINBREN,M. P. (1962a). S. AfT. -7. reed. Sci., 27, 77. ~., DICKINSON,D. B., SERAFINI,E. T. & DE SOOSA,J. (1962b). Ibid., 27, 87. , JuPP, P. G. & DE SOUSA,J. (1972). J. reed. Ent., 9, 155. MAlmlca~, Y. & PROVOST,A. (1969). Revue Elev. Mdd. vdt. Pays trop., 22, 179. METSELAAR,D., VAN SOMEREN,E. C. C., OUMA,J. H., KOSKEI,H. K. & GEMERT, W. (1973) Bull. ent. Res., 62, 597. , WILLIAMS,M. C., SIMPSON, D. I. H., WEST, R. & MIITERE, F. A. (1969). Arch. ges. Virusforsch., 26, 183. OKIA, N. O. & TuKEI, P. M. (1972). Abstr. annu. Sci. Conf. E.A. med. Res. Counc., Dar-es-Salaam. PELLISSIER,A. & ROUSSELET,R. (1954). Bull. Soc. Path. exot., 47, 228. RODHAIN, F., HANNOUN,C. & METSELAAR,D. (1972). Bull. IVld Hlth Org., 47, 295. SAWYER,W. A. & WHITMAN,L. (1936). Trans. R. Soc. trop. Med. Hyg., 29, 397. SCHMIDT, J. R., WILLIAMS,M. C., LULE, M., MIVlJLE, A. & MtlJOMBA, E. E.A. Virus Res. Inst., Annual Report of the year 1965. p. 24. SCOTT, G. R., COACKLEY,W., ROACH,R. W. & COWDY,N. R. (1963). -7. Path. Bact., 86, 229. & HEISCH, R. B. (1959). E. Afr. reed. -7., 12, 665. SIMKOVA,A. (1966). Cslkd. Epidem. 2klikrobiol. Imunol., 15, 304. SIMPSON, D. I. H., KNIGHT, E. M., COURTOIS,G., WILLIAMS,i . C., WEINBREN,i . e. & KIBUKAMUSOKE, J. W. (1967). E. Afr. med. -7, 44, 87. SMITHBURN, K. C. (1949). Am..7. trop. Med., 29, 389. & HADDOW,A. J. (1944). -7. Immunol., 49, 141. --, - & MAHAFFY,A. F. (1946). Am. -7. trop. Med., 26, 189. ,- & GILLETT,J. D. (1948). Br. -7. exp. Path., 29, 107. ~ , KOKERNOT,R. H., HEYMAN,C. S., WEINBREN,M. P. & ZENTKOWSKY,n . (1959a). S. Afr. med. -7., 33, 555. ~ , PATERSON,H. E., HEYMAN,C. S. & WINTER,P. A. D. (1959b). Ibid., 33, 959. STONES, P. B. (1960). Trans. R. Soc. trop. 214ed. Hyg., 54, 132. TAYLOR, R. M. (1967). Catalogue of Arthropod-Borne Viruses of the Worldj Pub. No. 1760. 1st Ed., U.S. Government Printing Office, Washington, D.C.
D. METSELAAR,B. E. HENDERSON,G. B. KIRYA,P. M. TUKEIAND A. DE GEUS
123
WEINBREN, M. P., KNIGHT, E. M., ELLICE,J. M. & HEWITT, L. E. (1959). Annual Report East Afr. Virus Res. Inst., 1958-59, p. 7. WEISZ, K. E., HAIG, D. A. & ALEXANDER,R. A. (1956). Onderstepoort J., vet. Res., 27, 183. WOLFF, H. L. & CROON,J. J. A. B. (1967). Antonie van Leeuwenhoek~ 33, 235. WOODALL,J. P., WILLIAMS,M. C., SANTOS,D. F. & ELLICE,J. M. (1962). Annual Report East Aft. Virus Res. Inst., 1961-62, p. 21. WORTH, C. B., PATERSON,H. E. & DE MEILON, B. (1960). Am. ft. trop. Med. nyg., 10, 583.
ISOLATION OF ARBOVIRUSES IN KENYA, 1966-1971 D. METSELAAR
(Medical Research Centre, Nairobi, Department of the Royal Tropical Institute of Amsterdam) B. E. H E N D E R S O N
Department of Pathology, USC School of Medicine, Los Angeles, California, G. B. KIRYA, P. M. T U K E I
East African Virus Research Institute, Entebbe A. DE GEUS
Introduction In 1966 of more than 200 arboviruses described from all over the world, 48 had been isolated in Africa, but only 7 of them were from Kenya. Outbreaks of disease in domestic animals had led to the isolation of Rift Valley Fever, African Horse Sickness, Bluetongue and Nairobi Sheep disease viruses. Continued interest in diseases and disease vectors of domestic animals by veterinarians in Kenya resulted in the isolation of Thogoto virus from ticks. In addition O'nyong-nyong virus was isolated from febrile patients and mosquitoes and Nyando virus from mosquitoes (quoted in Taylor's catalogue, 1967). More recently the isolation of Congo virus from a cow and Mount Elgon Bat virus from the salivary glands of a bat were reported (SIMPSON et al., 1967; /~£ETSELAARet al., 1969). That other arboviruses were also being transmitted within Kenya was suggested by the results obtained from serological surveys carried out in 1936 (SAWYER and WHITMAN) and 1952 (HAvDOW) when humans with yellow fever antibodies were observed. More evidence was obtained when antibodies against Wesselsbron and West Nile (HENDERSON et al., 1970a) and Sindbis and Bunyamwera viruses (G~.S~.Ret al., 1970) were detected in human sera. Although these past investigations have yielded valuable information, systematic surveillance of arbovirus activity in Kenya was not started until recently. The present work was initiated in 1966 to provide distributional information on viruses being transmitted by biting arthropods; an attempt was made to isolate viruses from mosquitoes and from the serum of patients suffering from fever. The present communication is a brief review of this work.
Mosquito breeding, climate and collection areas In most of Kenya, there are 2 rainy seasons. The main season is from April to June, and a second season is from October to November. In much of the coastal plain, mosquito collections can be organized the whole year round because of permanent marshes and occasional showers in the dryer periods. Part of the marshes are tidal and may produce large numbers of Aedes (Skusea) pembaensis Theobald, which breeds in association with the crab Sesarma meinerti de Man ( G o n ~ et al., 1957). On the plateau of Kenya, peri-annual mosquito breeding occurs in the marshy borders of the great lakes. Away from these lakes, mosquito collections are more directly dependent on the rainy season. 5 coastal areas and 2 plateau areas were chosen for the mosquito catches; in one of the coastal areas human blood was also collected (Map). Port Reitz is located at sea level near Mombasa; the region contains extensive tidal marshes. Collections were made between 29 June and 5 August, 1966. Mtwapa and Kikambala are located along the coast 16-24 km. north of Mombasa. Mtwapa is surrounded by extensive marshes, partly tidal; near Kikambala, there is a patch of broken forest. Collections were made between 4 October and 10 December 1967. Thanks are due to the Senior Parasitologist and staff members of the Division of Vector-Borne Diseases of the National Public Health Laboratory Services of the Kenya Government, to the Senior Research Officer and staff members of the Veterinary Department and to the Medical Officers at Msambweni Hospital for hospitality received in field laboratories and on premises. The assistance of Mrs. E. C. C. van Someren, who verified the identifications of the mosquitoes and corrected some, is gratefully acknowledged.
D. METSELAAR, B. E. HENDERSON, G. B. KIRYA, P. M. TUKEI AND A. DE GEUS
115
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Malindi is located 120 kin. north of Mombasa. The area is surrounded by many marshes. Marshes are present also in Sokoke Forest, an extensive broken forest west and south of the township. Collections were made during 15 November-19 December 1966; 9 March-23 April 1968; 7 December 1968-5 February 1969 and 7 November-19 December 1969. Garsen is located on the TaRa River, 110 kin. north of Malindi and 45 km. inland from the Indian Ocean. Downstream from Garsen the river flows through extensive permanent marshes. Collections were made between 2 and 8 March 1968. Kwale District is a coastal district south of Mombasa. The landscape is like that at Mtwapa, but with only a few marshes. Until 1969 human sera were collected in Kwale District as part of a comprehensive study of patients with febrile disease of unknown origin (DE GEUS et al., 1969). After termination of this study the area was visited during 7-30 July 1970; 8 November-2 December 1970; 19 February-11 March 1971 and 17 June-30 July 1971. During the latter 4 visits mosquitoes were collected over a wide area, and human blood was collected in one of the hospitals. Lake Naivasha is a fresh water lake with a marshy border zone located in the Rift Valley, 2,000 m. above sea level. The lake has an abundant bird population, including migrant species. Collections were made between 1 August and 20 September 1967. Langata and Ngong Forests, 2 adjacent forests, are part of an extensive forested area located near Nairobi at an altitude oI 1,800 m. Collections were made periodically during the wet seasons of 1967 and 1968. The coUections of mosquitoes in Sokoke Forest were initiated because of the previous finding of yellow fever immunity in non-human primates in this forest (SMITHBURN, 1949). The collections at Ngong and Langata Forest were organized because of strong indications that in 1943 a yellow fever patient had contracted infection in this forest (H~DoW, 1964). Materials and methods
Collection and storage of mosquitoes The majority of mosquitoes were collected from man at ground level. Catches were conducted in the
116
ISOLATION OF ARBOVIRUSES IN KENYA, 1966--1971
afternoon, in the evening, or occasionally throughout the night. Additional collections were made from man on tree platforms. Mosquitoes were identified in the field, killed with chloroform and pooled according to species for attempted virus isolation. Those species in which the females could not be differentiated were combined in groups. The mosquitoes pooled during 1966-1967 were kept frozen at --20°C. until inoculated into infant mice brought to the field; from 1968 onwards, however, the pools were sealed in plastic bags, stored in liquid nitrogen (WOLFF and CROON, 1967) and sent to Nairobi for inoculation.
Virus isolationsfrom mosquito pools and human sera Mosquito pools were triturated with a cooled mortar and pestle and suspended in cooled diluent (0.75% bovine plasma albumin--Armour Fraction V--in phosphate-buffered saline, pH 7.4). The pool sizes varied with the availability of mouse litters and the numbers of mosquitoes collected of each species. The mosquito suspensions were clarified by centrifugation at 4°C. and inoculated into litters of 8 newborn mice by 3 routes: intracerebrally (IC) (0.01 ml.), intraperitoneally (0.02 ml.) and subcutaneously (0.03 ml.). The remaining suspension was stored at --20°C. or --70°C. for reisolation attempts. The mice were observed daily for 3 weeks. Brains of sick mice were diluted 1 : 500 and passaged by the IC route into newborn mice. With one exception, established virus strains were sent to the East African Virus Research Institute, Entebbe, Uganda for identification. 5 virus strains that could not be identified in Entebbe have been fowarded to the Yale Arbovirus Research Unit (YARU) at New Haven, U.S.A. If possible, blood was taken by venepuncture. Otherwise fmgerprick blood was collected in small tubes containing phosphate buffered saline. After centrifugation, sera were stored in liquid nitrogen and sent to Nairobi for inoculation into litters of newborn mice, after which they were kept for reisolation attempts. Sera were inoculated into mice irrespective ot the presence of malaria parasites in Giemsa stained thick films. Haemagglutination-inhibition (HI) and/or complement fixation (CF) tests were performed for screening isolates against antisera of viruses representative of East Africa and, if necessary, sera of viruses from other areas were also used. Final identification was done by means of cross-neutralization tests CHIT). Antigens were prepared by either fluorocarbon (Arcton 113) or sucrose acetone extraction of infected suckling mouse brain or liver. Hyperimmune mouse antiserum was prepared by 4 sequential intraperitoneal inoculations of a 10% suspension of infected suckling mouse brain on days 7, 14, 21 and 28. HI tests were performed according to the method of CLARKEand CASALS(1958) and CF tests according to CASEY (1965); both tests were performed in microtitre plates. N T were done by the constant serum-varying virus dilution technique. Equal volumes of serum and virus dilution were mixed, incubated at 37°C for 1 hour and inoculated by the IC route into litters of 5 newborn or into 6 adult mice. Results Approximately 146,300 mosquitoes were collected in the 7 areas of Kenya studied, comprising 2 subfamilies, 5 genera and 50 species or groups of species and were processed in 1,420 pools and inoculated into suckling mice. The results of the mosquito collections, the number of isolations and the identifications that have been made so far, are summarized in Tables I and II. From 14 species or groups of species, 1 or more virus strains were isolated, giving a total of 34 strains. Moreover, viruses were isolated from 4 of the 419 sera from patients with febrile diseases. Of the 38 strains isolated, 12 remain, so far, unidentified of which several probably are hitherto undescribed viruses. The remaining 26 isolates include strains of arbovirus group A (Middelburg and Semliki Forest), group B (Banzi and Wesselsbron), the Bunyamwera group (Btmyamwera and Biliefe), Simbu group (Sango), Bwamba group (Pongola), California group (Tahyna) and 2 tmgrouped viruses (AR 1169/64 and Rift Valley Fever). Apart from yellow fever vaccine virus, no arboviruses are kept in our laboratory other than from those isolated by us in Kenya. First and single isolates are, therefore, genuine even when no re-isolation was achieved. Of the others, the possibility of laboratory pick-up was considered by studying the protocols. 2 isolates that could be pick-ups were omitted from the descriptions.
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TABLEII. Summaryof isolations. Locality of isolation
Strain PRMP -MMP
Port Reitz --
Aedes pembaensis
Maiindi
Mansonia
---
30 37 47 158 179
--
249
--
---------
312 346 379 433 483 498 525 607 626 636 637 714 766 824 52 54
-----
-----
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63 117 131 160 26 49 67 68 71 4 36 449 462 62 335
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Nairobi --
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Culex antennatus Anopheles coustani Mansonia africana Culex zombaensis Mansonia
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Culex thalassius-trit-group Anopheles coustani Culex
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Culex sitiens Anopheles coustani Mansonia uniformis Anopheles coustani Eretmapodites subsimplicipes Aedes pembaensis ,, ,,
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Culex rubinotus Mansonia africana Aedes dentatus ,
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H u m a n serum ,,
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Date collected
180 425 450 100 100 20
26- 7-66 28- 7-66 5-12-66 30- 3-68 27- 3-68 2 0 - 3--68 4-- 4 - 6 8 11-12-68 10-12--68 29- 1-69 19- 1-69 30- 1-69 17- 1-69 11-11-69 12-11-69 12-11-69 27-11-69 6-12-69 7-12-69 16-12-69 19-10--67 21-10--67 30-10-67 9-11-67 21-11--67 30-11-67 13-12-67 6- 3-68 23-- 8 - 6 7 8- 9-67 5- 9-67 8- 9-67 22- 7-68 28-11-68 13-11-68 9- 1-69 10-11-70 1 9 - 6--71
100 100
Mansonia africana
--
Garsen Naivasha
umformis
Pool size
,,
15 75 90 95 40 65 100 85 5 100 100 95 5 140 140 200 110 150 40 60 145 290 230 385 100 65
Results of identification
Re-isolation No
Yes No No No No
Yes No Yes No No
Yes No N.D. No No
Yes N.D. N.D. N.D. No
Yes Yes
Yes No No No No No No No
Tahyna Tahyna Sango AR
1169/64
Btmyamwera Pongola new?* Bunyamwera new? Shokwe? new? Bunyamwera Biliefe Middelburg Middelburg
Middelburg in process new? AR
1169/64
new?* Btmyamwera Bunyamwera n e w ?* Bunyamwera new?*
Bunyamwera Bunyamwera A A A
Ungrouped Bunyamwera Bunyamwera Bunyamwera A
Pongola
Bwamba
Banzi Banzi Banzi
B B B B A B
Banzi Sendiki Forest Wesselsbron i n process
Yes
Bunyamwera
Middelburg
No N.D. Yes
California California Simbu Ungrouped Bunyamwera Bwamba
new?*
Yes Yes Yes
Virus group
Biliefe process Rift Valley Fever
Bunyamwera
in
Ungrouped
not done. to Yale Arbovirus Research U n i t .
Discussion
Middelburg virus was repeatedly isolated in South Africa from Aedes (Ochlerotatus) caballas Theobald during an epizootic amongst sheep in which the virus and Wesselsbron virus were involved (KoKERNOT et al., 1957a). On the same occasion and later the virus was isolated several times from a number of other Aedes species. In West and Central Africa pools of Mansonia (Mansonioides) africana (Theobald), Mansonia (Mansonioides) uniformis Theobald, several Aedes species and mixed pools of mosquitoes yielded the virus. We isolated Middelburg virus from Aedes cumminsii group mosquitoes, Anopheles (Anopheles) coustani group, Culex (Culex) antennatus (Becket) and Gulex (Culex) sitiens Wiedemann (Table II). We succeeded in biological transmission of this virus between suckling mice by means of Aedes (Stegomyia) aegypti (Linnaeus) mosquitoes. Most of the mosquitoes from which the virus was isolated are mosquitoes usually breeding and biting in open country, not in forests. This is in accordance with the finding that in Tongaland, South Africa, up to 24% of sera of cattle and sheep had neutralizing antibodies (NA) (KOKEmqOTet al., 1961). Biological transmission between Ae. caballas and sheep was achieved by KOKEmqOT et al. (1957a). Sheep, cattle and Ae. caballas may, therefore, play a r61e in the dissemination of the virus. So far, the virus was not isolated from other vertebrate hosts, including humans. Of hundreds of human sera from countries in the southern part of Africa only a few showed antibodies (quoted by McIntosh in Taylor's catalogue, 1967). Semliki Forest Virus (SFV), first isolated in Uganda from Aedes (Aedimorphus) abnormalis Theobald collected in a woodland belt (SMIT~mORN and HADDOW, 1944) was further isolated from Aedes (Aedimorphus) argemeopunctatus Theobald in Mozambique (McINToSH et al., 1961); from males of Eretmapodites grahami Edwards (STO~qES, 1960) in Cameroon; from Anopheles (Cellia) funestus Giles in Kenya (HENDERSON et al., 1970a) and from Aedes (Neomelaniconeon) palpalis in Central African Republic (Institute Pasteur, Bangui, 1970). Most ff not all of these species are associated with forest or the mosquitoes that yielded the virus had been collected at the forest edge. The isolation reported in this paper is from Ae. (Aedimorphus) dentatus group collected in Ngong Forest. Elsewhere in Africa the virus was isolated once from a hedgehog and twice from birds of different species one of which is associated with woodland. In Africa the occurrence of human sera positive for NA is generally low. However, in Zalre higher figures were found in inhabitants of bush savannah and river border forests. In these environments high values for NA
D. METSELAAR, B. E. HENDERSON, G. B. KIRYA, P. M. TUKEI AND A. DE GEUS
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were also found in chimpanzees (quoted by Woodall in Taylor's catalogue, 1967.) NA have also been found in domestic quadrupeds in South Africa (KOKERNOT et al., 1961). SFV has not been associated with naturally occurring human disease. It is frequently used as a laboratory agent but only 1--subclinical-laboratory infection has been reported (CLARKE,1961). The variety of mosquitoes capable of carrying the virus in nature and the various genera which are capable of transmitting it in the laboratory (quoted by WoodaU in Taylor's catalogue, 1967) may provide an explanation for its wide geographical distribution. From the isolation in nature and serology it may be concluded that Semliki Forest virus is more associated with woodland and forests than with open grassland. Banzi virus was originally isolated from blood of a patient with wdld fever and from a pool of Culex (Neoculex) rubinotus Theobald in South Africa (SMITHBURNet al., 1959b) subsequently from the blood of a feverish child in Tanzania (WoODALLet al., 1962). The mosquitoes from which Banzi virus was isolated in South Africa and Kenya (Table II) may indicate that the risk of infection is more associated with open country than with forests. So far only mild disease has been observed. Wesselsbron virus is the cause of epizo6tics in sheep and was first isolated in South Africa by WEISZ et al. (1956) from a lamb. Subsequently it was isolated from mosquitoes belonging to the Anopheles subfamily and Aedes, Mansonia and Culex genera. Isolations from mosquitoes were made in South and West Africa and in Thailand. More isolations were made in Africa from a camel and a gerbil (Desmodillus) species. Several natural and laboratory infections in man have been reported. Relatively mild illness was observed. Serological surveys in South Africa, Mozambique and Angola showed that 507 out of 4,293 (11.8%) human sera and 254 out of 762 (33.3%) sera of sheep, cattle and goats showed NA (quoted by Mclntosh in Taylor's catalogue, 1967; Institute Pasteur Dakar, 1971; MClNTOSH et al., 1972). MAURICE and PROVOST(1969) found high rates of haemagglutlnation inhibiting antibodies (HIA) in sheep, goats and antelopes in Chad and Cameroon, ROOHAINet al. (1972) found a kori bustard (Audeatis kori) with these antibodies in South Ethiopia. The sources of the isolations and serology indicate that the virus may be more associated with open country than with forest. The isolation in Kenya from Ae. dentatus in Ngong Forest should then be an exception. Ae. dentatus however, takes, an intermediate position and frequently rests and bites at the forest edge (METSELAARet al., 1973). Serological tests in Kenya indicated that the virus may circulate in the northern and eastern (Coastal) parts of Kenya, to a lesser extent in the western parts (HENDERSONet al., 1970a; GESERet al., 1970). Because in northern Kenya Wesselsbron immunity seemed to parallel that of West Nile virus--probably a bird virus--HENDERSON et al. (1970a) suggested that Wesselsbron virus is also disseminated by birds. GESERet al. (1970) expressed the opinion, supported by experiments with Wesselsbrou virus (HENDERSONet al., 1970b), that at the Kenya Coast immunity to arboviruses of group B could play a r61e in the protection of the population against manifest infections of yellow fever. Bunyamwera virus was first isolated in 1943 ill Uganda from a pool of 14 Aedes species (SMITHBURN et al., 1946). Since then it has been recovered from Aedes (Neomelaniconeon) circumluteolus Theobald (KOKERNOT et al., 1958; MClNTOSH et al., 1972) and Ae. pembaensis (quoted by Woodall in Taylor's catalogue, 1967) in South Africa, M. africana in Nigeria (DooRMANand DRAPER, 1968) from Culex species in the Central African Republic (Institute Pasteur Bangui, 1971) and South Africa (MclNTOSH et al., 1972) and from Aedes (Finlaya) ingrami Edwards in Cameroon (Institute Pasteur Dakar, 1971). Apart from some of these mosquitoes we isolated the virus from M. uniformis and Eretmapodites subsimplicipes Edwards (Table II). From man it has been isolated in South Africa (KOKERNOTet al., 1958); Nigeria (BEARCROFT et al., 1963) and Uganda (WEINBRENet al., 1959). Mild disease, once with neck stiffness, once with blurring of vision was reported. The natural vertebrate host(s) of Bunyamwera virus are still unknown. Several related viruses, grouped together in the Bunyamwera group (CASALSand WHITMAN,1960) have been found in and outside Africa and because of serological overlapping, results of serological surveys should be interpreted with caution. Taking this into account NA have been found in substantial proportions (sometimes more than 30%) of human sera from various parts of Africa, from Borneo and South America. Antibodies have also been found in chimpanzee sera from Zaire (Congo) artd sera of domestic animals from South Africa (quoted by Woodall in Taylor's catalogue, 1967). In West Kenya, BOWEN et al. (1973) found neutralizing antibodies in 18 out of 161 sera (11%) from people of 10 years of age and older. GESER et al. (1970) found HIA in 52 out of 822 (6%) of human sera of all ages from West Kenya, in 58 out of 1,042 (5-5%) from Central Kenya and in 221 out of 834 (26.5%) from a coastal area of Kenya. In and near the latter area we isolated 6 strains of Bunyamwera virus. The various mosquito species from which the virus was isolated point to a wide variety of habitats of the virus. Probably man frequently penetrates these habitats and risks infection with Bunyamwera virus. In view of the substantial proportion of human sera
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positive with Bunyamwera (or related) antibodies and the infrequent observation of disease, most infections are presumably mild or subclinical. Beliefe virus was isolated in Uganda from a pool of Aedes abnormalis/tarsalis. HENDERSONet al. (1968) are of the opinion that it is different from other members of the Bunyamwera group and intermediate between Bunyamwera and Shokwe viruses. We isolated the virus in the coastal area of Kenya, once from mosquitoes, once from a patient. The patient, an adult male, suffered a fever for 3 days with rigors and a temperature peak of 103.6°F. He complained of some joint pains 2 weeks after discharge from the hospital. The natural vertebrate host(s) of Beliefe virus are unknown. Sango virus was repeatedly isalated from cattle in Nigeria, the first time was from serum of a cow at the slaughter house of Ibadan (CAusw¢ et al., 1972). The same authors isolated the virus from a pool of Culicoides. Our strain was isolated from M. uniformis collected in an area where herds of cattle frequently pass when driven to the slaughter-house of Mombasa. Nothing is known about the possible r61e of Sango virus Jn human disease. It is likely that cattle play a r61e in its dissemination. CAUSEYet al. did not isolate the virus from thousands of other vertebrate specimens.
Pongola virus was first isolated from Ae. circumluteolus in South Africa (Kor~RNOT et al., 1957b). Further isolations in that country were made from the same mosquito, a pool of M. uniformis and M. africana, from a pool of M. uniformis and a mixed pool of mosquitoes. (WORTH et al., 1960; MCINTOSH et al., 1972). In Uganda, Nigeria and the Central African Republic the virus was isolated from M. africana (WooDALL et al., 1962; BOORMANand D~PER, 1968; Institute Pasteur Bangui, 1971). In North Kenya it was isolated from M. africana, Culex (Culex) zombaensis Theobald and Aedes dentatus (HENDERSON et al., 1970a). 2 more vectors were found in the coastal plain of Kenya (Table II). The large number of vector species may indicate that Pongola virus favours a variety of habitats. Virus isolation from man or possible vertebrate hosts have not been reported. Serological surveys showed NA in 55 out of 427 (13%) sera of domestic animals and in 470 out of 2,813 (16.7%) human sera from South Africa, Angola and Mozambique. In South Africa the residents with antibodies were predominantly found in tropical and lowland areas, less in highland areas. (SMITHBURNet al., 1959a; MCINTOSHet al., 1962a, b; KOKERNOTet al., 1965a, b). It is likely therefore, that Pongola virus is widespread in Africa and has a wide range of vectors. Nothing is known about the natural vertebrate host(s) of Pongola virus. The vector species and the place of 15 isolates in Kenya do not suggest association with forest habitats of the virus. Domestic animals may play a r61e in its dissemination. Notwithstanding serological evidence of human infections little is known of disease in man. The name Tahyna has recently been given priority over Lumbo for a member of the California group of viruses isolated in Africa. Tahyna virus from Europe and Lumbo virus from Africa had at first been regarded as different viruses, but subsequent examinations made it clear that differentiation is not justified. In Europe Tahyna virus was isolated from Aedes species (quoted by Bardos in Taylor's catalogue, 1967). In Africa the virus was first isolated from Ae. pembaensis at Lumbo in a coastal plain of Mozambique, later by us from the same species at the coast near Mombasa. In Europe hares and rabbits are probably vertebrate hosts of the virus. A high proportion of hares in Czechoslovakia have antibodies (SIMKOVA, 1966) and HANNOUN(1968) isolated the virus from a rabbit in south France. The virus was isolated twice from human sera during a survey of healthy people ia Central Europe. In that part of the world Tahyna virus has been associated with febrile illness and an influenza-like syndrome. Under these circumstances the diagnosis was made because of serum conversion (B~DoS and SLUI~, 1963). Serological surveys in Europe have shown that man and his domestic animals are frequently infected (quoted by Bardos in Taylor's catalogue, 1967). In Africa BARDOSand SEFCOVICOVA(1961 ) found NA in 6% of 107 human sera from Uganda; KOKERNOT et al. (1962) in 16 out of" 128 (12.5%) from Mozambique and Natal Province of South Africa, but only in 1 out of 736 human sera from other parts of South Africa and Angola; KuNz et al. (1964) found NA in some of an unstated number of sera from West Africa; RODHAINet al. (1972) detected HIA in 7 sera from adults out of 300 sera from all age groups of people living near the Omo River in South Ethiopia. In Africa virus isolation was not reported from vertebrates. From the above it may be concluded that Tahyna or a related virus probably occurs in areas wide apart in Africa, but has a somewhat patchy distribution. Ae. pembaensis may be an efficient vector and because of the peculiar breeding habits of this mosquito, the virus may be most easily found in coastal plains near tidal marshes. Disease in man is probably infrequent and mild.
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AR 1169/64 virus was first isolated from a pool of M. uniformis in the Sudan (SCHMIDT et al., 1965). We isolated the agent from the same species at Malindi. OKIA and TUKEI (1972) isolated 2 strains from organs of the rodent Praomys jacksoni in Uganda. Other particulars of the virus or its r61e in human disease are not known to us. Rift Valley fever (R VF) virus was first isolated from sheep during a epizo6tic in domestic animals in the Rift Valley in Kenya (DAUBNEYet al., 1931). 4 investigators were infected when handling animals and virus. Later experiences have shown that infections in non-immune veterinarians and laboratory workers are quite common. Natural infections in humans have been reported frequently also, especially from South Africa at times of epizo6tics (GEAR et al., 1951, 1955). Typically, infected patients feel very ill, fever and joint pains are severe, blurring of vision and delirium not infrequent, The disease is, however, seldom if ever fatal. Epizo6tics in sheep and cattle are not uncommon in Africa. SCOTT et al. (1963) found a high level of antibodies in camels in North Kenya, MAURICE and PROVOST(1969) in wild antelopes and buffalo in Chad. These domestic and wild animals therefore, probably play a r61e in the dissemination of the virus. Other vertebrates may be reservoir hosts but these are still unknown. Rodents have been suspected to play a r61e in the natural cycle but SCOTTand HEISH (1959) neither isolated the virus nor detected antibodies in the sera of 285 rodents trapped in the Rift Valley of Kenya. During an epizo6tic in South Africa GEAR et al. (1955) did not find antibodies in 82 rodents either. Only a polecat was positive with NA. Sera of 13 birds and 27 wild animals of 6 species were all negative. PELLISlERand ROUSSELET(1954) examined 122 monkeys of 11 species in 2 institutions of Brazzaville but do not state whether these had been recently captured. 12 sera from 7 species of monkeys of which 113 individuals had been examined showed CF antibodies. On the other hand SMITIIBORNet al. (1948) did not find any NA in sera of 72 wild monkeys of 9 species in an area of Uganda where RVF virus was circulating. At least 10 of the animals had been taken from a forest where shortly before the virus had been isolated from mosquitoes. In Kenya DAVIES et al. (1972) tested sera from 333 baboons, 218 of which from an area where the year before an epizo6tic had been observed. All sera were negative for RVF antibodies. The virus was isolated from pools of Eretmapodites species and Aedes (Aedimorphus) tarsalis Newstead in Uganda (SMITHBURNet al., 1948); from Aedes (Ochlerotatus) caballus Theobald, Culex (Culex) theileri Theobald and Ae. circumluteolus in South Africa (GEAR et al., 1955). Isolations have, therefore, been made in association with forest and grassland country. Our isolate was from a male adult out-patient with mild illness and a temperature of 39.5°C. It was identified by Dr. F. G. Davies at the Veterinary Research Laboratory at Kabete. A serum sample taken a few months later had a neutralizing index of 4.0 logs. Examination of domestic animals in the area where the patient was living did not reveal an epizo6tic (F. G. DAVIES,pers. comm.). The isolate appears, therefore, to have been made from a sporadic case. Yellow fever virus was not isolated in the 2 areas of Kenya where it may have circulated in the past, nor did Ae. aegypti or Aedes (Stegomyia) simpsoni (Theobald), collected at the coast, yield yellow fever or any other virus. We have attempted to differentiate the viruses according to association with forest or more open country. With the information available, more precise definition of the ecology of the viruses was not possible.
Summary The isolation in Kenya of arboviruses from mosquitoes and patients is reported. Middelburg and Semliki Forest virus (group A), Banzi virus (group B), Bunyamwera and Beliefe virus (Bunyamwera group), Sango virus (Simbu group), Pongola virus (Bwamba group), Tahyna virus (California group), ungrouped AR 1169/64 and a number of viruses still to be identified were isolated from mosquitoes. Of the latter some may be hitherto undescribed viruses. Bunyamwera, Rift Valley Fever and 2 unidentified viruses were isolated from patients. The isolates are discussed against the background of information from isolations and serology performed elsewhere. Attempts are made to define their r61e in human disease. Some supertidal suggestions about the ecology are made. REFERENCES BARDOS, V. & SEFCOVlCOVA,L. (1961). J. Hyg. Epidem., Microbiol. Immun., 5, 501. & SLUKA,T. (1963). Cas. Ldk. cesk., 102, 394. BEARCROFT,W. G. C., PORTERFIELD,J. S. • SUTTON,R. N. P. (1963). Trans. R. Soc. trop. Med. Hyg, 57, 308.
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BOORMAN,J. P. T. & DRAPER, C. C. (1968). Ibid., 62, 269. BOWEN, E. T. W., SIMPSON,D. I. H., PLATT,G. S., WAY,H., BRIGHT,W. F., DAY, J., ACHAPA,S. & ROBERTS, J. M. D. (1973). Ibid., 67, 702. CASALS,J. & WHITMAN,L. M. (1960). Am. ft. trop. filed. Hyg., 9, 73. CASEY, H. L. (1965). Publ. Hlth Monogr., No. 74, pp. 31-34. Washington D.C. : U.S. Government Printing Office. CAUSEY,O. R., KEMP, G. E., CAIJSEY,C. E. & LEE, V. H. (1972). Ann. trop. A4ed. Parasit., 66, 357. CLARKE,D. H. (1961). Am. -7. trop. IVied. Hyg., 10, 67. CLARKE,P. H. & CASALS,J. (1958). Ibid., 7, 561. DAImNEY, R., HUDSON,J. R. & GARNI-IAM,P. C. C. (1931). -7. Path. Bact., 34, 545. DAVIES, F. G., CLAUSEN,B. & LUND, L. J. (1972). Trans. R. Soc. trop. Med. Hyg., 66, 363. GEAR, J., DE MEILLON,B., MEASROCH,V. & DAVIES,D. H. (1951). S. AfT. med. -7, 25, 908. ---, - - , LE ROUX, A. F., KOFSKY, R., INES, R. R., STEYN, J. Y., OLEFF,W. D. & SCHULTZ,K. H. (1955). Ibid., 29, 514. GESER, A., HENDERSON,B. E. & CHRISTENSEN,S. (1970). Bull. Wld Hlth Org., 43, 539. GEUS, A. DE, KRANENDONK,O. & BOHLANDER,H. J. (1969). E. Afr. reed. -7, 46, 491. GOINY, H., VAN SOMEREN,E. C. C. & HEISH, R. H. (1957). Ibid., 34, 1. HADDOW,A. J. (1952). Ann. trop. 214ed. Parasit., 46, 135. (1964). Trans. R. Soc. trop. 2tffed. Hyg., 59, 436. HANNOUN, CL. (1968). 8th Int. Congr. trop. ivied. 2~Ialar., Teheran, p. 674. HENDERSON,B. E., KIRYA,G. B., LULE, M. & SEKYALO,E. (1968). E.A. Virus Research Inst., Annual Report, 1968, p. 29. , METSELAAR,n., KIRYA, G. B. & TIMMS, G. L. (1970a). Bull. IlTld Hlth Org., 42, 787. , CHESHIRE,P. P., KIRYA, G. B. & LULE, M. (1970b). Am. -7. trop. Med. Hyg., 19, 110. INSTITUTEPASTEURBANGUI.Annual Reports for the years 1970 and 1971. INSTITUTEPASTEURDAKAR.Annual Report for the year 1971. KOKERNOT,R. H., DE MEILLON,B., PATERSON,H. E., HEYMAN,C. S. & SMITImlmN, K. C. (1957a). S. Aft. J. med. ScL, 22, 145. , SMITI-mURN,K. C., WEINBREN,M. P. & DE MEILLON,B. (1957b). Ibid., 22,~1. - - , --, DE M~ILLON, B. & PATERSON,H. E. (1958). Am. J. trop. Med. Hyg., 7, 579. , - & KLIIOE,E. (1961). Ann. trop. Med. Parasit., 55, 73. •, MCINToSH, B. M., WORTH,C. B., DE MORALS,T. & WEINBREN,M. P. (1962). A m . J . trop. Med. Hyg., 11, 678. ~., SZLAMP,E. L., LEVlTH, J. & MCINTOSH, B. M. (1965a). Trans. R. Soc. trop. Med. Hyg., 59, 553. ~ , CASACA,V. M. R., WEINBREN,M. P. & MClNTOSH, B. M. (1965b). Ibid., 59, 563. KrJNZ, CH., BUCKLEY,S. M. & CASALS,J. (1964). Am. -7. trop. Med. Hyg., 13, 738. MCINTOSH, B. M., WORTH, C. & KOKERNOT,R. H. (1961). Trans. R. Soc. trop. Med. Hyg., 55, 192. , SERAFINI,E. T., DICKINSON,D. B. & WEINBREN,M. P. (1962a). S. AfT. -7. reed. Sci., 27, 77. ~., DICKINSON,D. B., SERAFINI,E. T. & DE SOOSA,J. (1962b). Ibid., 27, 87. , JuPP, P. G. & DE SOUSA,J. (1972). J. reed. Ent., 9, 155. MAlmlca~, Y. & PROVOST,A. (1969). Revue Elev. Mdd. vdt. Pays trop., 22, 179. METSELAAR,D., VAN SOMEREN,E. C. C., OUMA,J. H., KOSKEI,H. K. & GEMERT, W. (1973) Bull. ent. Res., 62, 597. , WILLIAMS,M. C., SIMPSON, D. I. H., WEST, R. & MIITERE, F. A. (1969). Arch. ges. Virusforsch., 26, 183. OKIA, N. O. & TuKEI, P. M. (1972). Abstr. annu. Sci. Conf. E.A. med. Res. Counc., Dar-es-Salaam. PELLISSIER,A. & ROUSSELET,R. (1954). Bull. Soc. Path. exot., 47, 228. RODHAIN, F., HANNOUN,C. & METSELAAR,D. (1972). Bull. IVld Hlth Org., 47, 295. SAWYER,W. A. & WHITMAN,L. (1936). Trans. R. Soc. trop. Med. Hyg., 29, 397. SCHMIDT, J. R., WILLIAMS,M. C., LULE, M., MIVlJLE, A. & MtlJOMBA, E. E.A. Virus Res. Inst., Annual Report of the year 1965. p. 24. SCOTT, G. R., COACKLEY,W., ROACH,R. W. & COWDY,N. R. (1963). -7. Path. Bact., 86, 229. & HEISCH, R. B. (1959). E. Afr. reed. -7., 12, 665. SIMKOVA,A. (1966). Cslkd. Epidem. 2klikrobiol. Imunol., 15, 304. SIMPSON, D. I. H., KNIGHT, E. M., COURTOIS,G., WILLIAMS,i . C., WEINBREN,i . e. & KIBUKAMUSOKE, J. W. (1967). E. Afr. med. -7, 44, 87. SMITHBURN, K. C. (1949). Am..7. trop. Med., 29, 389. & HADDOW,A. J. (1944). -7. Immunol., 49, 141. --, - & MAHAFFY,A. F. (1946). Am. -7. trop. Med., 26, 189. ,- & GILLETT,J. D. (1948). Br. -7. exp. Path., 29, 107. ~ , KOKERNOT,R. H., HEYMAN,C. S., WEINBREN,M. P. & ZENTKOWSKY,n . (1959a). S. Afr. med. -7., 33, 555. ~ , PATERSON,H. E., HEYMAN,C. S. & WINTER,P. A. D. (1959b). Ibid., 33, 959. STONES, P. B. (1960). Trans. R. Soc. trop. 214ed. Hyg., 54, 132. TAYLOR, R. M. (1967). Catalogue of Arthropod-Borne Viruses of the Worldj Pub. No. 1760. 1st Ed., U.S. Government Printing Office, Washington, D.C.
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WEINBREN, M. P., KNIGHT, E. M., ELLICE,J. M. & HEWITT, L. E. (1959). Annual Report East Afr. Virus Res. Inst., 1958-59, p. 7. WEISZ, K. E., HAIG, D. A. & ALEXANDER,R. A. (1956). Onderstepoort J., vet. Res., 27, 183. WOLFF, H. L. & CROON,J. J. A. B. (1967). Antonie van Leeuwenhoek~ 33, 235. WOODALL,J. P., WILLIAMS,M. C., SANTOS,D. F. & ELLICE,J. M. (1962). Annual Report East Aft. Virus Res. Inst., 1961-62, p. 21. WORTH, C. B., PATERSON,H. E. & DE MEILON, B. (1960). Am. ft. trop. Med. nyg., 10, 583.