Yellow fever and Zika virus epizootics and enzootics in Uganda

552

TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE, VOL. 76, No. 4, 1982

Yellow fever and Zika virus epizootics and enzootics in Uganda

A. W. R. MCCRAE’ AND B. G. KIRYA'

Formerly East African Virus Research Institute, Entebbe, Uganda

Summary Data of monkey serology are presented which, together

with past evidence, support the view that yellow fever (YF) virus circulates in its primary sylvan host populations, i.e.., forest monkeys, in an enzootic state in Bwatnba County m western Uganda but as a series of epizootics in the forest-savanna mosaic zone of central Uganda. Evidence of an epizootic of Zika virus at the Zika Forest near Entebbe is described which occurred in two episodes, the first (in 1969) apparently following the build-up of non-immune monkey populations since a previous epizootic of 1962-63 and the second (in 1970) when Aedes afianus biting densities rose. This was followed only 18 months later by an intensive epizootic of YF virus, contradictory to the hypothesis that Zika virus alone would suppress subsequent epizootics of YF virus in nature, at least when redtail monkeys are involved. Conclusions are finally reviewed iu the light of more recent evidence of transovarial flavivirus transmission in mosquitoes, pointing out that phlebotomine sandflies also require fresh attention.

Introduction A masterly review of yelloti fever (YF) in Africa,

including the classic studies leading to the elucidation of its forest amplification cycle in Bwamba County of western Uganda, was provided by HADDOW (1968). One of the purposes of the present paper is to assess subsequent evidence for interactions between YF and Zika viruses in the field.

Much of Bwamba County is relatively low-lying compared to other forested area of Uganda, with extensive forest contiguous with the great Ituri Forest of the Zaire basin, and possessing high densities and exceptionally diverse assemblages of wild primate species. On the basis of antibody tests on monkey sera, HADDOW et al. (1947) concluded that YF virus circulated more or less constantly as an enzootic in the vears 1942-47. the over-all rate of conversion to YF &mmity being 27% per annum in forest monkeys. On further data of wild monkey immunity a similar conclusion was later reached for other areas of Uganda wherever the mosquito Aedes (Stegunyia) africanus (Theo.) was present; HADDOW et al. (1951) stating that w&h the exception of epizootics on’ one 0; nerhaus two islands of L. Victoria “... the picture &e&here has been of an enzootic disease?. either constantly present or (more probably) occurrmg as a series of frequently repeated episodes”. As the years proceeded, however, no actual isolations of YF virus were obtained outside Bwamba for more than a decade and it was clear that the small numbers of monkeys (often 20 to 50) in many small isolated forests (e.a. see BUXTON. 1952) would be insufficient to ma&a&r virus circulation ior long. Transovarial transmission of the virus in A. africaks was investigated bv GILLETT et al. (1950) with negative results. &d possible alternative ‘maintenance systems were therefore investigated. These included ectoparasitic dinogamasid mites from nests of the bushbaby Galago

smg&nsis (see LUMSDEN et al., 1955; LUMSDEN & ELLICE, 1956), reduviid bugs and spiders (potential mosquito predators) from forest (GILLET, 1958) and protozoa of a kind which might occur m tree-hole water in which Aedes develon (HADDOW et al.. 1963). None were shown capable of‘maintaining YE virus.

In 1964, in the follow-up of a single tangential human YF case only 30 miles (ca 50 km) NNW of the Entebbe peninsula, a pattern of isolations from A. africanus and serological results from monkeys was obtained in keeping with an intensive YF epizootic of only a few months’ local duration (WILLIAMS et al., 1965; SIMPSON et al., 1965 . In discussing this, HADDOW et al. (1965) pointe d out that YF immunity rates in samples of monkeys from central Uganda had fallen since 1950 to half of their pre-1950 level, from 42% to 21%. This posed the question of whether an epizootic wave of YF might have been in the process of sweeping across central Uganda.

R&tine monitoring of the human population and of mosquitoes (see Fig. 1) on the Entebbe peninsula continued, and appropriate studies were also conducted in Mawokota County of western Buganda in 1967-68 (HENDERSON et al., 1975), but still no further YF isolations were obtained. A large sample of 204 monkeys from several parts of Uganda (mainly northerly localities and not including Bwamba) in 1967-68 meanwhile showed an extremely low over-all incidence of neutralizing antibody against YF (3%), but very high against Zika (68%) (HENDERSON et al., 1969). This suggested that perhaps Zika virus, which like YF is not only a flavivirus (i.e. of Casals’ arbovirus Group B) but in central Uganda evidently shares the same primary sylvan host-vector system as YF (HADDOW et al., 1964), might in some way impede or even block the circulation of YF virus. Good evidence to this effect was nevertheless lacking. Epizootics of Zika virus had been apparent on the Entebbe peninsula in 1947-48, 1956 and 1962-63, but although no strictly dateable evidence of YF activity had been obtained in this area since the conversion to YF immunity of one sentinel rhesus monkey at the Zlka Forest in September 1947 (HADQOW et aE., 1948). details of monkey serology nevertheless suggested that isolations of kF might simply have been missed during intervals of monitoring, or for some other reason, rather than failing to circulate at all. In the large 1967-68 sample of-monkeys referred to above (HENDERSON et al.. 1969) onlv 13 adult/ subadult redtall monkeys ‘were irom *the central Ugandaforest-savanna mosaic belt and two of those

‘Present address: 19 Davenant Rd., Oxford OX2 8BT, England; reprint requests to: The Librarian, I.C.I.P.E., P.O. Box 30772, Nairobi, Kenya GY Uganda Vtrus Research Institute, P.O. Box 49, Entebhe, Uganda. ‘Present address: Makerere University Medical School, Dept. of Medical Microbiology, Kampala, Uganda.

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A. W. R. MCCRAE AND B. G. KIRYA 553

monkeys showed YF neutralizing antibody, one from Masaka District and one from the Entebbe penihsula. Experimental evidence has been even more equivocal. Studies of interaction between flaviviruses by HEN- DERSON et al. (1970) showed that two vervet monkeys recently immunized against Zika virus responsed td a challenge with YF virus by showing lower levels of viraemia than were seen in control non-immunized vervet monkeys. This reduction in the level of circulating YF virus could have resulted from short- lived comdement-fixing Zika virus antibodv but it was uncl&r whether -or not the reduction was sufficient to prevent biting A. ujiianus mosquitoes from becoming infected. One of the two Zika- immune monkeys showed a delayed peak of YF viraemia, possibly indicative of a prolonged viraemia, although this was not tested for. No such studies have been conducted on redtail monkeys, which are more important YF hosts in central Uganda than vervets (HADDOW et al., 1951).

In 1968 an epizootic of chikungunya virus (CHIK) evidently passed through the monkeys and mosquitoes of Zika Forest (MCCRAE et al., 1971), and because epizootics of Zika virus had occurred on the Entebbe peninsula in the year following CHIK on two if not three previous occasions, there was cause to question whether YF or Zika virus would occur in the fbllowing year.

No studv of Bwamba monkevs had been conducted for some ‘20 years. An iml;ortant question also outstanding was whether Bwamba might still repre- sent a possible type of enzootic source area for travelling epizootics of YF for central Uganda or indeed of any virus utilizing monkeys and Aedes as its primary sylvan host-vector system. The following account therefore includes results of serological studies of Bwamba monkeys sampled in 1%9, as well as of long-term monitoring activities on the Entebbe peninsula, and their interpretation.

Materials and Methods Mosquitoes

All mosquitoes referred fo in this paper were collected from the Zika Forest, 10 km north-east of Entebbe. Relevant details of this forest area are provided by BUXTON (1952), CORSET (1%4), HAD- DOW et al. (1964) and M&RAE et al. (1971, 1976).

Pairs of trained men caught all mosquitoes landing on themselves in individual glass tubes, the catches being segregated hourly according to sun time in which 1800 h coincided precisely with sunset. The most important of various catching schedules em- ployed was an ongoing weekly programme of routine two-hour (1800-2000 h) catches at all of the 20 ft (6.1 m) levels from ground level upwards on the 120 ft (36.6 m) Zika steel tower (detailed descri tion of the tower with relation to its surrounding orest P are given by CORB~T, 1964 and HADWW et al., i964). Once this tower had been erected in the Zika Forest (HADDOW et al., 1961b) substantial numbers of mosquitoes were collected from its platforms in every month from March l%l onwards, the only total breaks being July-October 1961, January-February 1962 and June-August 1964.

After collection and identification, mosquitoes were usually held alive overnight at ambient temperatures before being killed by freezing at -20°C.

The full nomenclature of all monkeys referred to in this paper is: Vervet monkey-Cerco@hezus uethiops (L.); Redtail monkey<ercopithecus ascunius schmidti Matschie; Mona monkey-Cercopithecus mona denti Thomas; Black Mangabey4kmcebus ulbigenu john- szoni Lydekker; Lowland Colobus-Cofobus ubyssini- cus ue1Lmi.s Matschie.

After unsuccessful attempts to trap redtail monkeys at the Zika Forest with the intention of live-bleeding and release, monkevs had to be sampled by means of 12-bore shbtguns. -

_ -

The 22 redtails from Kisubi Forest were all shot between 9th Seotember and 2nd October 1969 within 3 km of the Z&a Forest. Monkeys of the Zika Forest itself were left unmolested so that this vital part of the ecosystem under surveillance would remain intact. No monkeys had been shot in the Zika Forest since 1946 when the single adult redtail obtained was found to be YF immune (Prof. A. J. Haddow, personal commuhication) .

The 68 monkeys from Bwamba were shot between 6th and 20th November I%9 from six forested ‘localities between Bubukwanga and Mamirimiri in the northerly part of the county, all below loo0 m in altitude. The forest to the west or north-west of the main Bwamba road was then a Forest Reserve and remained intact, but to the east the forest had been reduced greatly since the studies reported by HAD- DOW et al. (1947! 1951). Vervet monkeys had not extended into tis cleared land to any noticeable extent, and redtails seemed to be thriving under the changmg conditions. Most of the 52 redtails and the single mona monkey in the sample were from forest patches surrounded by cultivation, whereas the 11 colobus were all from forest outliers contiguous with the main forest or from the main forest itself. The four mangabeys were from *e main forest.

Blood samples were taken as rapidly as possible from each fallen monkey by cutting open the rib cage and pericardium and syringing cardiac blood direct from an auricle. The samples, in pre-labelled McCart- ney bottles, were put immediately on to water ice in vacuum flasks. Durina field work of more than one day’s duration, serum-from clotted blood was drawn off and the clot discarded to avoid excessive haemoly- sis. On return to the laboratory the samples were cold-centrifuged and the sera stored at -20°C.

All monkeys were age-graded with the greatest care according to the criteria of HADDOW et al. (1951). Weight and measurements were recorded and ‘breed- ing condition was assessed externally from the appearance of genitalia, mammae etc. as well as internally from dissection. Field notes were made of dentition, pelage and any unusual features. The skull of each monkey was prepared as a museum specimen for more exact examination of dentition and cranial ossification.

Virus Isolation Mosquitoes were pooled according to species or

genus, A. africanus always separately; isolation procedures for these and for monkey sera are described by HADWW et al. (1964) and HENDERSON et al. (1975).

Virus Zdentifiution Arcton-extracted antigen was prepared in every


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554 YELLOW FEVER AND ZIKA VIRUS IN UGANDA

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Fig. 1. Monthly arithmetic means of A. afriionw taken in weekly routine t-hour (1800-2ooO h) cat&es at all 7 levels on the Zika Forest tower. Months when DO routine catches were conducted but when A. africamcr from other types of catch at the forest were processed are indicated by +. Months when mosquitoes were collected from which CHIK, Zika or YF virus were isolated are denoted by C, Z or Y respectively. In all, more than 70,ooO A. ofriconur from the Zika Forest were processed in the years l%l-73. Detailed catch records are not available thereafter.

PHASE PHASE ; I f LAPSE ;lI;

6 years + 73 days .i 74 I, 192 days - &&&evious isolation “$G$’

i 129 i h-y-----+

i ! i days :

i : : : i : 1 ; : i 4 f : i

J F MAMJJAS 0 N D n

r

J F MAMJ

1970

v

J A 5 0 N D

Fig. 2. The incidence of Zika virus from all mosquitoes collected in the Zika Forest in 1969-70, with the monthly biting densities of A. africonuc indicated by the histogram. Virus isolates from A. o/ric~~m~ taken in routine weekly post-sunset catches from tbc tower are shown by inverted triangles; virus isolated from A. ofncanuc taken in non-routine catches are shown by the black arrow-heads; the single isolate from A. apicoargmfeus is shown by the outline mow-head.


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A. W. R. MC(:RAB AND B. c;. KIRYA 555

Table I-Totals of A. africanus processed for attempted virus isolations from each catch at the Zii Forest ftom August 1%9 to December 1970. Catches from which Zii virus was isolated are in heavy type. Routine 2-hour post-sunset catches from the tower are shown in italics, others not

Total ~o~gcp.T (virus strairls)

Other

August 1969 23, 1, 15, 12, 27, 24 September 18, 12, 41, 34, 71, 48, 80, 61, 42 October 61, 41, 35, 34, 21, 43, 4, 20, 36

November 34, 27, 23, 123, 14, 17, 19 December 11, 12, 145, 1, 72, 136, 43, 6 January 1970 30, 29, 39, 1, 91 February 130, 49, 105, 38 March 34, 2, 36, 75, 120 April 63, 93, 160, 320, 77, 284 May 151, 18, 204, 151, 103, 76 June 116, 50, 162, 202, 62, 294, 58 July 14, 29, 9, 41, 30*, 2, 35

August 10, 18, 8, 42, 30, 54, 21 September 39, 27, 15, 48, 33, 15, 6

October 15, 36, 28, 12, 14 November 56, 58, 39, 41 December 20, 45, 12, 16, 10

*Isolation from 23 A. apicoargenteus taken in the same catch

77(2) : ;;y :

E 189 18

189 1 322 267 8

i 170 u;(l) 121

g(3) : y1

131 123 ;zl)

1E 103 8

Table II-Incidence and identification of the 15 isolates of Zika virus from A. uJium~~ and A. upicoargenreus from the Zii Forest, 1969-70

Date of

collection

Mosquitoes

Type Total in of catch (no.

catch* of pools)

No. in Strain positive reference

ml no. HA titre

VhUS

Re- Identification PH isolation method

4.8.69 R 23(l) 23 MI’ 9501 1:320 6.1 + HI, CF, NT 19.8.69 R 12(l) 12 MI’ 9521 1:80 6.2 + HI, CF, NT 4.9.69 S 12(l) 12 MI’ 9603 1:320 6.1 + HI, CF, NT

11.9.69 s 34(l) 34 MI’ 9620 1:1280 6.1 + HI, CF, NT 22.9.69 R W4 20 MP964O - -x + HI+, CF, NT 25.9.69 S 61(3) 21 MP 9645 1:320 6.1 -* HI, CF, NT

10.10.69 S 35(2) 15 MP %71 1:80 6.1 + HI, CF, NT 16.10.69 S 210) 21 MP %83 - - + HI+, CF, NT 24.4.70 R 2W) 91 Ml’ 10529 1640 6.3 + HI, CF, NT

1.6.70 R 116(2) 58 MP 10694 1:320 6.2 + HI, CF 314.6.70 E 50(l) 50 MP 10701 1:1280 6.3 - HI, CF, NT

8.6.70 R 162(3) 50 MP 10718 1:1280 6.2 - HI, CF, NT 15.6.70 R 202(2) 101 MP 10730 1:1280 6.2 f HI, CF 15.7.60 D 23(l)* 23 MP 10781 1:320 6.3 - HI, CF, NT l/2.9.70 E 39(l) 39 MP 10917 1:320 6.4 + HI, CF

*Type of catch: R = Routine weekly 2-hour post-sunset 7-level catch on tower S = Special weekly (28.7-1.12.69) 2-hour post-sunset catch at 4 positions at and neat the forest edge and at

ground level and 60ft on the tower E = Twice-monthly (14-7.7.69 and 21.4-15.10.70) 24-hour 4position catch at and near the forest edge D = Daytime 3%-hour 2-position ground level catch at swamp forest/dry forest ecotone

“Failed to haemagglutinate throughout the range of pH used +Tested on re-isolate *Could not he read owing to poor mouse litters “Pool of A. apicw~enteus. All others were of A. at?icanus


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case from lirst passage mouse brain by the method of PORTERFIELD & ROWE (1960). Haemagglutination (HA) tine for each antigen was then measured for its optimum over a range of pH values. Haemagglutina- tion-inhibition (HI) screening followed the method of CLARKE & CASALS (1958) against 16 antisera using a constant-serum: varying-virus technique in microtitre plates. These antisera were of the bunyaviruses Shokwe, Germiston and Olifantsvlei and the flaviviruses Zika (prototype strain MR766), YF (French neurotropic strain), West Nile, Wesselsbron, Ntaya, Kadam, Dengue 1, Ban& Spondweni, Uganda S, Usutu, Dakar bat and Entebbe bat salivary gland virus (IR30). Complement-fixation (CF) testing was by the method of CASEY (1965) against 12 flaviviruses including Zika prototype strain MR766. Neutraliza- tion testi (NT) followid procedures identical to those of HADDoW et al. (1964). Neutralization indices were those of REED &‘MU~NSCH (1938).

Morlkqr serology HI screening of kaolin-extracted sera was by the

method of CLARKE & CASALS (1958) against 12 reference antigens of the alphaviruses CHIK, Semliki Forest virus and Sindbis, the flaviviruses Zika, YF, West Nile, Wesselsbron, Ntaya, Kadam, Dengue I and Ban& and the bunyavirus Bynyamwera. The sera found positive by HI screening tests were then re-rested against CHIK, Sindbis, YF, Zilca, West Nile, Wesselsbron and Bunyamwera viruses, to deter- mine their antibody titres.

All monkey sera were tested for neutralizing antibodies against YF and Zika viruses, using an estimated 100 LD5o per 0.03 ml, or 63 LD50 per 0.03 ml in the case of YF tests on monkey sem from Bwamba.

Results Long-term surveillance of mosquitoes at the Zika Forest

Catching of mosquitoes for attempted virus isolation from the Zika Forest was intermittent from 1946 to 1960, with relatively few catches in 1949-54 and 1959-60. To place the present results in a long-term context, a summary of A. uftianus catch data is presented in Fig. 1, which also shows the months when mosauitoes were caught in this forest from which isoh&ons of CHIK CHADDOW et al., l%la; MCCRAE et al., 1971; hJLE et al., 1975), Zika MADDOW et al., , 1964) or YF (KIRYA et al.. 1972. i977) were obtai&d. fiosquito records since &e end of 1973 have not been a&able to us, but up to November 1976 no further isolations of anv of these viruses had been obtained from the Z&a Forest mosquitoes (Dr. P. A. K. Addy, personal com- munication).

Monthly tieans of the numbers of A. afn’canus taken in the weekly routine 1800-2000 h catches from the tower provide indices of biting density as shown in Fix. 1. These indices correlate most closely with &all of the preceding one to two months @&RAE 81 h%ANUhIA. 1970) as would be exacted in a tree-hole-b&ding s&es. Also noted was a tempor- ary reduction of the numbers of A. uf?icmur biting, esmciallv at canonv level and above, on the few o&&ions when eve-r&g temperatures feil below 13°C. The numbers of A. africanus taken in each of these routine catches rangid from two to 320.

Incidence of Zika virus isolatims 1969-70 The first isolation of Zika virus was from A.

afiicanur taken in a routine catch on 4th August 1969, Byears and 73 days after the previous isolaGon of thii virus from the forest (HADDoW et al., 1964). Details of the incidence of this and subsequent isolations in the 1969-70 series are given in Tables I and II. They are summarized in Fig. 2 which shows the two phases in which they occurred. 14 isolations were from A. africanus and one from Aedes (Stegomyia) apicoargm- as Cll~eo.). Few A. a&amxteus were taken in rout&e catches from tile tower, as this species is preponderantly diurnal in its biting habits (HADDOW & SSENKUBUGE, 1965). It was collected mostly in non-routine catches (see Table I and footnote, Table II); only ten during Phase I and nine during the lapse, but 223 in Phase II. Other mosquito swcies. from which Zika virus was not isolated,bccu&d in &milar proportions to those listed by HADDoW et d. (1964) and MCCRAE et al. (1971).

When A. uftianus from single catches were divided into two or more pools, no additional isolations were thus obtained (Table II). Even so, the minimum field infection rate /lo4 mosquitoes tested (MFIR) was initially high, 98-O for the months August to October 1%9, falling to 19.5 for April to September 1970. Allowing for an over-all MFIR of 36.0 for these two phases combined, some six isolations would have been expected from the 1603 A. africanus collected in the six calendar months between these phases had transmission persisted then.

It is notable that A. africanus biting densities indicated by the routine iower catches- were low throughout Phase I. The mean catch for August I%9 when isolations commenced was 19.25, the lowest monthly mean catch since records be&n in June 1%3. The mean for the full three months of Phase I was only 36.7. Biting densities in April 1970 suddenly rose to a level five times as great, the first isolation of Phase II occurring towards the end of that month, and biting rates remained high until the end of June by which time five of the six isolations of this phase from A. afticanus had occurred.

Virus Zsolation and Identification Preliminary reports of these results have been given

by KIRYA et al. (1970, 1971) and KIRYA & LULE (1971).

Table III-Results of cross complement fixation (CF) tests behveen MP 10529-10917 aod Zii (reference stxain MR 766). Titres expressed as the ratio of optimal dilutions of antigen to antiserum

Antigen or Homologous ZikS Zik3

aIltiserum antiserum antigen sntiscrum

ZikS 80 : 80 MP 10529 20: 80 20: 80 20 : 160 MP 10694 40: 40 40:40 20: 80 MP 10701 20 : 160 80: 80 20 : 160 MP 10718 20: 40 20 : 80 20: 80 MP 10730 20: 80 80: 80 20: 80 MI’ 10781 40: 320 20: 80 40: 320 MP 10917 20: 40 20: 80 20: 40


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All positive mosquito pools produced pathogenicity in newborn mice inoculated i.c. or i.p. and in adult mice inoculated i.c. (but not i.p.) in six to seven days. Arcton-extracted antigens of each strain haemagglu- tinated goose red cells maximally at the titres and pH values shown in Table II. Screening HI tests of all these antigens against the 16 togavirus hyperimmune sera (see above) showed inhibition by Zika but not by Spondweni or other sera. Cross CF tests were carried out aginst 12 flavivirus antigens/antisera and significant reactions were shown between all the isolated strains and Zika. Results of these CF tests comparing all Phase II strains in homologous or anti-Zika challenge are shown in Table III. In reciprocal mouse-protection NTs, all strains tested gave higher

neutralization indices against Zika than against YF or Banxi (KIRYA et al., 1970,197la; KIRYA & LULE, 1971).

Monkey serology The sampling dates of the monkeys from Kisubi

Forest spanned the period when mosquitoes were collected from the Zika Forest to yield the third to the seventh of the 15 Zika virus isolates. The pre-adult age grades of these 21 monkeys included only one infant, one juvenile and no subadults. HI and NT immunity rates are summarized in Table IV. HI testing for CHIK antibody resulted in 81% of the sera positive, 13 of these 17 (including that from the juvenile) giving strong reactions of at least 1:40. Both

Table Iv-Monkeys sampled fkom Kisubi Forest (near Zika Forest) 9 September-2 October 1969, and fmm six lowland forested localities in Bwamba 6-20 November 1969, showing the incidence by species and age grade of immunity by HI and by NT mouse-protection testing

Location Species Age

grade*

HI positive NT positive Total tested CHIK SIND YF ZIKA WN WESS BUN YF’ zKA**

Kisubi Redtails f

1 1 1 1

SA () ------- _ _ A 17 16 2 2 4 6 6

OA 2 1 2 ALL 21 17 3 2 4 7 8

Bwamba RedtailS f 4 1 1 1 1 1 1 9 4 2 1 1 2 2 3 2 1

SA 10 8 1 6 3 1 1 6 3 A 26 23 4 13 16 10 13 3 17 17

OA 3 3 1 3 2 1 3 3 3 ALL 52 38 8 24 23 15 20 6 29 25

9, Mona SA 1 1 1 1 2, Mangabeys I 1 1 1 1 1 1

A 3 2 3 1 1 2 1 3 2 ,, Colobus f 2 1

1 SA 1 1 1 1 1 1 1 1 A 7 7 1 5 4 3 4 7 5

“others” ALL 16 11 1 10 7 6 8 2 13 9

*After Haddow et 01. (1951). I = infant (c. O-O.5 years) J = juvenile (c. 0.5-1.5 years) SA = subadult (c. 1.5-3-O years) A = adult (c. 3.0-10 years) OA = old adult (c. lO-20? years). ‘YF challenge of an estimated 63 LDsd0.03tnl l *Ziks challenge of an estimated 100 LD&O.O3ml

Table V-YF and Zika virus NT immunity rates for alI monkey species combined in the Bwamba sample, as observed and as staodardized

Total YF NT pos. Zika NT pos.

Age grade NOS. tested

Obs. %

SMP %

Obs. %

SMP %

Obs. %

SMP %

Infant 7 10.3 3.5 28.6 28.6 Juvenile 10 14.7 7.4 20.0 10.0 Subadult 12 17.7 16.8 66.7 33.3 Adult 36 52.9 57.9 72,2 66.7 Old adult 3 4.4 14.4 1000 100.0

Total 68 100.0 loo.0 60.3 69.9 50-o 60.4 l SMP = standardized monkey population of Haddow et al. (1951) having an age-steucture as shown in this column.


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the old adults were HI CHIK negative. By contrast, HI nositive against YF (2/21 = 10%) and Zika anti&n (4/21 =’ 19%) all gave uniformlv weak reactions of 1: 10, while higher-percentage immunity rates were indicated bv NT than bv HI against both YF (7/21 = 33%) and Zika antigen (8/2‘i = 38%).

The sample of 68 monkeys from Bwamba shot in November 1969 provides a good representation of age-grades for redtails, showing a higher’percentage of the three youngest age-grades than those given by HADDOW et al. (1951) (see also comparison in Table V) indicating more effective persecution by local hunters and trappers in the 1960s than in the 1940s. As no significant differences were apparent in YF NT immum& rates according to locality (those rates ranged only from 57 to 73% in the subsamples from five localities; at the sixth only one monkey was obtained) or according to monkey species (in con- formity with the findings of HADDOW et al. 1947, 1951 for forest monkey;), the data for all Bwamba monkeys are presented together in Table V.

For Bwamba, more of the YF or Zika NT-positive monkey sera were also HI-positive than was the case for Kisubi (Table IV), and the Bwamba sera also gave a spread of HI titres and frequency distribution according to age appropriate to a more or less continuous enzootic status for both these viruses. The increasing incidence of CHIK HI-immunity with age indicated a similar status for this virus also. Sera which were NT-positive to either YF or Zika (i.e., excluding sera positive to both) showed HI antibody (if any) only of the kind positive by NT.

Discussion The repeated recurrence in the Entebbe area of

CHIK followed in the same year or the next by Zika virus at intervals of five to eight and possibly up to 10 years has been discussed by MCCRAE et al. (1971), who regard such congruent periodicity as a function of the time taken for the monkeys involved (N.B. as primary rather than secondary or tangential hosts) to replace themselves with sufficient densities of non- immunes. Such congruence implies efficiency, and there is good evidence that infected A. ufricanus may pass readily from one forest to another, either when Ihey fly above the canopy in a state appropriate for blood feeding (HADDOW et al., 1964) or at ground level for oviposition (CORBET, 1964). The operation of a simple common factor controlling this periodicity is particularly indicated by the many differences between the two viruses, underlined by findings at the Zika Forest that CHIK circulates for only a few months whereas Zika virus has scanned 11. 12 and 13 months in three separate series of isolations. Since CHIK is an alphavirus and Zika is a flavivirus the two would not interact antigenically, but the absence of YF isolations meanwhile lent credence to the view that the presence of Zika virus within the primary host svstem had interfered with this efficiencv. Ieaving’YF to Iill in the gaps left by Zika virus, or else that YF might have been suppressed to lower and more sporadic levels of incidence.

Serological results of the 1969 Kisubi monkey sample (Table IV) confirm that these animals had been involved in the 1968 CHIK epizootic, but indicate that they had not by then become involved in the Zika virus epixootic then beginning at the Zika

Forest. The results nevertheless provide evidence that some of the older of these monkeys had been involved with both Zika and YF viruses at a similar time in the past.

An important implication here is that if viruses occur as periodic epizootics rather than as continuous enzootics it is not valid to convert immunity rates to those expected from a standard monkey population (HADDOW er al., 1947, 1951).

As Zika virus had previously occurred in 1962-63 (HADDOW et al., 1964) it could be suggested that (a) YF had been missed during the gap in surveillance of A. africanus in June to August 1964 (see Fig. l), (b) YF had been missed at some other time by sheer misfortune. (c) strains of YF had occurred which were not detectable’by routine methods of isolation, or (d) YF had occurred at Kisubi but not at Zika Forest. It ‘is also possible, but statistically highly improbable, that YF had been masked by the occurrence of CHIK or Zika virus in the same mosquito pools, since mice are affected more slowlv bv YF than bv these other viruses (CORNET et al.,* 1979a). Whatever the case, argument could follow several lines other than that Zika virus had hampered the dynamics of YF transmission.

The full series of Zika virus isolations of 1969-70 from the Zika Forest (Fig. 2) suggests on several counts that a high proportion of the resident redtail monkey population of some 40 to 50 animals (MCCRAE et al., 1971) would have become immune by the end of it. Phase I gave rise to the extremely high MFIR of 98.0 from A. ufricanus over three months (or 113.6 over 74 days; Table I) when biting densities of these mosquitoes were low, indicating that a low immunity rate in the monkeys was a factor leading to initiation of this phase and therefore that a raised immunity rate brought about its extinction. The virus did not reappear until more than six months later when A. africanus biting densities had risen dramati- cally, and’the lower MUIR* and longer duration of Phase II again indicates a dwindling number of non-immune monkeys. Figures for immunity rates to Zika virus from Bwamba (Tables IV, V) and else- where (HENDERSON et al., 1969) as well as the 1969 Kisubi CHIK data (Table IV) suggest that by the end of the epizootic at least 80% of the Zika Forest monkeys would have become immune.

A series of YF isolations from the Zika Forest, 16 from A. afticunus (MFIR of 49.8) and, surprisingly, one from Coouilkttidia fuscotmnutu (Theo.) was finally reported for 13th March-8th June (KI~YA et al., 1972, 1977). That. this could happen within 18 months of the cessation of an apparently intensive enizootic of Zikavirus at the same site is evidence enough that Zika virus would not impede the build-m) of YF to a full enizootic, but doubt on this *The role of A. upicoargenreus in actual transmission of Zika virus remains doubtful. HADDOW et al. (1964) reported no isolations from 1,045 A. upicourgenteus taken during the 1962-63 epizootic when the MFIR of A. uficunus was 14.8. SEMPALA et al. (1970) found that Zika virus remained erratically detectable in A. upidoargenteur for 10 days after infection without the clear eclipse of virus on days 1 to 6 shown by A. aji-icanus, and transmission by bite was not achieved. Failure to re-isolate the virus from Ml’ 10871 (Table II) makes it possible that this was a laboratory contaminant, although unlikely since Zika virus was not otherwise “off the shelf’ at the time.


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A. W. R. MCCRAE AN11 R. G. KIRYA 559

point was maintained by the report (KIRYA & OKIA, 1977) that of 18 redtails shot in various forests on the Entebbe peninsula (seven of them from the Zika Forest itself) in July 1972, i.e., in the month following cessation of YF at Zika, only two (both from Zika Forest) showed protective antibody against Zika virus by NT. However, while the NT challenge dose of Zika virus is given by KIRYA & OKIA (1977) as 100 LDqn. in the orevious report on the same monkeys by

_“ ,

KIRYA et al.- (1972) th; dose was given as 3&m LD,,. Furthermore. the YF and Wesselsbron NT challenge dose was briginally given as 790, not 100 LDso, and in keeping with this all HI-positives were found to be more numerous that NT-positives against each of these viruses. We now accept the strong circumstantial evidence that immunity rates to Zika virus bv 1972 were in fact probably high.

Further field evidence contrary td the hypothesis that enizootics of YF are blocked bv Zika virus _--- -r-----~~

includes (a) the conversion to YF i&unity of a sentinel rhesus monkey at the Zika Forest in Septem- ber 1947 (HADDOW et al., 1948) while Zika virus isolations were obtained from there from another sentinel rhesus in April 1947 and from A. ufricunus in January 1948 (DICK et al., 1952) implying coincident epizootics of both viruses, (b) the report of SBRIB et al. (1968) that the 1960-62 Ethiopian YF epidemic appeared to have spread in forests by a cycle involving A. africanus and monkeys following in the earlier path of Zika virus, (c) the serological evidence from Bwamba (Tables IV, V) that both viruses may co-exist as high-incidence enzootics, even in the presence of furth& fiaviviruses, and (d) fmdings in eastern Seneeal indicating that Zika virus circulated in Aedes mosquitoes in October to December 1976, overlap- ping with YF in the linal month (CORNET et al., 1979~). This comparative delay in appearance of YF was attributed by CORNET et al. (1979a) to the longer incubation period and less persistent and efficient transmission of YF than of Zika virus by mosquitoes.

Finally, the periodicity of Zika, YF and CHIK epizootics needs to be considered in the light of recent laboratory evidence of transovarial transmission of flaviviruses by mosquitoes (AITKEN et al., 1980; Coz et al., 1976; DUTARY & LEDUC, 1981; ROSEN et al., 1978), as supported by the circumstantial field evidence that YF virus has been isolated not only from females but from males of wild-caught Aedes (Diceromyiu) furcifer-taylori during an epizootic in Senegal (CORNET et al., 1979b). There is no such evidence for Zika virus, however, which persisted for only one wet season at a time in the eastern Senegal studv areas whereas YF virus recurred in three conskutive years (CORNET et al., 1979b; SALAUN et al.. 19811. It is of soecial interest here that TUPP et al. (1481) bbt&ed n’egative results when ittempting transovarial transmission of the alphavirus CHIK, a virus which also occurs in eastern Senegal (CORNET et al., 1976), in Aedes furcifzr, in South Africa. Even so, maintenance of flaviviruses in aestivating Aedes eggs cannot be ruled out for any area and might be crucial for virus survival where dry seasons are so prolonged that mosquitoes cannot survive as adults or larvae (CORNET et al., 1979~; GERMAIN et ul:, 1981). It need not be invoked, however, to explam the situation described in the present paper. A furciferllt&ni are apparently absent from the Entebbe area though

present in lower altitude woodlancis of Uganda including the outskirts of the Bwamba forests and A. uegypti, the experimental mosquito used by AITKEN et al. (1979) and BEATTY et al. (1980), is not involved in either area. If transovarian transrmssion in A. uficunus persisted over long periods of time, more scattered patterns of virus isolation from them would be expected than shown in Fig. 1. As it stands, the evidence that transovarian transmission in mosquitoes alone could account for persistence of flaviviruses at any one site for more than one year has not been established. At the Zika Forest the evidence therefore still points to the replacement rate of monkey populations as the prime determinant of the periodicity of Zika and YF in the form of travelling epizootics, in common with CHIK (MCCRAE er al., 1971).

One of the most intriguing findings in all the YF studies in East Africa, and one which has never been followed up, has b&n the isolation of YF from nhlebotomine sandflies taken in Bwamba ~SMITF~ r---m I-

BURN et al., 1949). In a wider context, SP~ELMAN (1975) points out that since viruses of at least three other diverse groupings appear readily to infect the ovaries of phlebotomines, these insects may possess some unique anatomic vulnerability to virus infection in general. Few phlebotomines have been processed for virus isolation in the course of African investigations into flaviviruses, but many of them often occur in hollow trees, for example, where savanna bush- babies (G&go spp.) typically nest. Now that phlebotomines are better established as insectary animals it seems constructive to suggest they they should be investigated in connection-&h u&xplained patterns of YF immunity among wild bush-babies (see HAD- DOW, 1%8 for summary; see also KIRYA et al., 1971c).

The role of Aedes (Stegomyia) simpsoni (Theobald), sensu Edwards (1912) in extendine svlvan YF euizoe- tics to peridomes& epidemic; in Uganda’ and Ethiopia is summarized by HADDOW (1%8); a similar potential for extending other A. @canus-borne sylvatic epizootics should not be overlooked. In western Uganda strongly anthropophilous populations of A. simpsuni s.1. were formerly thought to be restricted to Bwamba* but have since been found in the Nyamaga- sani Valley (M&RAE & MAWEIJE, 1969) and the Mubuku-Sebwe Valley (P. Manuma, personal com- munication) of the southern and eastern Ruwenzori foothills respectively. In the latter area their appearance in great numbers is a recent phenomenon, a cause for concern should they spread further afield.

In summary, evidence of congruent but not coinci-

*HUANG (1979) raises A. simpsai to the status of a species complex, tentatively ascribing Bwamba material to A. (S.) bromeliae (Theobald, 1911) or possibly A. (S.) lilii (Theobald, 1910). However, the type locality ofA. brumeliae is Kampala, an area where only non-anthropophilous populations of A. simpsoni sensu lato occur (MUKWAYA et al., 1%9). HUANG (1979) cites only lilii from Ethiopia where A. simpsoni s.1. is regarded as anthropophilous throughout its range where it was the principal vector of the devastating YF epidemics of the l%os (SBRIB et al., 1968). Thus if Bwamba simpsai s.1. are not Iilii (and are clearly not simpsoni senw _- s&to), then zoophily-anthropophily would not Gregate in keeping with these three morphotypes alone. Further problems of interpreting these behavioural attributes in field populations of A. simpsuni s.1. in West Africa are raised by PAJOT (1977) and BANG et 01. (1979).


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dent periodicity of discrete CHIK and Zika virus epizootics at the Zika Forest suggest only simple bite-to-bite transmission by individual mosquitoes in the forest-savanna mosaic there, and the same may be the case for YF virus, which is not evidently orevented from circulatinn bv prior incidence of Zika &us. How this might be l%zd with apparently more persistent circulation of all these three viruses in large forests and more erratic circulation in drier eco- systems, with or without alternative maintenance systemM.g. for YF involving bush-babies (see above) or ticks (GERMAIN et al., 1979)-or long- distance introduction+.g. for CHIK by bats (BRBs & CHAMBON, 1964) or birds (CAUSEY, 1969)-remain topics of potentially fruitful research.

Causey, 0. R. (1969). Ibadan Arbovirus Laboratory Annual

We are particularly indeb6d to Dr. G. W. Kafuko, former Director, EAVRI, for support and encourage- ment. to Mr. 1. S. Kinadon for considerable heln and advice in coll&ng and age-grading monkeys and to Mr. L. E. Hewitt for carrying out most of the NT mouse-protection tests on monkey sera by techniques identical to those used in previous years at the Institute.

Most of the staff of the EAVRI Sections of Entomology and Vertebrate Ecology and of Arbovir- ology took part in these studies, notably Messrs. Y. Ssenkubuge, P. Manuma, C. Mawejje, A. Kitama, D. Kaya, M. Lule, E. Sekyalo, A. Mukuye and E. Mujomba, and we thank them all. To our colleagues Drs. B. E. Henderson, P. M. Tukei, L. G. Mukwaya and S. D. K. Sempala and to the late Professor A. J. Haddow, Dr. C. E. Gordon Smith and Dr. P. A. K. Addy we are grateful for help in many ways. Dr. J. S. Porterfield gave valuable criticism of the manuscript. Finally, we thank the Chief Game Warden of the Uganda Government Ministry of Agriculture and Animal Resources for permission to shoot monkeys and for providing the services of marksmen, and the World Health Organization for permission to cite the cyclostyled documents WHOMR72.7 and WHO/ VBC79.725.

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Smithburn, K. C., Haddow, A. J. & Lumsden, W. H. R. (1949). An outbreak of sylvan yellow fever in Uganda with Aedes (Szegomyiu) africanus Theobald as principal vector and insect host of the virus. Annals of Tropical Medicine and Parasitology, 43, 74-89.

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Accepted 6th June, 1979. Revised manuscript received 29th March, 1982.

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AHRTAG publications include Diarrhoea Dialogue-a free quarterly newsletter on all aspects of the prevention and control of diarrhoeal diseases which is available in English, French and Spanish.

For list of publications and further information on facilities available and project areas write to:

AHRTAG, 85, Marylebone High Street, London WIM 3DE, UK.


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Yellow fever and Zika virus epizootics and enzootics in Uganda

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Transcript of Yellow fever and Zika virus epizootics and enzootics in Uganda