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Geographic Aedes aegypti strains and dengue-2 virus: Susceptibility, ability to transmit to vertebrate and transovarial transmission
Ann. Virol. (Inst. Pasteur) 1981, 132 tl, 357-370
GEOGRAPHIC AND
AEDES
DENGUE-2
ABILITY AND
TO
AEGYPTI
VIRUS:
TRANSMIT
TRANSOVARIAL
STRAINS
SUSCEPTIBILITY, TO
VERTEBRATE
TRANSMISSION
by Fran~oise-Xavi~re Jousset Unitd d'Ecologie Virale, Institut Pasteur, 75724 Paris Cedex 15
SUMMARY Belations between dengue-2 virus (New Guinea C strain) and its most common vector, Aedes aeggpti, were investigated in 5 geographical strains of mosquitoes originating from endemic areas for dengue fever. Four strains were recently collected from regions with high (Hanoi strain, Vietnam), rather high (Guadeloupe strain, Carribean), low (Lagos strain, Nigeria), or no incidence for dengue virus (Kari strain, Upper Volta). The fifth strain was colonized in the laboratory for several years (Queensland strain, Australia). Following infection by intrathoracic inoculation,, the susceptibility of the strains to dengue-2 virus, their ability to transmit the virus to suckling mice (SM) by bite and the possibility of transovarial transmission were compared. The virus replicated in the 5 strains at the same rate averaging 4.1 loglo PFU/mosquito, and the 50 % mosquito infection dose was similar for each strain (0.001 PFU). The ability of infected females to transmit the virus to SM by bites varied from 37 to 56 % according to the strain. Transovarial transmission was demonstrated in 4 strains. The Kari strain from non-endemic region did not transmit the virus. The minimal filial infection rate was rather low ranging from 0.3 to 1.2 %0Infected progeny of both sexes were detected only in the first or second ovarian cycle from eggs ovoposited between the 14th and the 16th day post inoculation of parent females. KEY-WORDS: Arbovirus, Aedes aeggpli, Dengue virus; Susceptibility, Transmission to vertebrate, Transovarial transmission, Mouse.
Manuscrit re~u le 20 m a r s 1981, acceptd lc 18 s e p t e m b r e 1981.
358
F.-X. JOUSSET
INTRODUCTION Dengue fever infections, caused b y at least 4 viruses classified as a subgroup of the group B (Flavivirus) of Togaviridae, are among the most severe h u m a n viral diseases in tropical and subtropical areas. They are characterized b y fever, aches, pains and sometimes b y haemorrhagic manifestations, shock and death of infected children [30]. These diseases are t r a n s m i t t e d to man b y mosquitoes of the genus Aedes, principally A. aegypli and A. albopictus. The susceptibility of both Aedes species to infection with the 4 dengue serotypes has been investigated [13, 14, 15, 33]. Furthermore, the mechanisms b y which most arbovirnses reappear in enzootic areas following the period of interrupted active transmission (i. e. winter in the t e m p e r a t e zone, dry season in the tropics) remain still obscure. Among the several hypotheses reviewed b y Reeves [25] the possibility of transovarial transmission in the vector has been p u t forward. This mechanism of virus maintenance in nature has been shown to occur in ticks for tick-borne encephalitis virus [26], in sand-flies for vesicular stomatitis virus [31] and since 1973 in mosquitoes for some B u n y a viruses of the California encephalitis and B u n y a m w e r a groups [10, 18] and, as recently demonstrated, for some flaviviruses such as Koutango, Japanese encephalitis, yellow fever and St Louis encephalitis viruses [2, 4, 11, 17, 291 .
The purpose of the present s t u d y was to compare t h e susceptibility to dengue-2 virus of 5 strains of A. aegypti collected from areas where dengue viruses are endemic or not, the ability of the mosquitoes to transmit the virus under l a b o r a t o r y conditions and to investigate and demonst r a t e the possible transovarial transmission of the virus in A. aegypti.
MATERIALS
AND METHODS
Virus slrain. The virus used in these studies was the prototype New Guinea C strain of dengue type 2, mouse passage level 25. Stock virus was prepared from 5 to 6-dayinfected suckling mice (SM) as a 10 % brain homogenate in Hanks medium, pH 7.2, containing 7.5 % of bovine serum albumin, 200 units of penicillin and 50 ~g of streptomycin/ml. The supernatant obtained after clarification at 2,000 rpm for 15 min was stored at -- 70 ~ C as 1.0 ml aliquots in sealed ampoules.
FCS i.c. i.t. LAC MFIR MIDso o r . c.
= = --= ---
foetal calf serum. intracerebrally. intrathoracic(ally). La Crosse. minimal filial infection rate. m o s q u i t o i n f e c t i o n d o s e 50 %. ovarian cycle.
PFU p.i. PS
= = =
SM = SMLD~ o ~
plaque-formiug unit. post inoculation. a c o n t i n u o u s p i g k i d n e y celA c u l tures. suckling mouse. s u c k l i n g m o u s e l e t a | d o s e 50 %.
AEDES
AEGYPTI
AND DENGUE-2 VIRUS
359
Virus assay. Virus titrations of stock suspensions and infected mosquitoes were done by plaque assay on continuous pig kidney cell cultures (PS) according to the procedure described by Rager-Zisman and Merigan [23]. Serial 5-fola dilutions of the test material were prepared in Leibovitz growth medium, pH 7.4, supplemented with 3 % foetal calf serum (FCS) and antibiotics. From each dilution (0.2 ml) was inoculated into 4 microplate wells, and 0.4 ml of overlay medium (Leibovitz: 47 %; FCS: 3 %; 2,3 % carboxymethylcellulose: 50 %) was added after 3 h of incubation. Plaques were counted 5 days later, after staining with naphtalene blue black. The virus titre was expressed as the number of plaqueforming units (PFU) per ml of suspension or per insect. Dengue virus was also titrated by inoculating intracerebrally (i. c.) ten 2-3 days old SM with 0.02 ml of 10-fold dilutions prepared in Hanks medium. The virus titre was estimated b y the end point dilution method according to Reed and Muench [24] and expressed as suckling mice letal dose 50 % (SMLDso) per ml.
Mosquitoes. The following strains of A. aeggpti were chosen according to t h e i r origin: 1 colonized strain from Queensland, Australia (Queens strain) reared for numerous generations in the laboratory, and 4 field strains recently collected (2 or 3 generations in. colony) in Vietnam (Hanoi strain) where dengue infections are very common, m Guadeloupe (Guad. strain) where dengue lever is frequent, in Nigeria (Lagos strain) where only some cases of dengue are reported, and in Upper Volta (Kari strain) where no dengue disease occurs. All mosquitoes were reared in an insectarium at 25 ~ C, with 80 % room humidity, under a photoperiod of 18 h of light. Larvae were given a diet of mice biscuits, and adult mosquitoes were maintained on 10 % sucrose solution. All experiments were conducted under the same conditions using, for infection, groups of 3-7 days old mosquitoes.
Inoculation o/ mosquitoes. Adult mosquitoes were infected by intrathoracic (i.t.) inoculation. After being anaesthetized by cold at 4 ~ C for 15 min, mosquitoes were transferred into a Petri dish placed over an ice bath. They were inoculated by inserting, under binocular, a glass microneedle between the two first segments [6, 27]. The equipment used to fill the microneedles with the virus suspension and to inject it into the body cavity was similar to t h a t described by L'H~ritier [19] for injecting drosophila, with some improvements. The mean volume injected was determined by weighing the needle before and after a series of inoculation. It was about 0.6 tzl per female and 0.3 71 per male, corresponding to half the weight of the mosquitoes. The mortality of insect due to i.t. inoculation was low,
Screening /or susceptibilitg o] A. aegypti /rom different areas to dengue-2 virus. The susceptibility of the 5 strains of A. aegypti to dengue-2 was tested by establishing the virus growth curves or kinetics of virus replication in mosquitoes and by comparating the titre of a same viral suspension in each strain. a) Kinetics o/ replication. - - Females and males of each strain of A. aegypti were infected by i.t. inoculation with a 1/10 suspension of dengue-2 virus stock. Samples of 5 females and 5 males were collected at regular intervals and stored at -- 70 ~ C. Prior to viral titration in PS cells, each pool of 5 mosquitoes was triturated in 1.0 ml of Leibovitz medium, and suspensions were clarified at 2,000 rpm for 15 rain.
360
F.-X. J O U S S E T
b) Titration o[ virus in mosquitoes. - - Serial 10-fold dilutions of a suspension of dengue-2 virus, previoulsy titrated in PS cells, were injected i.t. into groups of A. aeggpti females of the 5 strains. These insects were held for 10 days, then generally at least 16 mosquitoes for each dilution were ground individually in 1.0 ml of medium and tested for the presence or absence of virus in PS cells. Infection rate was expressed as the percentage of virus positive mosquitoes in each dilution, and the titre of the virus suspensions was calculated b y the method of Reed and Muench [24] and expressed in mosquito infectious dose 50 % (MIDso). Transmission to SM. For transmission attempts, pools of about 20 i.t. infected females were allowed to engorge individually on normal SM. Each mosquito, deprived of a sugar source for at least 48 h, was placed into a cylindric plastic container (7 cm lenght, 3,5 cm in diameter) covered with a gauze. An SM was maintened for 1 h 30 min, at the top of each container, in darkness. Then, engorged and not engorged mosquitoes were returned separately into cages and bitten SM to their mothers. The transmission a t t e m p t s were started 4 days post inoculation (p.i.) of females and continued until their death (73 days). Females might engorge 3 times, the first time from 4 to 13 days p.i., the second time from 17 to 45 days p.i. and the third time from 52 to 73 days p.i. The bitten SM were observed for l0 days for signs of illness or death. Brains from all a p p a r e n t l y normal SM were tested for the presence of virus by reinoculation to five SM. Viral identification was confirmed b y the complement fixation test (LBCF) [7] using antigens prepared from SM brains b y the sucrose acetone method [9], reference dengue t y p e l, 2, 3 and 4, and Chikungunya hyperimmune ascitic fluids prepared according to B r a n d t et al. [5]. The transmission rate was expressed as the percentage of engorged females transmitting the virus to susceptible animals. Transovarial transmission. To investigate the possibility of vertical transmission, m a t e d and i.t. infected females were allowed to ovoposit 2 days after a blood meal on SM. Eggs from 3 ovarian cycles (ov. c.) were collected at 9 and 14 days p.i. for the first ov. c., at 16 and 25 days p.i. for the second and at 30 and 40 days p.i. for the third. Eggs were held at least 8 days at room t e m p e r a t u r e in order to inactivate the viruses which might contaminate their surface. Then, they were hatched. After larval and pupal development at 25 ~ C, 5-8 days old adults were sexed, pooled by lots of about 40 females or 50 males, and tested for virus. For Queens strain, the 4th instar larva progeny were also examined. In order to increase the virus titre assumed to be rather low, each pool of F1 progeny was triturated in 1.5 ml of H a n k s medium and inoculated i.t. into 15 uninfected A. aegypti. After l0 days, the mosquitoes were ground and assayed for virus in PS cells. If plaques occurred, suspensions of progeny mosquitoes were inoculated i.e. into a litter of SM. Viral recovery and identification were confirmed as previously described.
RESULTS Titration of virus stock. T h e m e a n t i t r e of t h e v i r u s s t o c k a s s a y e d s e v e r a l t i m e s , b o t h in SM a n d P S cells, w a s a b o u t 6.6 loglo SMLDso a n d 5.2 loglo P F U / m l , resp e c t i v e l y . T i t r a t i o n in SM w a s t h e r e f o r e 25 t i m e s m o r e s e n s i t i v e t h a n in P S cells.
AEDES AEGYPTI AND DENGUE-2 VIRUS
36I
Multiplication of dengue-2 virus in five A. aegypti strains. Each mosquito was inoculated with a p p r o x i m a t i v e l y 10 P F U for a female and 5 P F U for a male. The kinetics of virus replication are summarized in table I, and figure 1 shows the typical growth curve of the virus in the Hanoi strain. Each point represents the mean virus titre of 5 mosquitoes. Following inoculation, no infectivity could be detected in PS cells or in SM. Thi s corresponds to the eclipse phase. The virus content per insect, 24 h p. i. was already equal or superior to the inoculum except for males of Queens and Guadeloupe strains. The virus titres increased until the 10th day and then stabilized at a p p r o x i m a t e l y 4.1 loglo P F U per insect. A slight decrease was observed later until the death of mosquitoes, b u t the virus titre remained high. The p a t t e r n of virus replication in both sexes was similar b u t the virus titres were a p p r o x i m a t e l y 2.5 times less in males than in females at the maximal phase. This difference results from the fact t h a t males are smaller than females. The 5 mosquito strains were similar in their ability to s u p p o r t dengue-2 virus replication after inoculation. "]'ABLE I. - - Comparative virus content of ~ and ~ ~ A aegypti >> from 5 geographic strains after parenteral infection with dengue-2 virus (*). T i m e a f t e r i n o c u l a t i o n (in d a y s ) Mosquito strain
Females
Males
3h
24 h
2
6
10
15
20
25
30
35
40
50
55
63
Queens Hanoi Guad. Lagos Karl
0 0 0 0 0
-1.7 1.0 1.7 f.7
1.8 2.7 2.8 2.4 3.7
3.1 3.3 3.9 3.5 4.8
4.1 4.4 4.0 4.6 3.9
4.4 4.4 4.0 4.6 4.3
4.5 4.3 3.7 4.3 4.5
3.8 4.3 4.1 4.0 4.2
4.3 4.1 4.0 4.1 3.9
4.2 4.1 4.0 4.0 4.0
3.7 3.9 3.7 4.0 4.7
3.8 3.8 3.5 3.9 4.1
3.5 3.3 3.9 --
3.7 -3.7 4.0
Queens Hanoi Guad. Lagos Kari
0 0 0 0 0
0.2 1.0 0.1 1.5 1.6
1.3 2.3 --2.3 --
2.0 2.6 2.5 3.1 4.4
3.5 3.7 -3.4 4.2
4.1 3.3 3.5 3.7 --
4.3 3.4 --3.5
-3.3 3.5 3.2 3.6
3.4 --
3.4
3.8
(*) I n o c u l a t i o n w a s 1 log10 P F U p e r f e m a l e a n d 0.7 loglo P F U p e r m a l e . T h e d a t a a r e e x p r e s s e d in loglo P F U a n d r e p r e s e n t t h e m e a n f r o m 5 m o s q u i t o e s .
Since no significant difference in the dengue-2 virus content could be detected in these 5 strains, an experiment was carried out to reveal a possible variation in the infection rate of female mosquitoes following inoculation of serial 10-fold dilutions of virus stock. The result of this experiment are summarized in table II. At the 1/10,000 dilution, i. e. 0.001 P F U inoculated per insect, the Lagos and Kari strains had more t h a n 50 % of infected females whereas the other 3 had less, with a significative difference. However, t h e a m o u n t of dengue-2 virus able to infect 50 ~ / o f
362
F.-X. JOUSSET
mosquitoes was estimated to be between 0.002 and 0.0005 P F U according to the strain of Aedes. The titre of the stock virus was 7.9 to 8.5 lOgl0 MIDso/ml. The difference was only 4-fold, and no significative variation in susceptibility in these 5 strains could be detected. It should be noted t h a t the virus
E
3 {3.
2
~ _
5
J
I
i
i
I
t
i
10
15
20
25
30
35
40
days
after
II
.d" I
J
I
50
60
70
inoculation
FIG. 1. - - Dengue-2 virus content in female and male mosquitoes from H a n o i strain, following i. t. inoculation. Each points represents the m e a n virus titre of mosquitoes.
TABLE II. - - Percentage of infected ~ mosquitoes 10 days after inoculation of various doses of dengue-2 virus (*). Dilutions of virus suspension (in loglo) ( n u m b e r of P F U inoculated) (**) Mosquito strain
2 (1)
3 (0.1)
4 (0.01)
5 (0.001)
6 (0.0001)
Queens Hanoi Guad. Lagos Karl
100 100 100 100 100
100 100 100 100 100
94 95 100 100 95
37 23 33 58 79
10 8 8 17 5
7 (0.00001) 0 0 0 0 0
Titre of suspension in MIDso 8.0 7.9 7.9 8.5 8.2
(*) Calculated w i t h 12 to 16 insects. (**) Titre of inoculated virus suspension was 5.2 P F U / m l . E a c h female received a b o u t 0.6 ~l of virus suspension.
A E D E S A E G Y P T I AND DENGUE-2 VIRUS
363
content of all infected mosquitoes assayed in PS cells was very similar and close to 4 log~o P F U , whatever m a y be the a m o u n t of virus inoculated. Transmission to S M .
The attractiveness of SM for female mosquitoes was 49, 53, 67, 76 and 69 % for Queens, ttanoi, Guadeloupe, Lagos and Kari strains, respectively. An overall total of 1,302 transmission a t t e m p t s were carried out; 826 females took a full blood meal and 258 SM were infected by bite of a single female, of which 233 died following bite. The transmission rate for each strain is reported in table III and figure 2. No transmission occurred on the 4th day p. i.; the first transmission began on the 5th day p. i. for Queens (1/8), Lagos (2/16) and Karl (1/15) strains. This corresponds v e r y likely to incubation time for the virus to replicate in the salivary gland. The transmission rates increased until t h e 10th day p. i. and were fluctuant at each attempt. However, transmission rates were maintened until the death of females. E x c e p t for one case where the 10 females tested for the Lagos strain t r a n s m i t t e d the virus, in no other instance could all t h e females transmit: the vector percentage reached 56 % (54/97), 45 % (43/95), 37 % (43/117), 45 % (48/106) and 4 1 % (53/129) for Queens, Hanoi, Guadeloupe, Lagos and Kari strains, respectively, from the 10th day p. i. to death. These results did not v a r y significantly enough to distinguish the transmission efficiency of females originating from endemic or nonendemic areas, at least after this mode of infection of mosquitoes. The n u m b e r of females used for transmission a t t e m p t s was too i m p o r t a n t (108, 139, 146, 102 and 112 for each strain) to keep each female individually t h r o u g h o u t the experiment and follow its ability to t r a n s m i t at each bite.
TABLE I I I . - -
of i.t. infected females transmitting dengue-2 virus by bite to SM by incubation time.
Percentage
I n c u b a t i o n t i m e in d a y s 4-7 d a y s p. i. (*)
10-45 d a y s p. i. (**)
52-73 d a y s p. i, (***)
Mosquito strain
Nb ~ engorged
Nb 9 transmitting
percenrage
Nb 9 engorged
Nb 9 transmitting
percentage
Nb ~ engorged
Nb ~ transmitting
percentage
Queens Hanoi Guad. Lagos Karl
43 38 83 60 58
1 0 1 10 5
2 0 1 17 9
65 85 87 90 84
37 39 36 42 35
57 46 41 47 42
32 10 30 16 45
17 4 7 6 18
53 40 23 37 40
(*) 1st feeding. (**) 1st a n d 2rid feeding. (***) 3rd feeding.
36~.
F.-X. JOUSSET
c
i i
,
,
,
,
i
,
,
o
,
8o ~q90~
uol~s,wsu~J~
~uoaJad
f
s f c4
8 i
,
,
~.~
i
o~
uor~sluJsu~,J~
~uasJaa
~
Ol
UOpSSI~gU~J ~
lu8sJad
AEDES A E G Y P T I AND DENGUE-2 VIRUS
365
Transovarial transmission experiments. The results of experiments to determine transovarial transmission rates of dengue-2 virus in experimentally infected females of A. Aeggpli are summarized in table IV. W i t h the exception of the Kari strain, transovarial transmission occurred in the 4 other strains. Despite t h a t difference, perhaps not significant, the minimal filial infection rate (MFIR) was r a t h e r low with a m a x i m a l rate of 1.2 %o in Guadeloupe strain. A total of 16,590 a d u l t progeny from 3 or. c. (ovarian cycles) were tested in 406 pools. F r o m the 8 positive pools for dengue-2 virus, 6 were from male and 2 from female lots. T h e y came from eggs ovoposited on the first or second ov. c., b u t always after at least 14 days p. i. of females. It thus appears t h a t the incubation period prior to ovoposition is essential for suecessfull transmission. No infected progeny could be recovered in the adults from ovoposition on the 25th day p. i. or later. The mean M F I R for t h e 5 strains was inferior to 0.5 %o. E x a m i n a t i o n of 2,494 larva progeny from Queens strain tested in 29 pools gave negative results. TABLE IV. - - Dengue-2 virus in r l progeny from 3 ov.c of i.t. infected (~ A. aegypti >) females 9 to 50 days p.i. No. progeny tested
Strains
Queens
?
Pools+/ no tested
Origin of positive cases ov.e.
d.p.i.
MFIR
Total MF1R
1 400 1 642 2 494
0/39 1/40 0/29
2nd
16
1/586
1/3 042
1 903 I 833
1/54
1/43
2nd 2nd
16 16
1/748 1/739
1/1 868
~ c~
1 773 1 478
1/45 3/31
1st 1st
14 14
1/664 1/598
1/813
Lagos
9
1 461 1 494
0/38 1/33
2nd
16
1/178
1/2 955
Kari
~
1 725 I 881
0/44 0/39
larvae Hanoi Guad.
2
0/3 606
DISCUSSION Five geographic strains of A. aeggpti including one colonized (Queens) and four recently collected from dengue-2 virus endemic or non-endemic areas (Hanoi, Guadeloupe, Lagos and Kari) were compared for (1) their susceptibility to infection with dengue-2 virus, (2) their ability to contam i n a t e a vertebrate host, and (3) the possibility of a transovarial transmission of the virus as a mechanism of m a i n t e n a n c e of the virus in n a t u r a l conditions.
366
F.-X. JOUSSET
Concerning the first point of comparison, the results indicate t h a t the five strains were alike in their ability to replicate the virus after parenteral infection. In this respect, it is interesting to note t h a t the titration of the virus stock b y inoculating mosquitoes proved to be the most sensitive, averaging 8.2 loglo MIDs0/ml. Mosquitoes are 1,000 times more sensitive than PS cells (5.2 loglo P F U / m l ) and 40 times more sensitive than SM (6.6 log10 SMLDso/ml). B o t h males and females were susceptible to the virus with a slight difference in the a m o u n t of virus as already reported for A. albopictus [27]. No variation was observed between mosquito strains in the virus content of infected insects after a standard incubation period, w h a t e v e r the a m o u n t of virus inoculated might be. The q u a n t i t y of virus required to infect 50 % of mosquitoes varied only 4-fold between the strains and averaged 0.001 P F U . Similar results showing no difference in the susceptibility to parenteral infection with a single arbovirus among mosquito strains of the same species from different areas, have been reported [13, 15, 16]. On the contrary, a variation of 1,000- to 1,500-fold in the infection rate or in the virus content among such strains could be demonstrated when the virus was ingested [1, 12, 13, 15, 16, 32]. In our study, a t t e m p t s were carried out to infect mosquito females b y feeding on viremic SM or on pledgets soaked with a mixture of virus ~ guinea-pig blood Jr sucrose 10 % (equal volume). The titres of infectious meals were 4.2 and 5.9 lOglo SMLD~o/ml, respectively. But, none of 265 females from the 5 strains fully engorged on SM was infected; out of the 160 females from the 5 strains fed and engorged on pledgers, only 2 of Queens strain were infected, with a virus content of 4.1 log~o P F U . This failure to infect adults orally could result from various factors: insufficient viremia titres in SM and/or the effect of a possible barrier which would take place before virus replication in the mosquito such as a gut cellular resistance [22]. Obviously, the fact t h a t the virus strain used in these experiments had undergone 25 consecutive mouse passages, m a y have modified its ability to infect mosquitoes b y oral route. Whatever, Gubler et al. [15] succeeded in orally infection of several A. aeggpli strains with this New Guinea C dengue-2 virus strain. Concerning the biological transmission of dengue-2 virus to a verteb r a t e host, the SM, no significant variation could be detected between the five geographic strains. In the best period, from the 10th day p. i., the mean transmission rates varied from 37 (Guadeloupe strain) to 56 % (Queens strain). This similarity can be explained b y the fact t h a t after parenteral infection, all mosquitoes finally had the same virus content, and the infection of salivary glands was similar in all strains. There is, of course, no assurance t h a t similar results would have been obtained using mosquitoes infected b y oral route. However, it has been reported t h a t resistant mosquito strains from non-endemic areas to oral infection with dengue-2 virus are just as able to t r a n s m i t the virus, once t h e y become infected as those from susceptible strains from endemic areas [15]. Oil the contrary, with the yellow fever-A, aegypli and the La Crosse (LAC, a m e m b e r of the California subgroup of Bunyaviridae)-A. triserialus
AEDES A E G Y P T I AND DENGUE-2 VIRUS
367
systems, there was a variation in transmission rate: the strains more susceptible to oral or parenteral infection showed the highest ability to t r a n s m i t the virus by bite [1, 3, 12]. Finally, the transovarial transmission of dengue-2 virus was demonst r a t e d in 5 different strains of A. aeggpti mosquitoes after parenteral infection, in F1 adult progeny. The Karl strain from non-endemic region did not transmit. In positive strains, both F1 females and males were found infected as in the ease of other transovarially t r a n s m i t t e d flaviviruses [2, 4, 17, 29]. However, the frequency of the phenomenon was r a t h e r low from 0.03 to 0.12 ~ in the best ease (Guad. strain). The M F I R were similar or about 10 times lower t h a n those reported for other flaviviruses. No q u a n t i t a t i v e data were given for K o u t a n g o virus t r a n s m i t t e d by A. aeggpti and for dengue viruses t r a n s m i t t e d by A. albopictus [11, 29]. Yellow fever was t r a n s m i t t e d by A. aeggpti and A. mascarensis in a d u l t progeny from the first ov. c. with an M F I R from 0.14 to 0.16 % [2, 4]. St Louis encephalitis virus was t r a n s m i t t e d transovarially in first or. e. by A. albopidus and A. epadius with a higher M F I R in F, larvae t h a n t h a t in adult progeny when larval development occurred at 27 ~ C (0.52 vs 0.07 % and 0.45 vs 0.16 ~ respectively). The M F I R increase in adult progeny when larval development took place at 18 ~ C [17]. Japanese encephalitis virus was t r a n s m i t t e d by A. albopiclus, A. togoi and Culex tritaeuiorhgnchus. In the first two eases, the virus was detected in a d u l t progeny with an M F I B of 0.37 and of 0.70 %, respectively. In C. trilaeniorhgnchus, no adult was infected b u t the virus was recovered in 0.47 ~ of larva progeny [28, 29]. In our study, no increase in the transovarial transmission rate of dengue-2 virus could be detected in A. aeggpti larvae as compared to a d u l t progeny, in contrast with results obtained with St Louis and Japanese encephalitis viruses [17, 28]. The transovarial transmission of dengue-2 virus was d e m o n s t r a t e d in the two first or. e. of infected females b u t t h e time interval from infection to the day of ovoposition seemed more i m p o r t a n t t h a n the rank of or. c. In all eases, infected progeny was ovoposited on day 14 or 16 p. i., b u t no transmission was detected from progeny laid on days 25 to 50 p. i. The absence of transovarial transmission in the late second or third ov. c. was probably not due to the stop of multiplication of the virus in the ovaries since the titre of the virus in adults remained constant, but resulted more likely from insufflsant n u m b e r of ovoposited eggs at the time. Similar results were reported in the case of yellow fever virus which was transm i t t e d to a low level in the 3rd ov. e. at 23 days and not in 4 and 5th ov. c. at 35 and 45 days [4]. Contrarily to flaviviruses, viruses of the California subgroup of B u n y a viridae exhibit a very high transovarial transmission rates in the first ov. c. by their mosquito vectors. Thus, 98 ~ of infected females t r a n s m i t t e d LAC virus to their offspring, and the mean percentage of infected offspring from an infected female was 7 1 % [201. Seemingly, 55 % of the infected A. lrivillatus tested t r a n s m i t t e d transovarially Triviltatus virus which
368
F.-X. JOUSSET
was isolated from 16 % of the F1 generation progeny examined [8]. On the other hand, in the LAC-A. triseriatus system, the rank of ov. c. was important: larvae from first ov. c. following infection of females were never infected; LAC virus was not detected in the second ov. c. larvae in which the blood meal for the first ov. c. was not infectious [21]. Since transovarial transmission of dengue-2 virus b y A. aegypti was demonstrated, it is likely t h a t the 3 other t y p e s of dengue viruses can also be t r a n s m i t t e d transovarially, as it has been shown with A. albopictus (L. Rosen, unpublished work). Althought transovarial transmission appears under laboratory conditions at low rates, it can exist in nature and it has to be investigated under field situations, b u t its epidemiologic significance is still unknown.
RESUME R E L A T I O N S E N T R E CINQ S O U C H E S G E O G R A P H I Q U E S D E (( A E D E S A E G Y P T I D ET L E V I R U S D E LA D E N G U E : S E N S I B I L I T E , C A P A C I T E Y E C T R I C E P O U R U N tt()TE VERTt~13RI~ ET TnANSMISSION TnANSOVARIENNE
Cinq souches g~ographiques de Aedes aeggpti, vecteur des virus de la dengue, out 6t6 compar6es ~ l'~gard de ce virus (du t y p e 2) q u a n t ~ ]eur sensibilitY, leur capacit~ veetrice pour un h6te vert~br~ sensible - - le souriceau - - et leur capacit6 ~ t r a n s m e t t r e le virus ~ leur descendance. Les diff~rentes souches de moustiques provenaient de pays off la dengue, maladie humaine, est relativement fr~quente (Asie du Sud-Est, Guadeloupe) ou non (Afrique, Australie). AprSs inoculation du virus 5 des moustiques par voie intrathoraeique, aucune diff6rence significative a'a ~t~ d~cel~e entre les souehes, qu'elles viennent ou non de pays end~miques de la dengue. La replicatioa virale atteint environ 4,1 loglo P F U par insecte ; les doses p e r m e t t a n t 50 % d'infection de moustiques sont de l'ordre de 0,001 P F U . Selon les souches, 37 ~ 56 % des femelles infect~es t r a n s m e t t e n t par piqfire le virus aux souriceaux. Enfin, la transmission transovarienne a 5t~ raise en 6vidence : ~ l'exception de la soucbe d'Afrique de l'Est, r~gion non end6mique pour la dengue, les quatre autres souches transm e t t e n t le virus a u n e faible proportion (0,03 ~:~ 0,12 %) de leur descendance, le 14 et le 163 jour apr~s inoculation. MOTS-CLI~S : Arbovirus, Aedes ae.qgpti, Virus de la dengue ; Sensibilit6, Capacit6 vectrice, Transmission transowwienne, Souris.
REFERENCES T. H. G., Downs, W. G. & SHOPE, R. E., Aedes aeggpli strain fitness for yellow fever virus transmission. Amer. J. trop. Med. Hgg., 1977, 26, 985-989.
[1] A I T K E N ,
A E D E S A E G Y P T I AND DENGUE-2 VIRUS
369
[2] AITKEN, T. H. G., TESH, R. B., BEATY, B. J. & ROSEN, L., Transovarial transmission of yellow fever virus by mosquitoes, Aedes aeggpti. Amer. J. trop. Med. Hyg., 1979, 28, 119-121. [3] BEATY, B. J. ~r AITKEN, T. H. Ca., In vitro transmission of yellow fever virus by geographic strains of Aedes aeggpti. Mosquito News, 1979, 39, 232-237. [4] BEATY, B. J., TESH, R. B. 8r AITKEN, T. H. Ca., Transovarial transmission of yellow fever virus in stegomya mosquitoes. Amer. J. trop. Med. Hgg., 1980, 29, 125-132. [5] BRANDT,W. E., BUESCHER, E. L. & HETRICK, F. M., Production and characterization of arhovirus antibody in mouse ascitic fluid. Amer. J. trop. Med. Hy.q., 1967, 16, 339-347. [6] CARLEY, J. G., STANDVAST, H. A. & KAY, B. H., Multiplication of viruses isolated from arthropods and vertebrates in Australia in experimentally infected mosquitoes. J. reed. Entomol., 1973, 10, 244-249. [7] CASEV, H. L., Standardized diagnostic complement fixation method and adaptation to microtest. Public health monograph 74 (34 p.), U.S. Governement Printing Office, Washington D. C., 1965. [8] CHRISTENSEN,B. M., RO'~VLEY,W. A., gONG, Y. M., DORSEY, D. C. ~r HAUSLER, W. J., Laboratory studies of transovarial transmission of trivittatus virus by Aedes trivittatus. Amer. J. lrop. Med. Hyg., 1978, 27, 184-186. [9] CLARKE,D. H. ~ CASALS, J., Techniques for hemagglutination and hemagglutination inhibition with arthropod borne viruses. Amer. J. lrop. Med. Hyg., 1958, 7, 561-573. [10] CORNER, L. C., ]:{OBERTSON,A. K., HAYLES, L. B. & IVERSEN, J. 0., Cache Valley virus: experimental infection in Culiseta inornata. Canad. d. Microbiol., 1980, 26, 287-290. [11] Coz, J., VALADE, M., CORNET, M. & ROUlN, Y., Transmission transovarienne d'un flavivirus le virus Koutango chez Aedes aegypli. C. R. Acad. Sci. (Paris) (Sbr. D), 1976, 283, 109-110. [12] GRIMSTAD,P. R., CRAIG, G. B., Ross, Q. E. & YUILL, T. M., Aedes triserialus and La Crosse virus: geographic variation in vector susceptibility and ability to transmit. Amer. J. trop. Med. Hyg., 1977, 26, 990-996. [la] GVBLER, D. J. & ROSEN, L., Variation among geographic strains of Aedes albopictus in susceptibility to infection with dengue viruses. Amer. J. trop. Med. Hyg., 1976, 25, 318-325. [14] GUBL~R, D. J. & ROSEN, L., Quantitative aspects of replication of dengue viruses in Aedes albopictus after oral and parenteral infection. J. reed. Entomol., 1977, 13, 469-472. [15] GUBLER, D. J., NALIM, S., TAN, R., SAIPAN, H. ~r SAROSO, J. S., Variation in susceptibility to oral infection with dengue viruses among geographic strains of Aedes aegypti. Amer. J. trop. Med. Hyg., 1979, 28, 1045-1052. [16] HARDY, J. L., REEVES, W. C. & SJi)GREN, R. D., Variation in the susceptibility of field and laboratory populations of Culex tarsalis to experimental infection with western equine encephalomyelitis virus. Amer. J. Epidemiol., 1976, 10a, 498-505. [171 HARDY, J. L., ROSEN, L., KRAMER, L. D., PRESSER, S. B., SHROYER, D. A. & TURELL, M. J., Effect of rearing temperature on transovarial transmission of St Louis encephalitis virus in mosquitoes. Amer. J. trop. Med. Hy9., 1980, 29, 963-968. [18] LE Duc, J. W., The ecology of California group viruses. J. meal. Entomol., 1979, 16, 1-17. [19] L'HEmTIER, Ph., A convenient device for injecting large number of flies Drosophila. In/ecl. Sere., 1952, 26, 131. [20] MILLER, B. R., DE FOLIART, G. R. ~r YUILL, T. M., Vertical transnfission of La Crosse virus (California encephalitis group) transovarial and filial infection rates in Aedes triseriatus. J. reed. Entomol., 1977, 14, 437-440.
370
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[211 MILLER, B. R., DE FOLIART, G. R. &; YUILL, T. M., Aedes triseriatus and [22] [231
[24] [25] [261 [27] [28]
[29] [30] [31]
[32] [33]
La Crosse virus: lack of infection in eggs of the first ovarian cycle following oral infection of females. Amer. d. trop. Med. Hyg., 1979, 28, 897-901. MURPHY, F. A., Cellular resistance to arbovirus infection. Path. Invertebr. Vectors Dis., 1975, 266, 197-203. RAGER-ZISMAN,B. • MERIGAN,T. C., A useful quantitative semi-micromethod for viral plaque assay. Proc. Soc. exp. Biol. (N. Y.), 1973, 142, 1174-1180. REED, L. J. & MUENCH, H., A simple method of estimating fifty per cent and point. Amer. d. Hyg., 1938, 27, 493-497. REEVES, W. C., 0verwintering of arboviruses. Progr. meal. Virol., 1974, 17, 193-220. REHACEK, J., Transovarial transmission of tick-borne encephalitis virus by ticks. Acta virol., 1972, 6, 220-226. ROSEN, L. & GUBLER, D. J., The use of mosquitoes to detect and propagate dengue viruses. Amer. J. trop. Med. Hyg., 1974, 25, 1153-1160. ROSEN, L., SHROYER, D. A. & LIEN, J. C., Transovarial transmission of Japanese enceFhalitis virus bv Culex tritaeniorhynchus mosquitoes. Amer. J. lrop. Meal. Hyg., 1980~, 29, 711-712. ROSEN, L., TESH, R. B., LIEN, J. C. & CROSS, J. H., Transovarial transmission of .Japanese encephalitis virus by mosquitoes. Science, 1978, 199, 909-911. SCHLESINGER, R. W., Dengue viruses, in ~ Virology monographs ))(S. Gard & C. HALLAUER)16, Springer-Verlag, Wien, New York, 1978. TEsn, R. B., CHIANOTIS, B. N. & JOHNSON, K. M., Vesicular stomatitis virus (Indiana serotype): transovarial transmission by phlebotomine sandflies. Science, 1972, 175, 1.177-1479. TESH, R. B., GUBLER,D. J. & I21OSEN,L., Variation among geographic strains of Aedes albopictus in susceptibility to infection with chikungunya virus. Amer. J. trop. Med. Hyg., 1976, 25, 326-335. WHITEHEAD, R. H., YUILL, T. M., GOULD, D. J. & SIMASATHIEN, P., Experimental infection of Aedes aegypti and Aedes albopiclus with dengue viruses. Trans. roy. Soc. trop. Med. Hyg., 1971, 65, 661-667.
GEOGRAPHIC AND
AEDES
DENGUE-2
ABILITY AND
TO
AEGYPTI
VIRUS:
TRANSMIT
TRANSOVARIAL
STRAINS
SUSCEPTIBILITY, TO
VERTEBRATE
TRANSMISSION
by Fran~oise-Xavi~re Jousset Unitd d'Ecologie Virale, Institut Pasteur, 75724 Paris Cedex 15
SUMMARY Belations between dengue-2 virus (New Guinea C strain) and its most common vector, Aedes aeggpti, were investigated in 5 geographical strains of mosquitoes originating from endemic areas for dengue fever. Four strains were recently collected from regions with high (Hanoi strain, Vietnam), rather high (Guadeloupe strain, Carribean), low (Lagos strain, Nigeria), or no incidence for dengue virus (Kari strain, Upper Volta). The fifth strain was colonized in the laboratory for several years (Queensland strain, Australia). Following infection by intrathoracic inoculation,, the susceptibility of the strains to dengue-2 virus, their ability to transmit the virus to suckling mice (SM) by bite and the possibility of transovarial transmission were compared. The virus replicated in the 5 strains at the same rate averaging 4.1 loglo PFU/mosquito, and the 50 % mosquito infection dose was similar for each strain (0.001 PFU). The ability of infected females to transmit the virus to SM by bites varied from 37 to 56 % according to the strain. Transovarial transmission was demonstrated in 4 strains. The Kari strain from non-endemic region did not transmit the virus. The minimal filial infection rate was rather low ranging from 0.3 to 1.2 %0Infected progeny of both sexes were detected only in the first or second ovarian cycle from eggs ovoposited between the 14th and the 16th day post inoculation of parent females. KEY-WORDS: Arbovirus, Aedes aeggpli, Dengue virus; Susceptibility, Transmission to vertebrate, Transovarial transmission, Mouse.
Manuscrit re~u le 20 m a r s 1981, acceptd lc 18 s e p t e m b r e 1981.
358
F.-X. JOUSSET
INTRODUCTION Dengue fever infections, caused b y at least 4 viruses classified as a subgroup of the group B (Flavivirus) of Togaviridae, are among the most severe h u m a n viral diseases in tropical and subtropical areas. They are characterized b y fever, aches, pains and sometimes b y haemorrhagic manifestations, shock and death of infected children [30]. These diseases are t r a n s m i t t e d to man b y mosquitoes of the genus Aedes, principally A. aegypli and A. albopictus. The susceptibility of both Aedes species to infection with the 4 dengue serotypes has been investigated [13, 14, 15, 33]. Furthermore, the mechanisms b y which most arbovirnses reappear in enzootic areas following the period of interrupted active transmission (i. e. winter in the t e m p e r a t e zone, dry season in the tropics) remain still obscure. Among the several hypotheses reviewed b y Reeves [25] the possibility of transovarial transmission in the vector has been p u t forward. This mechanism of virus maintenance in nature has been shown to occur in ticks for tick-borne encephalitis virus [26], in sand-flies for vesicular stomatitis virus [31] and since 1973 in mosquitoes for some B u n y a viruses of the California encephalitis and B u n y a m w e r a groups [10, 18] and, as recently demonstrated, for some flaviviruses such as Koutango, Japanese encephalitis, yellow fever and St Louis encephalitis viruses [2, 4, 11, 17, 291 .
The purpose of the present s t u d y was to compare t h e susceptibility to dengue-2 virus of 5 strains of A. aegypti collected from areas where dengue viruses are endemic or not, the ability of the mosquitoes to transmit the virus under l a b o r a t o r y conditions and to investigate and demonst r a t e the possible transovarial transmission of the virus in A. aegypti.
MATERIALS
AND METHODS
Virus slrain. The virus used in these studies was the prototype New Guinea C strain of dengue type 2, mouse passage level 25. Stock virus was prepared from 5 to 6-dayinfected suckling mice (SM) as a 10 % brain homogenate in Hanks medium, pH 7.2, containing 7.5 % of bovine serum albumin, 200 units of penicillin and 50 ~g of streptomycin/ml. The supernatant obtained after clarification at 2,000 rpm for 15 min was stored at -- 70 ~ C as 1.0 ml aliquots in sealed ampoules.
FCS i.c. i.t. LAC MFIR MIDso o r . c.
= = --= ---
foetal calf serum. intracerebrally. intrathoracic(ally). La Crosse. minimal filial infection rate. m o s q u i t o i n f e c t i o n d o s e 50 %. ovarian cycle.
PFU p.i. PS
= = =
SM = SMLD~ o ~
plaque-formiug unit. post inoculation. a c o n t i n u o u s p i g k i d n e y celA c u l tures. suckling mouse. s u c k l i n g m o u s e l e t a | d o s e 50 %.
AEDES
AEGYPTI
AND DENGUE-2 VIRUS
359
Virus assay. Virus titrations of stock suspensions and infected mosquitoes were done by plaque assay on continuous pig kidney cell cultures (PS) according to the procedure described by Rager-Zisman and Merigan [23]. Serial 5-fola dilutions of the test material were prepared in Leibovitz growth medium, pH 7.4, supplemented with 3 % foetal calf serum (FCS) and antibiotics. From each dilution (0.2 ml) was inoculated into 4 microplate wells, and 0.4 ml of overlay medium (Leibovitz: 47 %; FCS: 3 %; 2,3 % carboxymethylcellulose: 50 %) was added after 3 h of incubation. Plaques were counted 5 days later, after staining with naphtalene blue black. The virus titre was expressed as the number of plaqueforming units (PFU) per ml of suspension or per insect. Dengue virus was also titrated by inoculating intracerebrally (i. c.) ten 2-3 days old SM with 0.02 ml of 10-fold dilutions prepared in Hanks medium. The virus titre was estimated b y the end point dilution method according to Reed and Muench [24] and expressed as suckling mice letal dose 50 % (SMLDso) per ml.
Mosquitoes. The following strains of A. aeggpti were chosen according to t h e i r origin: 1 colonized strain from Queensland, Australia (Queens strain) reared for numerous generations in the laboratory, and 4 field strains recently collected (2 or 3 generations in. colony) in Vietnam (Hanoi strain) where dengue infections are very common, m Guadeloupe (Guad. strain) where dengue lever is frequent, in Nigeria (Lagos strain) where only some cases of dengue are reported, and in Upper Volta (Kari strain) where no dengue disease occurs. All mosquitoes were reared in an insectarium at 25 ~ C, with 80 % room humidity, under a photoperiod of 18 h of light. Larvae were given a diet of mice biscuits, and adult mosquitoes were maintained on 10 % sucrose solution. All experiments were conducted under the same conditions using, for infection, groups of 3-7 days old mosquitoes.
Inoculation o/ mosquitoes. Adult mosquitoes were infected by intrathoracic (i.t.) inoculation. After being anaesthetized by cold at 4 ~ C for 15 min, mosquitoes were transferred into a Petri dish placed over an ice bath. They were inoculated by inserting, under binocular, a glass microneedle between the two first segments [6, 27]. The equipment used to fill the microneedles with the virus suspension and to inject it into the body cavity was similar to t h a t described by L'H~ritier [19] for injecting drosophila, with some improvements. The mean volume injected was determined by weighing the needle before and after a series of inoculation. It was about 0.6 tzl per female and 0.3 71 per male, corresponding to half the weight of the mosquitoes. The mortality of insect due to i.t. inoculation was low,
Screening /or susceptibilitg o] A. aegypti /rom different areas to dengue-2 virus. The susceptibility of the 5 strains of A. aegypti to dengue-2 was tested by establishing the virus growth curves or kinetics of virus replication in mosquitoes and by comparating the titre of a same viral suspension in each strain. a) Kinetics o/ replication. - - Females and males of each strain of A. aegypti were infected by i.t. inoculation with a 1/10 suspension of dengue-2 virus stock. Samples of 5 females and 5 males were collected at regular intervals and stored at -- 70 ~ C. Prior to viral titration in PS cells, each pool of 5 mosquitoes was triturated in 1.0 ml of Leibovitz medium, and suspensions were clarified at 2,000 rpm for 15 rain.
360
F.-X. J O U S S E T
b) Titration o[ virus in mosquitoes. - - Serial 10-fold dilutions of a suspension of dengue-2 virus, previoulsy titrated in PS cells, were injected i.t. into groups of A. aeggpti females of the 5 strains. These insects were held for 10 days, then generally at least 16 mosquitoes for each dilution were ground individually in 1.0 ml of medium and tested for the presence or absence of virus in PS cells. Infection rate was expressed as the percentage of virus positive mosquitoes in each dilution, and the titre of the virus suspensions was calculated b y the method of Reed and Muench [24] and expressed in mosquito infectious dose 50 % (MIDso). Transmission to SM. For transmission attempts, pools of about 20 i.t. infected females were allowed to engorge individually on normal SM. Each mosquito, deprived of a sugar source for at least 48 h, was placed into a cylindric plastic container (7 cm lenght, 3,5 cm in diameter) covered with a gauze. An SM was maintened for 1 h 30 min, at the top of each container, in darkness. Then, engorged and not engorged mosquitoes were returned separately into cages and bitten SM to their mothers. The transmission a t t e m p t s were started 4 days post inoculation (p.i.) of females and continued until their death (73 days). Females might engorge 3 times, the first time from 4 to 13 days p.i., the second time from 17 to 45 days p.i. and the third time from 52 to 73 days p.i. The bitten SM were observed for l0 days for signs of illness or death. Brains from all a p p a r e n t l y normal SM were tested for the presence of virus by reinoculation to five SM. Viral identification was confirmed b y the complement fixation test (LBCF) [7] using antigens prepared from SM brains b y the sucrose acetone method [9], reference dengue t y p e l, 2, 3 and 4, and Chikungunya hyperimmune ascitic fluids prepared according to B r a n d t et al. [5]. The transmission rate was expressed as the percentage of engorged females transmitting the virus to susceptible animals. Transovarial transmission. To investigate the possibility of vertical transmission, m a t e d and i.t. infected females were allowed to ovoposit 2 days after a blood meal on SM. Eggs from 3 ovarian cycles (ov. c.) were collected at 9 and 14 days p.i. for the first ov. c., at 16 and 25 days p.i. for the second and at 30 and 40 days p.i. for the third. Eggs were held at least 8 days at room t e m p e r a t u r e in order to inactivate the viruses which might contaminate their surface. Then, they were hatched. After larval and pupal development at 25 ~ C, 5-8 days old adults were sexed, pooled by lots of about 40 females or 50 males, and tested for virus. For Queens strain, the 4th instar larva progeny were also examined. In order to increase the virus titre assumed to be rather low, each pool of F1 progeny was triturated in 1.5 ml of H a n k s medium and inoculated i.t. into 15 uninfected A. aegypti. After l0 days, the mosquitoes were ground and assayed for virus in PS cells. If plaques occurred, suspensions of progeny mosquitoes were inoculated i.e. into a litter of SM. Viral recovery and identification were confirmed as previously described.
RESULTS Titration of virus stock. T h e m e a n t i t r e of t h e v i r u s s t o c k a s s a y e d s e v e r a l t i m e s , b o t h in SM a n d P S cells, w a s a b o u t 6.6 loglo SMLDso a n d 5.2 loglo P F U / m l , resp e c t i v e l y . T i t r a t i o n in SM w a s t h e r e f o r e 25 t i m e s m o r e s e n s i t i v e t h a n in P S cells.
AEDES AEGYPTI AND DENGUE-2 VIRUS
36I
Multiplication of dengue-2 virus in five A. aegypti strains. Each mosquito was inoculated with a p p r o x i m a t i v e l y 10 P F U for a female and 5 P F U for a male. The kinetics of virus replication are summarized in table I, and figure 1 shows the typical growth curve of the virus in the Hanoi strain. Each point represents the mean virus titre of 5 mosquitoes. Following inoculation, no infectivity could be detected in PS cells or in SM. Thi s corresponds to the eclipse phase. The virus content per insect, 24 h p. i. was already equal or superior to the inoculum except for males of Queens and Guadeloupe strains. The virus titres increased until the 10th day and then stabilized at a p p r o x i m a t e l y 4.1 loglo P F U per insect. A slight decrease was observed later until the death of mosquitoes, b u t the virus titre remained high. The p a t t e r n of virus replication in both sexes was similar b u t the virus titres were a p p r o x i m a t e l y 2.5 times less in males than in females at the maximal phase. This difference results from the fact t h a t males are smaller than females. The 5 mosquito strains were similar in their ability to s u p p o r t dengue-2 virus replication after inoculation. "]'ABLE I. - - Comparative virus content of ~ and ~ ~ A aegypti >> from 5 geographic strains after parenteral infection with dengue-2 virus (*). T i m e a f t e r i n o c u l a t i o n (in d a y s ) Mosquito strain
Females
Males
3h
24 h
2
6
10
15
20
25
30
35
40
50
55
63
Queens Hanoi Guad. Lagos Karl
0 0 0 0 0
-1.7 1.0 1.7 f.7
1.8 2.7 2.8 2.4 3.7
3.1 3.3 3.9 3.5 4.8
4.1 4.4 4.0 4.6 3.9
4.4 4.4 4.0 4.6 4.3
4.5 4.3 3.7 4.3 4.5
3.8 4.3 4.1 4.0 4.2
4.3 4.1 4.0 4.1 3.9
4.2 4.1 4.0 4.0 4.0
3.7 3.9 3.7 4.0 4.7
3.8 3.8 3.5 3.9 4.1
3.5 3.3 3.9 --
3.7 -3.7 4.0
Queens Hanoi Guad. Lagos Kari
0 0 0 0 0
0.2 1.0 0.1 1.5 1.6
1.3 2.3 --2.3 --
2.0 2.6 2.5 3.1 4.4
3.5 3.7 -3.4 4.2
4.1 3.3 3.5 3.7 --
4.3 3.4 --3.5
-3.3 3.5 3.2 3.6
3.4 --
3.4
3.8
(*) I n o c u l a t i o n w a s 1 log10 P F U p e r f e m a l e a n d 0.7 loglo P F U p e r m a l e . T h e d a t a a r e e x p r e s s e d in loglo P F U a n d r e p r e s e n t t h e m e a n f r o m 5 m o s q u i t o e s .
Since no significant difference in the dengue-2 virus content could be detected in these 5 strains, an experiment was carried out to reveal a possible variation in the infection rate of female mosquitoes following inoculation of serial 10-fold dilutions of virus stock. The result of this experiment are summarized in table II. At the 1/10,000 dilution, i. e. 0.001 P F U inoculated per insect, the Lagos and Kari strains had more t h a n 50 % of infected females whereas the other 3 had less, with a significative difference. However, t h e a m o u n t of dengue-2 virus able to infect 50 ~ / o f
362
F.-X. JOUSSET
mosquitoes was estimated to be between 0.002 and 0.0005 P F U according to the strain of Aedes. The titre of the stock virus was 7.9 to 8.5 lOgl0 MIDso/ml. The difference was only 4-fold, and no significative variation in susceptibility in these 5 strains could be detected. It should be noted t h a t the virus
E
3 {3.
2
~ _
5
J
I
i
i
I
t
i
10
15
20
25
30
35
40
days
after
II
.d" I
J
I
50
60
70
inoculation
FIG. 1. - - Dengue-2 virus content in female and male mosquitoes from H a n o i strain, following i. t. inoculation. Each points represents the m e a n virus titre of mosquitoes.
TABLE II. - - Percentage of infected ~ mosquitoes 10 days after inoculation of various doses of dengue-2 virus (*). Dilutions of virus suspension (in loglo) ( n u m b e r of P F U inoculated) (**) Mosquito strain
2 (1)
3 (0.1)
4 (0.01)
5 (0.001)
6 (0.0001)
Queens Hanoi Guad. Lagos Karl
100 100 100 100 100
100 100 100 100 100
94 95 100 100 95
37 23 33 58 79
10 8 8 17 5
7 (0.00001) 0 0 0 0 0
Titre of suspension in MIDso 8.0 7.9 7.9 8.5 8.2
(*) Calculated w i t h 12 to 16 insects. (**) Titre of inoculated virus suspension was 5.2 P F U / m l . E a c h female received a b o u t 0.6 ~l of virus suspension.
A E D E S A E G Y P T I AND DENGUE-2 VIRUS
363
content of all infected mosquitoes assayed in PS cells was very similar and close to 4 log~o P F U , whatever m a y be the a m o u n t of virus inoculated. Transmission to S M .
The attractiveness of SM for female mosquitoes was 49, 53, 67, 76 and 69 % for Queens, ttanoi, Guadeloupe, Lagos and Kari strains, respectively. An overall total of 1,302 transmission a t t e m p t s were carried out; 826 females took a full blood meal and 258 SM were infected by bite of a single female, of which 233 died following bite. The transmission rate for each strain is reported in table III and figure 2. No transmission occurred on the 4th day p. i.; the first transmission began on the 5th day p. i. for Queens (1/8), Lagos (2/16) and Karl (1/15) strains. This corresponds v e r y likely to incubation time for the virus to replicate in the salivary gland. The transmission rates increased until t h e 10th day p. i. and were fluctuant at each attempt. However, transmission rates were maintened until the death of females. E x c e p t for one case where the 10 females tested for the Lagos strain t r a n s m i t t e d the virus, in no other instance could all t h e females transmit: the vector percentage reached 56 % (54/97), 45 % (43/95), 37 % (43/117), 45 % (48/106) and 4 1 % (53/129) for Queens, Hanoi, Guadeloupe, Lagos and Kari strains, respectively, from the 10th day p. i. to death. These results did not v a r y significantly enough to distinguish the transmission efficiency of females originating from endemic or nonendemic areas, at least after this mode of infection of mosquitoes. The n u m b e r of females used for transmission a t t e m p t s was too i m p o r t a n t (108, 139, 146, 102 and 112 for each strain) to keep each female individually t h r o u g h o u t the experiment and follow its ability to t r a n s m i t at each bite.
TABLE I I I . - -
of i.t. infected females transmitting dengue-2 virus by bite to SM by incubation time.
Percentage
I n c u b a t i o n t i m e in d a y s 4-7 d a y s p. i. (*)
10-45 d a y s p. i. (**)
52-73 d a y s p. i, (***)
Mosquito strain
Nb ~ engorged
Nb 9 transmitting
percenrage
Nb 9 engorged
Nb 9 transmitting
percentage
Nb ~ engorged
Nb ~ transmitting
percentage
Queens Hanoi Guad. Lagos Karl
43 38 83 60 58
1 0 1 10 5
2 0 1 17 9
65 85 87 90 84
37 39 36 42 35
57 46 41 47 42
32 10 30 16 45
17 4 7 6 18
53 40 23 37 40
(*) 1st feeding. (**) 1st a n d 2rid feeding. (***) 3rd feeding.
36~.
F.-X. JOUSSET
c
i i
,
,
,
,
i
,
,
o
,
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o~
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AEDES A E G Y P T I AND DENGUE-2 VIRUS
365
Transovarial transmission experiments. The results of experiments to determine transovarial transmission rates of dengue-2 virus in experimentally infected females of A. Aeggpli are summarized in table IV. W i t h the exception of the Kari strain, transovarial transmission occurred in the 4 other strains. Despite t h a t difference, perhaps not significant, the minimal filial infection rate (MFIR) was r a t h e r low with a m a x i m a l rate of 1.2 %o in Guadeloupe strain. A total of 16,590 a d u l t progeny from 3 or. c. (ovarian cycles) were tested in 406 pools. F r o m the 8 positive pools for dengue-2 virus, 6 were from male and 2 from female lots. T h e y came from eggs ovoposited on the first or second ov. c., b u t always after at least 14 days p. i. of females. It thus appears t h a t the incubation period prior to ovoposition is essential for suecessfull transmission. No infected progeny could be recovered in the adults from ovoposition on the 25th day p. i. or later. The mean M F I R for t h e 5 strains was inferior to 0.5 %o. E x a m i n a t i o n of 2,494 larva progeny from Queens strain tested in 29 pools gave negative results. TABLE IV. - - Dengue-2 virus in r l progeny from 3 ov.c of i.t. infected (~ A. aegypti >) females 9 to 50 days p.i. No. progeny tested
Strains
Queens
?
Pools+/ no tested
Origin of positive cases ov.e.
d.p.i.
MFIR
Total MF1R
1 400 1 642 2 494
0/39 1/40 0/29
2nd
16
1/586
1/3 042
1 903 I 833
1/54
1/43
2nd 2nd
16 16
1/748 1/739
1/1 868
~ c~
1 773 1 478
1/45 3/31
1st 1st
14 14
1/664 1/598
1/813
Lagos
9
1 461 1 494
0/38 1/33
2nd
16
1/178
1/2 955
Kari
~
1 725 I 881
0/44 0/39
larvae Hanoi Guad.
2
0/3 606
DISCUSSION Five geographic strains of A. aeggpti including one colonized (Queens) and four recently collected from dengue-2 virus endemic or non-endemic areas (Hanoi, Guadeloupe, Lagos and Kari) were compared for (1) their susceptibility to infection with dengue-2 virus, (2) their ability to contam i n a t e a vertebrate host, and (3) the possibility of a transovarial transmission of the virus as a mechanism of m a i n t e n a n c e of the virus in n a t u r a l conditions.
366
F.-X. JOUSSET
Concerning the first point of comparison, the results indicate t h a t the five strains were alike in their ability to replicate the virus after parenteral infection. In this respect, it is interesting to note t h a t the titration of the virus stock b y inoculating mosquitoes proved to be the most sensitive, averaging 8.2 loglo MIDs0/ml. Mosquitoes are 1,000 times more sensitive than PS cells (5.2 loglo P F U / m l ) and 40 times more sensitive than SM (6.6 log10 SMLDso/ml). B o t h males and females were susceptible to the virus with a slight difference in the a m o u n t of virus as already reported for A. albopictus [27]. No variation was observed between mosquito strains in the virus content of infected insects after a standard incubation period, w h a t e v e r the a m o u n t of virus inoculated might be. The q u a n t i t y of virus required to infect 50 % of mosquitoes varied only 4-fold between the strains and averaged 0.001 P F U . Similar results showing no difference in the susceptibility to parenteral infection with a single arbovirus among mosquito strains of the same species from different areas, have been reported [13, 15, 16]. On the contrary, a variation of 1,000- to 1,500-fold in the infection rate or in the virus content among such strains could be demonstrated when the virus was ingested [1, 12, 13, 15, 16, 32]. In our study, a t t e m p t s were carried out to infect mosquito females b y feeding on viremic SM or on pledgets soaked with a mixture of virus ~ guinea-pig blood Jr sucrose 10 % (equal volume). The titres of infectious meals were 4.2 and 5.9 lOglo SMLD~o/ml, respectively. But, none of 265 females from the 5 strains fully engorged on SM was infected; out of the 160 females from the 5 strains fed and engorged on pledgers, only 2 of Queens strain were infected, with a virus content of 4.1 log~o P F U . This failure to infect adults orally could result from various factors: insufficient viremia titres in SM and/or the effect of a possible barrier which would take place before virus replication in the mosquito such as a gut cellular resistance [22]. Obviously, the fact t h a t the virus strain used in these experiments had undergone 25 consecutive mouse passages, m a y have modified its ability to infect mosquitoes b y oral route. Whatever, Gubler et al. [15] succeeded in orally infection of several A. aeggpli strains with this New Guinea C dengue-2 virus strain. Concerning the biological transmission of dengue-2 virus to a verteb r a t e host, the SM, no significant variation could be detected between the five geographic strains. In the best period, from the 10th day p. i., the mean transmission rates varied from 37 (Guadeloupe strain) to 56 % (Queens strain). This similarity can be explained b y the fact t h a t after parenteral infection, all mosquitoes finally had the same virus content, and the infection of salivary glands was similar in all strains. There is, of course, no assurance t h a t similar results would have been obtained using mosquitoes infected b y oral route. However, it has been reported t h a t resistant mosquito strains from non-endemic areas to oral infection with dengue-2 virus are just as able to t r a n s m i t the virus, once t h e y become infected as those from susceptible strains from endemic areas [15]. Oil the contrary, with the yellow fever-A, aegypli and the La Crosse (LAC, a m e m b e r of the California subgroup of Bunyaviridae)-A. triserialus
AEDES A E G Y P T I AND DENGUE-2 VIRUS
367
systems, there was a variation in transmission rate: the strains more susceptible to oral or parenteral infection showed the highest ability to t r a n s m i t the virus by bite [1, 3, 12]. Finally, the transovarial transmission of dengue-2 virus was demonst r a t e d in 5 different strains of A. aeggpti mosquitoes after parenteral infection, in F1 adult progeny. The Karl strain from non-endemic region did not transmit. In positive strains, both F1 females and males were found infected as in the ease of other transovarially t r a n s m i t t e d flaviviruses [2, 4, 17, 29]. However, the frequency of the phenomenon was r a t h e r low from 0.03 to 0.12 ~ in the best ease (Guad. strain). The M F I R were similar or about 10 times lower t h a n those reported for other flaviviruses. No q u a n t i t a t i v e data were given for K o u t a n g o virus t r a n s m i t t e d by A. aeggpti and for dengue viruses t r a n s m i t t e d by A. albopictus [11, 29]. Yellow fever was t r a n s m i t t e d by A. aeggpti and A. mascarensis in a d u l t progeny from the first ov. c. with an M F I R from 0.14 to 0.16 % [2, 4]. St Louis encephalitis virus was t r a n s m i t t e d transovarially in first or. e. by A. albopidus and A. epadius with a higher M F I R in F, larvae t h a n t h a t in adult progeny when larval development occurred at 27 ~ C (0.52 vs 0.07 % and 0.45 vs 0.16 ~ respectively). The M F I R increase in adult progeny when larval development took place at 18 ~ C [17]. Japanese encephalitis virus was t r a n s m i t t e d by A. albopiclus, A. togoi and Culex tritaeuiorhgnchus. In the first two eases, the virus was detected in a d u l t progeny with an M F I B of 0.37 and of 0.70 %, respectively. In C. trilaeniorhgnchus, no adult was infected b u t the virus was recovered in 0.47 ~ of larva progeny [28, 29]. In our study, no increase in the transovarial transmission rate of dengue-2 virus could be detected in A. aeggpti larvae as compared to a d u l t progeny, in contrast with results obtained with St Louis and Japanese encephalitis viruses [17, 28]. The transovarial transmission of dengue-2 virus was d e m o n s t r a t e d in the two first or. e. of infected females b u t t h e time interval from infection to the day of ovoposition seemed more i m p o r t a n t t h a n the rank of or. c. In all eases, infected progeny was ovoposited on day 14 or 16 p. i., b u t no transmission was detected from progeny laid on days 25 to 50 p. i. The absence of transovarial transmission in the late second or third ov. c. was probably not due to the stop of multiplication of the virus in the ovaries since the titre of the virus in adults remained constant, but resulted more likely from insufflsant n u m b e r of ovoposited eggs at the time. Similar results were reported in the case of yellow fever virus which was transm i t t e d to a low level in the 3rd ov. e. at 23 days and not in 4 and 5th ov. c. at 35 and 45 days [4]. Contrarily to flaviviruses, viruses of the California subgroup of B u n y a viridae exhibit a very high transovarial transmission rates in the first ov. c. by their mosquito vectors. Thus, 98 ~ of infected females t r a n s m i t t e d LAC virus to their offspring, and the mean percentage of infected offspring from an infected female was 7 1 % [201. Seemingly, 55 % of the infected A. lrivillatus tested t r a n s m i t t e d transovarially Triviltatus virus which
368
F.-X. JOUSSET
was isolated from 16 % of the F1 generation progeny examined [8]. On the other hand, in the LAC-A. triseriatus system, the rank of ov. c. was important: larvae from first ov. c. following infection of females were never infected; LAC virus was not detected in the second ov. c. larvae in which the blood meal for the first ov. c. was not infectious [21]. Since transovarial transmission of dengue-2 virus b y A. aegypti was demonstrated, it is likely t h a t the 3 other t y p e s of dengue viruses can also be t r a n s m i t t e d transovarially, as it has been shown with A. albopictus (L. Rosen, unpublished work). Althought transovarial transmission appears under laboratory conditions at low rates, it can exist in nature and it has to be investigated under field situations, b u t its epidemiologic significance is still unknown.
RESUME R E L A T I O N S E N T R E CINQ S O U C H E S G E O G R A P H I Q U E S D E (( A E D E S A E G Y P T I D ET L E V I R U S D E LA D E N G U E : S E N S I B I L I T E , C A P A C I T E Y E C T R I C E P O U R U N tt()TE VERTt~13RI~ ET TnANSMISSION TnANSOVARIENNE
Cinq souches g~ographiques de Aedes aeggpti, vecteur des virus de la dengue, out 6t6 compar6es ~ l'~gard de ce virus (du t y p e 2) q u a n t ~ ]eur sensibilitY, leur capacit~ veetrice pour un h6te vert~br~ sensible - - le souriceau - - et leur capacit6 ~ t r a n s m e t t r e le virus ~ leur descendance. Les diff~rentes souches de moustiques provenaient de pays off la dengue, maladie humaine, est relativement fr~quente (Asie du Sud-Est, Guadeloupe) ou non (Afrique, Australie). AprSs inoculation du virus 5 des moustiques par voie intrathoraeique, aucune diff6rence significative a'a ~t~ d~cel~e entre les souehes, qu'elles viennent ou non de pays end~miques de la dengue. La replicatioa virale atteint environ 4,1 loglo P F U par insecte ; les doses p e r m e t t a n t 50 % d'infection de moustiques sont de l'ordre de 0,001 P F U . Selon les souches, 37 ~ 56 % des femelles infect~es t r a n s m e t t e n t par piqfire le virus aux souriceaux. Enfin, la transmission transovarienne a 5t~ raise en 6vidence : ~ l'exception de la soucbe d'Afrique de l'Est, r~gion non end6mique pour la dengue, les quatre autres souches transm e t t e n t le virus a u n e faible proportion (0,03 ~:~ 0,12 %) de leur descendance, le 14 et le 163 jour apr~s inoculation. MOTS-CLI~S : Arbovirus, Aedes ae.qgpti, Virus de la dengue ; Sensibilit6, Capacit6 vectrice, Transmission transowwienne, Souris.
REFERENCES T. H. G., Downs, W. G. & SHOPE, R. E., Aedes aeggpli strain fitness for yellow fever virus transmission. Amer. J. trop. Med. Hgg., 1977, 26, 985-989.
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