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Northern Territory Department of Health Library Services Historical Collection

A PRELIMINARY INVESTIGATION OF POSSIBLE ORAL AND TRANSOVARIAL TRANSMISSION OF ROSS RIVER VIRUS BY AEDES NORMANENSIS <TAYLOR) FROM THE NORTHERN TERRITORY.

P. I. Whelan A and J. Shorthose +

46276

ANorthern Territory Department of Health, Darwin NT + Department of Primary Production, Darwin NT

CSNTAAL LIBRARY

- 6 MAR · 1998

~muronv

HEALTH SER'lfCES

SUMMARY

Wild caught females of Aedes normanensis from Mataranka in the Northern Territory were fed on new born mice intracerebrally innoculated with Ross River ( RR) virus isolated from the Northern Territory. Classic RR virus symptoms were produced in litters of new born mice f rom fi ve to eight days after exposure to the infected mosquitoes. Tests on the mice showed cytopathic effects and the presence of alpha ·..rirus. Eggs laid by the caged mosquitoes reared to adults failed to show any evidence of transovari al transmission at least at the . 46% level of individuals examined.

INTRODUCTION

Relatively high isolation rates of RR virus have been made from wild caught ~ normanensis at Matqranka, approximately 380km south of Darwin in the Northern Territory ( Whelan and Shorthose, unpublished) , suggesting that this species is a potential vector of RR virus in the Northern Territory. RR vi rus has been previously isolated f r o m ~ normanensis in Queensland ( Doherty et al 1 979). but there have been no reports on the vector competence o f ~ normanensis and RR virus.

~ normanensis is a very widespread mosquito species in the Northern Territory, from the sub coastal areas~ inland to at least Tennant Creek, approximately 800km south of Darwin, and can reach very high populatton le~els soon after th~ start of the rainy season ( Nocthecn Ter·rLtory Health Depac·t ment cecordsi I f A e . no r ma n e n s i s i s c o n f ^{1 t,} me d a s a ·, e c t o 1 · o t' P. R v i ^t' u s , i t w o u 1 ct have important significance to t he transmission of human disease in the Northern Territory and neighbouring states. Part of this paper examines the possible oral tranmission of RR virus by Ae. normanensis under laboratory conditions.

The high rate of RR virus i s olations from the semi -arid, seasonal rainfall area around Mataranka were made in January, 1984 soon after the beginning of the rain;;' season. This was dt a t i me when numbers o f ~ n o t' rn a n e n s 1 :::. -...: l-' r e c i-.d a t i ·: e 1 .. ' h i 9 h a n d t h e nurnbe rs of most ot her s pec10.s LncludLnq ^{f' -{ __} ~I.t_n_~l_Lc..:::_st_~_:_L~~- '-"P.C'e r·ela.ti ve l y

Transovarial transmission of arboviruses have been well documented, with La Crosse virus by Aedes atropalpus (Freier and Beier 1984), St Louis Encephalitis virus by Aedes atropalpus and Aedes epactius ( Hardy et al 1984) , Yellow Fever virus by Aedes aegypti and Aedes mascarensis ( Beaty et al 1980) and Dengue virus by Aedes albopictus and ~ aegypti ( Rosen et al 1983)

Preliminary data suggesting transovarial transmission of RR virus by Aedes vigilax in Queensland has been reported ( Kay 1982), and suggestions of arbovirus survi val in Australia in drought resistant Aedes eggs has been made ( Doherty 1 977), but there have been no reports of attempts to examine possible transovarial transmission of RR virus in ~ nor-manensis. Part of this paper documents a pr eliminary investigation of possible t ransovarial transmi ssion of RR vir-us b:; ~ normanensis.

MATERIALS AND METHODS

Wild caught f emale Ae. normanensis were collected by EVS dry ice baited light traps ( Rohe & Fall 1979) in May 1985 at Mataranka, approximately 380km south of Darwin in the Northern Territory and transported t o Darwin in catching containers held in moist t owel covered polystyrene foam boxes. The mosquitoes were sorted to species on refrigerated cold tables: and all obviously dead individuals were discarded. The mosquitoes were counted and transferred to two netting covered cages ( 30cm x 30cm x 30cm) in an insectary and maintained at 27-31 degrees C and 85-95%

relati ve humidity. Daily moistened cotton towels were placed over each cage.

The mosquitoes were given access to water and a 10% sucrose solution in cotton wool pads on a daily regime of sugar from 0900 to 1600 hours, and water for the remainder. An outbred strain of straw mice obtained from the Department of Agriculture in South Australia1 and maintained as a closed colony at the Department of Primary Production. Berri mah Farm, Northern Territory was utilized. A 0. 02 aliquot of two strains of RR virus ( V582) and ( V591 ) isolated from k _ normanensis collected at Mataranka in 1984, were innoculated intracerebrally into two litters of six mice, three to four days old. The litters were exposed to the wild caught A!L_ normanensis on the third day after inoculation for four one hour peri ods each day over two days. Four hundred and twenty-six mosquitoes contained in Cage 1 were allowed to feed on Litter 1, i nnoculated with RR virus strain V582, while the seventy-fi ve mosquitoes contained in Cage 2 were allowed to feed on Litter 2 innoculated with RR virus strain V591.

The l i t t e r s were restrained in nylon stockings during exposure t o mos q u 1 t oes, ::1. n d c et u t' rH' d a.ft e c' expos u t' e to th -2 1 c mothers 1 n n e t: t 1 n q c o v ~ t' ~ ct m 1. c · ~ , ^{.-t .J ~; ::; .} A n um b e r o f i n d i ·' 1 d u ci L s o f L i t t e ^r-~' 3 a n d -l w ~ r e c ^L'Hl d o rn l. ~:·. e l ..., <' t-. P. d a n d p ^{1 cl} c e d i n e i t he c C ,-i. g e 1 o r • C .1 '-:1 ~

21 while Li t t er 5 was plac:ed onl:/ in Cage 2 and Li t t er ,.5 onl-:; 1n

Cage 1.

2

c o , \\

The mice were monitored for disease symptoms and killed approximately two to four days after the f i r s t symptoms of hind leg paralysis in each l i t t e r became e '..rident. The mice were examined for cytopathic effects and the presence of alpha viruses by methods described elsewhere ( Whelan and Shorthose1

unpublished) .

Samples one day examined taking assaying

of blood fed mosquitoes were taken on the same day after i n i t i a l exposure t o t he innoculated mice.

i n d i ,1 i d u a 1 1 J f o r t h e p r e s e n c e a n d 3. mo u n t o f ',, i r u s a 0. 2 aliquot of supernatant fcom eac h insect sample

in Vero cells and cal culating l og 10 PFU/ ml.

and and b-; and

Moribund or dead mosquitoes from each cage were removed four t i me s e a c h d a y a n d h e 1 d i n 1 a b e 1 1 e d '! i a 1 s i n 1 i q u i d n i t c o g e n . Mosquitoes were removed for up t o nineteen days after i n i t i a l exposure to innoculated mice and daily samples from each cage were pooled and examined for the presence of virus as described elsewhere (Whelan and Shorthose, unpublished). At day 15 after i n i t i a l feeding, the mosquitoes were denied access to blood for 24 hours and samples of nine or ten l i ve mosquitoes from the respective cages wepe sampled and tested individually f or the presence and amount of virus.

Some positive plaque assays of the pools of moribund and dead or live individuals were assayed by plaque reduction tests as described by Gorman et al (Gorman et al 1975) to confirm the presence of RR virus.

The caged mosquitoes were allowed to lay eggs on moistened from Mataranka, and eggs were removed every second day.

adult mosquitoes reared from each egg batch were held for least 24 hours after emergence, and then pooled separately each sex. Each pool was tested for virus by taking a 0.

aliquot of supernatant from each insect pool and innoculating mud The at for 1 ml to

BHK-21 cells and observed for cytopathic effects. Pools we re

passaged twice before being discarded as negative.

RESULTS

Oral Transmission The numbers of number of pools Table 2.

~ normanensis present in each positive for virus are shown in

cage and Table 1

the and

The days of exposure of each l i t t e r to the caged mosquitoes and the days of f i r s t observation of symptoms and evidence for the presence of virus are also shown in Tables and 2.

oo, '\

The amounts of virus detect ed in indi vidual mosquitoes are shown in Table 3.

The pools of moribund and dead mosquitoes showed the presence of virus from 3 t o 8 days after initial exposure to the cerebrally inoculated mice.

Litters of new born mice showed classic RR virus hind l eg paralysis fi ve t o eight days after first exposure to the caged mosquitoes: and t hese l i t ters exhibited cytopathic effects and a number test ed were pusi t i ~e f or alpha virus.

The indi vidual samples of blood fed mosquitoes showed amount s of vi r us r a n g i n g f r' om 5 t o 2 0 P F U / m 1 .

Samples of li ve mosquitoes taken 16 days after initial exposure to innoculated mi ce showed 5/ 10 positive for virus in one cage and 8/ 9 positive for virus i n the other cage, with virus amounts ranging from 5PFU/ ml to log 10-5. 1 PFU/ ml.

Transovarial Transmission

The days of egg produc t ion and t he number of subsequent progeny reared from each cage of mosquitoes are shown in Table 4 and Table 5. All male and female progeny of the eggs produced by the caged mosquitoes were negati ve for virus. There were 215 and 31 progeny produced f rom each respective cage after day 8

following the initial exposure of the mosquitoes to innoculated mice. This indi cates that no transovarial transmission was evident at least at the 0. 46% level of indi viduals examined.

DISCUSSION

Oral Transmission

In this preliminary investigation there are a number of problems that became evident, but which could be overcome with improved design. These problems include the unknown number of mosquitoes which fed on the inoculated mice initially, and hence the number of infected mosquitoes in t he cages at any one time is unknown. The random sharing of both l i tter 3 and l i t t e r 4 between the two cages meant that the presence of mor e than one strain in each cage was poss ible. This is not critical as both strains were isolated from ~ normanens is at Mataranka at the same t ime.

This investigation has shown oral transmission of RR virus in

~ normanensis and that virus levels of up to log 10-5. 45

PFU/ ml were present in the infected mosquitoes. The i!'1vestigation has al so demonstrated that the i nf ected ~

norm an e n s i s c an prod u c e P R 'l l r us s y mp t oms and c i cc u 1 a t i n g ·; 1 1. • u c::

in new born mice in a.s l i tt le as fi ve days . The apparent

c e d u c t i o n i n t h e n u m b fl c • ·) f ,j a :/ s a. f t e r f i r s t e x p o s u r e t o p c o d u c e

v i r u s s J mp t o ms i n t h e 1 a t ^<2 c l 1 t t 8 t' s ( i e .. 5 a n d 6 ) ma y b e r e 1 a t e ct

t o a p o s s i b 1 e i n c r e a s i n g n um b e r· o f i n f e c t e d mo s q u i t o e s i n e a c h cage. There is evidence that even at day 15 there are both non- infected mosquitoes and individuals that had possibly taken their first virus infected blood meal ( Table 3).

Transovarial Transmission

The investigation of possible transovarial transmission of RR virus ink:__ normanensis cannot be conclusi ve. The eggs laid and progeny subsequently tested may not have come from infected female mosquitoes. The suggestion that the majority of the progeny were not from infected females does howe ver seern unlikely, when 8 out of 9 indi viduals in one cage sampled at day 16 were positi ve for virus ( Table and ⁷ of these indi viduals had relati vely high amounts of virus. Only the 215 progeny produced after day 8 ( Table 4) were included in the calculation to arrive at the . 46% level of progeny tested for transovarial transmission. This would increase the probability that the progeny tested were from infected females.

The report transmission mosquitoes at

by Hardy et al ( Hardy et al 1984) of transovarial St. Louis encephalitis virus by Culex and Aedes 18 degrees C but not at 27 degrees C indicates that tcansovarial transmission 1n some species may only occur at either a lower average temperature or possibly a low night temperature. Kay CKay 1982> however, reported evidence of transovarial transmission of RR virus i n ~ v1gilax with females maintained at 28 degrees C. Hardy et al reported the relati ,ely low minimal infection rates for adult progeny in Culex tarsalis of 1: 1 989. Freier and Beier ( Freier and Beier 1984) reported relatively the high filial infection rates for La Crosse virus in F1 adult progeny of parenterally infected Aedes atropalpus and Aedes triseriatus females of 13. 9% and 10. 7% respecti vel y. Kay ( Kay 1 9 8 2) rep o ct e d t he r e 1 at i 'le 1 y high t' ates of t cans o ,, aria 1 transmission of RR v1cus in two of 51 females of RR virus inoculated females of k:__ vigilax.

This investigation.does suggest that if transovarial transmission of RR virus i n ~ normanensis does occur at the temperatures tested, i t may not occur at the relati vely high levels of some other species. Further work with a larger number of known infected individuals is necessary to more fully in·,estigate the probability of transovarial transmission of RR virus in ~

normanensis.

. .

Acknowledgements

Mr G. P. Davis and Ms. H. A. Kelton of the Northern Territory Health Department Medical Entomology Branch carried out the adult collection, identification maintenance and rearing of mosquitoes. Their dedicated assistance is gratefully acknowledged.

Ms. Kelton maintained the insectary, organized and maintained the mice, kept the records, assisted with the tabulation of the results and carr 1ed out the t,yping and word processing. Her invaluable organizat i on and assistance is gratefully acknowledged.

Acknowledgement is also gi ~en t o Mr. Stuart Smith of the Medi cal Entomology Branch, Nor t her n Terri tor y Department of Health, who assisted with the tabulation of the results.

The assistance and guidance of Dr. Territory Department of Primary acknowledged.

G. Gard of Production

the is

Northern gratefully

The dedicated assistance of Mr. R. Heir and Mrs. S. Walsh of the A. L. Rose virol ogy Laboratory, Northern Territory Department of Primary Production, wi t h t he virus isolations i s also gratefully acknowledged.

Part of the work has been carried out with the assistance of the Commonwealth National Disease Control Program.

References

Beaty B. J. , Tesh R. B. & Aitken T. H. C 1980)

transmission of Yellow Fever virus in Stegomyia Am. J. Trop. Med. Hyg. 29( 1) : 1 25-1 32

Transovarial mosquitoes.

Doherty R. L.

1973-1976.

1977) Ar t hr opod-borne vi ruses 1 n Australia, AJE3AK 55 C Pt . 2) : 1 03-1 30

Doherty R. L. , Carley J. G. Kay B. H. , Fillipich C. , Frazier C. K. ( 1979) Isolation of virus mosquitoes collected in Queensland, 1972-76.

< Pt. 5) 509-520

Freier J.E. & .3eier transmiss i on of Am. J. Trop. Med. Hyg.

J. C. ^{( 198 4)} La Crosse virus

33( 4; : 708-71 4

Oral by

and Aedes

Marks E. N. &

strains from AJEBAK 57

transovarial at r~opal pus.

Gorman B. M., Leer J. R. . Fillipich C. , Goss P. D. & Doherty M. D.

( 1975) Plaquing and neutralization of arboviriuses in the PS-EK line of cells. Aus t . J. Med. Technol. 6 65-71

Ha r d y J . L . , R o s e n L . , R e e ·: e s W . C . , S c r i v a n i R. P . & P r e s s e r S . B .

~ 1984) Experime nt al transo'lar1al transmissi on of St .

L o u i s e n c e p h a 1 i t i s · .· 1 c u ~-'. b .-- C u 1 e x a n d A. e d e ~ mo s q u i t o e s . Am. J . T t' , o. He .J i; ·1 ; , 1 r: 6 - ¹ ~ S

G

ao,, \

baited light trap for mosquito borne encephalitis surveillance. Bull. Soc. Vector. Ecol. 4 24-27

Rosen L. , Shroyer D. A. , Tesh R. 8., Freier J. E & Jih Ching Lien ( 1983) Transovarial transmission of dengue 'liruses by mosquitoes: Aedes albopictus and Aedes aegypti.

Am. J. Trop. Med. Hyg., 32( 5) : 1108-1119

Whelan P. I. & Shorthose .J. •. unpublished) The isolation of alpha and flavi viruses in the Northern Territory, 1982-1985.

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T,;BLE 3. AMOUNT OF VIRUS DETECTED IN INOIVIGUAL Ae. norman~ns1s DAY AFTER INTRODUCTION

OF INOCULATED MICE 0

15

CAGE NO.

1 1 1 1 l 1 1 1 1 1 1 1 1 1 2

NO. !JF INDIVIDUALS

1 3 1 2 1 1 1 1 1 1 1 1 1 5

STOMACH \)IRUS ,~ OMME~H ,:Ot·HEW PF^1_1/ m:

bloodfed 5 1;-~gorged 20 bloodfed 0 engorged 10 engorged 0

starved 0

starvi:"d 15 starved log _10-5.1 starved 1 og 10-3.9 starved 1 og 10-S.45 starved 1 og 10-4. '.::

starved 1 og 10-5.S starved log 10-4.0 starved 1 og 10-3.6 starved 0

5 log 10-3.9 log 10-2.7 1 og 10-4.3 log 10-3.4

~ ·-,S:Lr 4. PROGENY OF CAGED Ae.normane-ns1s [,r, i' ,..,FTER : r·4TROOIJC TI ON 0 1 ^·7 c... 3 4 5

<1F I r·;(1C iJL A TED MICE

f.J,:,. " f ^{r11'~'~:'·l '.J} 1 toes ^{1 n C} ag '2 445 288 259 248

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~~ ^{~I ~J} r· ' l rr, ,:\ t t: number~ 5 10

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f.j ¹.l fl'! L ,.. .. :. t r11a l es tested 2

1 11: C ... ~' , .. \ +· +'c-rnal>2-s testt"d l

TESTED FOR "!JRU~; CAGE 1

6 7 ~; 9 10

187 1 73 157 13:, 1 ::?3

250 130 350 3[10

27 14 0 -> ^'7 14

23 19 49 11

(V5821

l l 12 ¹³

91 74 50

320 250

11 31

1 7

l ,_) 21

14 15 16

40 26 16 81

1

17

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1 •:

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0

;ABLE ^C J . PROGENY OF CAGED Aedes :J A y AFTER INTRODUCTION 0 1 2 ^{7 ._)} 4

;:1F

r

r·WC ULA TED MI CE

•' ^\_I• , T ;Iii squ1toes 1 n cage 75 75 72 59 51

r,~, r,. ~· ... j .,· 1 rrict t e number 5 20

·-,; ':' ) ] ·; 1 aid

,' . ; I J fl)~),-:

·=· f ^ma 1 ^t:='S tested 1 5

i ,; ·T1 t. •:-r ,.

females lestelj 1 7

• j t

normanensis TESTED FOR VIRUS

5 6 7 8 9

48 36 36 31 I 31

I

30 7 I ^{7 _IL ...}

I I

9 8 I 4

I

11 ⁹ I 10

CAGE 2 10 11

28 -,,

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40 47

1 4

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lq 15

74

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