Seasonal infection of Culex mosquitos and swine with Japanese encephalitis virus

Bull. Org. mond. Sante' 1973, 49, 347-352Bull. Wld Hlth Org. Seasonal infection of Culex mosquitos and swine with Japanese encephalitis virus T. OKUNO,1 C. J. MITCHELL,1 P. S. CHEN,1 J. S. WANG,2 & S. Y. L1N 2 A year-round study ofthe infection rates ofJE virus in Culex mosquitos was made during 1970-71 in 2 hamlets in Taoyuan County, China (Province of Taiwan). JE virus was recoveredfrom S of 314 pools of C. annulus, andfrom I of 22 pools of C. tritaeniorhyn- chus; these recoveries occurred during a 14-dayperiod in July 1971. None ofthe 288 pools of C. p. fatigans, which had been collected between October and April, was positive. In addition, sentinel swine were assessedfor antibody and virus. All the pigs became highly immune by 23 July; in each hamlet all the pigs had become infected within 1 week, virus being detected in them for only 1-2 weeks. Virus-positive mosquitos appeared to have obtained their infections at about the time that viraemia was occurring in the sentinel swine. These observations illustrate once again the lower infection rate and shorter duration of virus- positive mosquitos in China (Province of Taiwan) as compared with Japan. In China (Province of Taiwan), Japanese encepha- litis virus has been repeatedly isolated from 3 species of Culex mosquito: C. tritaeniorhynchus, C. fusco- cephalus, and C. annulus (Hu, 1958; Wang et al., 1962; Cates & Detels, 1969; Cates et al., 1969; Detels et al., 1970; Okuno et al., 1971). It was not known, however, whether or not C. p. fatigans, which repre- sents the species persisting in the island throughout the winter, is infected with JE virus. Such knowledge seemed necessary, since JE virus isolation from C. p. pallens has been reported in Korea as well as in Japan (Buescher et al., 1959; Hayashi et al., 1966; Matsuya- ma et al., 1968; Lee et al., 1969). During 1970-72, seasonal assessments of the popu- lation of known and potential JE vector mosquitos were made by Mitchell & Chen (1973) in Taoyuan County, China (Province of Taiwan). A portion of the mosquitos collected during the period August 1970-August 1971 was tested for JE virus in order to determine the seasonal infection rates for C. annulus, C. tritaeniorhynchus, and C. p. fatigans. The virus assessment of the mosquitos was coupled with that of sentinel pigs located on the premises I WHO Japanese Encephalitis Vector Research Unit, Taipei, China (Province of Taiwan). (Mailing address: c/o Regional Office for the Western Pacific, World Health Organization, P.O. Box 2932, 12115 Manila, Philippines. Present address: Department of Entomology, 322 Briggs Hall, University of California, Davis, Calif. 95616, USA. ' Provincial Serum and Vaccine Laboratory, Taipei, China (Province of Taiwan). where the mosquitos were collected. The objective was to confirm the already reported mosquito-pig- mosquito transmission cycle (Hurlbut, 1964; Konno et al., 1966), or at least to observe the amplifying effect of swine in increasing the population of virus- positive mosquitos (Scherer et al., 1959) at typical rural premises in Taoyuan County. MATERIALS AND METHODS Methodsfor collecting andpreserving the mosquitos The samples of female Culex mosquitos were taken in 2 hamlets similar in all essential respects except that the interiors of homes in hamlet 2 were treated with a residual spray of propoxur on 17 June 1971 (Mitchell & Chen, 1973). From 16 August 1970 to 21 August 1971, approximately equal numbers of pools of Culex mosquitos were collected from each hamlet every week except that of the lunar new year and during the month following the propoxur spray, when few pools were taken from hamlet 2. The total of 624 pools comprised 314 of C. annulus, 22 of C. tritaeniorhyn- chus, and 288 of C. p. fatigans. Most of the first two species were taken from buffalo bait, whereas C. p. fatigans was taken from human and livestock habita- tions. Preliminary processing of mosquitos During the course of each successive week the mosquito collections, from which the engorged speci- mens had been removed, were sorted under chloro- 3125 -347- T. OKUNO ET AL. form-induced anaesthesia into pools of the same species, physiological condition (deplete or gravid), and provenance (hamlet 1 or 2). The maximum pool size was 100 for C. annulus and C. tritaeniorhynchus but only 50 for C. p. fatigans because larger pools of this species were toxic to suckling mice. The sorted specimens were put into rubber-stoppered glass tubes and stored at -60°C until they were ground and suspended for injection into mice. Sentinel swine Ten 4-month-old Landrace hybrid swine of unspe- cified sex were placed in a pigsty at each hamlet on 2 June 1971. The pigsties were surrounded by the hamlets' normal populations of swine, cattle, and chickens (Mitchell et al., 1973). During the period 11 June to 23 July 1971, the swine were bled once a week. Sera were separated at the laboratory on the day of collection and stored at -60°C. The swine sera were first tested for haemagglutina- tion-inhibition (HI) antibody. When antibody was found, the presence of virus was sought in those samples taken 2 weeks previously. Testing was re- peated 3 times in order to minimize the influence of individual test fluctuations. To remove nonspecific haemagglutination (HA) inhibitors, the swine sera were first treated with acetone in 3 cycles, followed by absorption with packed goose erythrocytes and inac- tivation at 56°C for 30 minutes. HI tests for the treated sera were performed according to the method of Clarke & Casals (1958). The extracted JaGAr 01 antigen was used in dilutions ranging from 1: 800 to 1: 1600 to give 8 HA units. Procedures for virus isolation and identification A litter of suckling mice 3-5 days old was inocu- lated intracerebrally with each specimen of mosquito pool or swine serum. The inoculum remaining was refrozen and kept at -60°C until the results were known. Preliminary identification of positive isolates was made by testing their haemagglutination capaci- ty, utilizing the crude alkaline aqueous extract of the inoculated mouse brains. With swine sera, the tests were carried out directly; with mosquito pools, they were performed after 2-4 mouse passages. Final identification of isolates found HA-positive was achieved by the complement-fixation (CF) test (Okuno et al., 1971). RESULTS Mosquito infection rates Of the 624 pools tested, only 6, collected between 7 and 20 July, were positive (Table 1). Five were Table 1. Rates of virus isolation from C. annulus and C. tritaeniorhynchus at 2 study sites in Taoyuan County, 30 June-31 July 1971 Date No. of positive pools/No. tested (week ending) hamlet 1 hamlet 2 Totals 3 July 1971 0/10 0/0 0/10 10 July 3/15 0/2 3/17 17 July 1/15 1/5 2/20 24 July 0/9 1/14 1/23 31 July 0/10 0/11 0/21 Totals 4/59 2/32 6/91 C. annulus pools and the sixth was of C. tritaeniorhyn- chus. All the pools of C. p. fatigans were negative. The infection in hamlet 2, with an aggregate rate of 2/19, occurred a week later than in hamlet 1, where the rate was 4/30. When the remaining inocula of 4 of the 6 positive samples were re-tested, re-isolation was obtained in all four. Among the 60 pools taken during the critical period, virus isolations were obtained in 2 of the 29 taken from buffalo bait. With the other methods of collection, these proportions were as follows: dry-ice trap, 3/6; Magoon trap, 1/6; D-Vac vacuum insect net, 0/12; indoor resting collections, 0/6; and light trap, 0/1. Many of the denominators are too small for valid comparison, but it is note- worthy that the highest virus recovery rate was from dry-ice trap collections and the lowest from D-Vac collections. There were 30 pools each of deplete and gravid mosquitos, 2 of the isolates being from the former pools and 4 from the latter. Conversion of swine HI antibody One of the 10 sentinel pigs at hamlet 2 died at the end of June with symptoms of bronchopneumonia. Among the other 19 pigs (Table 2) antibody conver- sion occurred a week later at hamlet 2 (16 July) than at hamlet 1 (9 July). It was striking that at each hamlet practically all the sentinel pigs became posi- tive at the same time, after the appearance of low-titre antibody in about 20% of them. One of the sentinel pigs at hamlet 1 developed an antibody titre as high as 1: 10 240. By 23 July all 19 pigs were found to be highly immune against JE virus. The specificity of HI at the serum dilution of 1: 10 is doubtful since its appearance was inconsistent (e.g., pig 7 at hamlet 1). Viraemia of sentinel swine When 20 sera of the hamlet-I swine (codes G and H) and 18 sera of the hamlet-2 swine (codes H and I) 348 JE INFECTION RATES IN MOSQUITOS AND SWINE Table 2. Hi antibody titre and viraemia in sentinel swine bled in 4 successive weeks at 2 hamlets in Taoyuan County Specimen HI antibody titre Viraemia code 2 July (G) 9 July (H) 16 July (I) 23 July (J) 2 July (G) 9 July (H) 16 July (I) hamlet 1 1 < 10 < 10 320 1 280 (-) (-) 2 < 10 < 10 1 280 1 280 (-) (+) 3 < 10 < 10 1 280 1 280 (-) (+) 4 < 10 < 10 2560 1 280 (-) (+) 5 < 10 20 10 240 2 560 (-) (-) n.t.v. a 6 10 10 80 640 (-) (-) 7 10 <10 2560 1280 (-) (+) 8 < 1 0 20 2 560 640 (-) (-) 9 < 1 0 < 1 0 5 120 2 560 (-) (+) 10 < 10 < 10 < 10 1 280 (-) (-) hamlet 2 1 < 10 < 10 < 10 1 280 (-) (-) 2 < 10 < 10 < 10 640 (-) (+) 3 < 10 < 10 10 1 280 (-) (+) 4 < 10 < 10 < 10 640 (-) (+) 5 < 10 10 160 320 n.t.v.a () () 6 <10 <10 < 10 320 (-) (+) 7 < 10 10 80 1 280 (-) (-) 8 < 10 < 10 < 10 320 (-) (+) 10 < 10 < 10 < 10 1 280 (-) (+) a Not tested for virus. were inoculated into mice after almost 6 months' storage at -60°C, the virus recovery rate was strik- ingly high; 5 of 10 pigs were found to have been viraemic at hamlet 1 on 9 July, and 6 of 9 pigs at hamlet 2 were found to have been positive a week later (Table 2). No virus was recovered from the sera that had attained an antibody titre of more than 1: 20 (e.g., samples 5 and 7 from hamlet 2). Each inoculation was made without previous knowledge of the HI titre of the serum, and the results suggest a high credibility for the virus isolation test. HA characteristics of the isolates The HA titres of the isolates from the 6 positive mosquito pools were found to range from 1: 1 600 to 1 : 6 400 (Table 3). The HA titres of the 11 isolates from swine sera showed a wide variation, from 1: 200 to 1: 25 600, due perhaps to the use of first-passage virus. Most of the strains showed the highest HA titre in the pH range of 6.4-6.6, as compared with the known optimum pH range of 6.6-6.8 for the HA of the JaGAr 01 reference strain. Identification by CF test The results of a representative test are shown in Table 4; two other CF tests for the remainder of the isolates gave similar results. Differences in titre, such as those seen in the TaiAr 51271 strain, could have been due to a variation in CF potency between antigen batches. The CF test results indicate that all 17 isolates are at least closely related to, and presum- ably identical with, JE virus. DISCUSSION The virus assessments made on C. annulus and C. tritaeniorhynchus confirm the results obtained at Chunan in China (Province ofTaiwan) in the summer 349 T. OKUNO ET AL. Table 3. Haemagglutination characteristics of antigens v prepared from 17 JE-positive isolates at Taoyuan study sites in 1971 Passage Haemagglutination Site Date level in characteristicsStrain no. Source (hamlet) collected mouse ptimum brains Optirang Titre TaiAr 49471 C. annulus 1 8-9 July 3 6.4-6.6 6 400 TaiAr 49571 C. tritaeniorhynchus 1 9 July 2 6.4-6.6 1 600 TaiAr 49871 C. annulus 1 7 July 4 6.4-6.6 3 200 TaiAr 51271 C. annulus 1 12 July 2 6.4-6.6 6 400 TaiAr 52271 C. annulus 2 15 July 2 6.4-6.6 3 200 TaiAr 54571 C. annulus 2 20 July 2 6.4-6.6 3 200 TaiAn 271 swine serum 1 9 July 1 6.4-6.6 200 TaiAn 371 swine serum 1 9 July 1 6.6 400 TaiAn 471 swine serum 1 9 July 1 6.6 400 TaiAn 571 swine serum 1 9 July 1 6.4-6.6 6 400 TaiAn 671 swine serum 1 9 July 1 6.4-6.6 6 400 TaiAn 771 swine serum 2 16 July 1 6.4-6.6 12800 TaiAn 871 swine serum 2 16 July 1 6.6 12 800 TaiAn 971 swine serum 2 16 July 1 6.4-6.6 25 600 TaiAn 1071 swine serum 2 16 July 1 6.6 12 800 TaiAn 1171 swine serum 2 16 July 1 6.6 12 800 TaiAn 1271 swine serum 2 16 July 1 6.0-6.8 400 a Io % crude alkaline aqueous antigen prepared from infected mouse brains. Table 4. Complement-fixation titres of the antigens for the 6 isolates from Taoyuan mosquitos with a reference serum PrepaationPasslevli Serum titre/ Antigen titre ReferenceStrain no. PreparatioinPa e antigentitre vs 4 homolog. HA titre of ofantigen ~mouse brains atgnite units of serum each antigen JaGAr 01 25 % SAFD a 4 b 64/128 128 6 400 TaiAr 49471 10 % crude wet 3 64/ 64 32 6400 TaiAr 49571 10% crude wet 2 64/128 64 1 600 TaiAr 49871 10 % crude wet 4 64/64 32 3 200 TaiAr 51271 10 % crude wet 2 32/32 16 6 400 TaiAr 52271 10 % crude wet 2 64/32 32 3 200 TaiAr 54571 10 % crude wet 2 64/64 64 3 200 Normal brain 25 % SAFD a - < 4/< 4 < 4 - Normal brain 10 % crude wet - <4/< 4 < 4 a Freeze-dried, sucrose-acetone-extracted antigen. b No. of passages after the plaque-cloning in the Regional Reference Laboratory for Arboviruses, Tokyo. 350 JE INFECTION RATES IN MOSQUITOS AND SWINE of 1969 by Okuno et al. (1971), who found that these mosquitos were positive for the virus only between 20 June and 6 July. The present assessments estab- lished that at Taoyuan (both hamlets) these species were positive for the virus only between 7 and 20 July, and negative during the remainder of the year. The 78 pools tested in June and the 85 pools tested in August were more than twice as numerous as those in the 1969 investigations, and the 30 pools taken in September were sufficient to suggest the absence of virus in that month. The process of swine infection observed at each study site was striking in its synchrony. When 1 or 2 of the sentinel pigs were converting HI antibody, a majority of the antibody-negative pigs (5 of 8 at hamlet 1, 6 of 7 at hamlet 2) were found to be viraemic. Assuming that swine 3.5 months of age develop HI antibody to a detectable level approxi- mately 6 days after infection, following the viraemia on the third and fourth days (Okuno et al., unpub- lished data), the greatest prevalence of viraemic sentinel swine at Taoyuan (both hamlets) is presumed to have existed between 3 and 18 July. No infected mosquito was detected forerunning swine viraemia. The dates for the 2 hamlets consid- ered individually are as follows: hamlet 1: mosquitos positive 7-12 July, swine deduced positive 3-13 July; hamlet 2: mosquitos positive 10-24 July, swine deduced positive 10-18 July. This observation ap- pears to be at variance with previous ones (Buescher & Scherer, 1959; Konno et al., 1966) indicating a gradual or cyclic build-up of a relatively dense population of infected mosquitos. Our sampling scale for mosquitos during June 1971, however, seems comparable to those employed by the above-men- tioned workers, and recent studies in China (Province of Taiwan) by Detels et al. (1970) and Okuno et al. (1971) as well as in Japan (Nakamura et al., 1968) also failed to prove that the virus appeared in mosquitos before swine. That JE virus was detected at about the same time in the pigs and the mosquitos suggests the possibility of virus transmission to several pigs by a single transmissive mosquito through multiple probing, as has been demonstrated experimentally on chicks by Muangman et al. (1972). In such a case, the density of swine-infecting mosquitos might well be below any detectable level. Four of the 6 positive mosquito pools showed a low virus titre, suggesting that any infected mosquito in these 4 pools contained virus to an amount not exceeding the level of 103. It seems likely that mosquitos with such a level of virus multiplication were the result, rather than the cause, of swine viraemia. The apparent disappearance of infected mosquitos before the full growth of virus in them, and before they became transmissive, accords with the relatively short period of JE virus activity in China (Province of Taiwan) observed repeatedly (Wang et al., 1962; Detels et al., 1970; Okuno et al., 1971) and confirmed in this investigation. In sharp contrast, the periods of virus activity observed by many investigators in various parts ofJapan (Buescher et al., 1959; Hayashi et al., 1965; Hayashi et al., 1966; Konno et al., 1966; Yamamoto et al., 1966; Yamamoto & Manako, 1968; Matsuyama et al., 1968; Shichijo et al., 1968) were usually 1 month and occasionally longer than 2 months. Moreover, the recovery rates of virus from mosquito pools in Japan have reached 15/17 (Buescher et al., 1959), 10/10 (Hayashi et al., 1966), and 8/11 (Shichijo et al., 1968), whereas in China (Province of Taiwan) the rate has been only 9/37. In the search for virus among mosquitos during the non-epidemnic season, a total of 288 pools of C. p. fatigans were collected in Taoyuan between 18 Octo- ber and 17 April; all proved negative. An additional 13 pools of C. p. fatigans collected at Peitou, sub- urban Taipei, during February-March 1971 were also negative. These results suggest that the role of C. p. fatigans as a JE vector in Taoyuan County, China (Province of Taiwan) is probably negligible. 0UMe INFECTION SAISONNIEiRE DE MOUSTIQUES CULEX ET DE PORCS PAR LE VIRUS DE L'ENCIEPHALITE JAPONAISE On a recherche' les taux d'infection par le virus de 1'enc6phalite japonaise (JE) chez Culex annulus, C. tri- taeniorhynchus et C. pipiens fatigans au cours d'une enquete entomologique, en 1970-71, dans deux hameaux du comte de Taoyuan, Chine (province de Taiwan). Le virus JE a ete isole a partir de 5 lots sur 314 de C. annulus et d'un lot sur 22 de C. tritaeniorhynchus. Ces isolements ont ete reussis exclusivement durant la periode du 7 au 20 juillet 1971. Le virus est apparu dans un hameau une semaine avant d'etre d6cele dans le second, 351 352 T. OKUNO ET AL. et dans les deux cas des moustiques positifs ont ete trou- v6s pendant 1-2 semaines. Le taux d'isolement maximal a 6te enregistr6 pendant la semaine se terminant le 23 juillet, dans un des hameaux. On n'a pas isole de virus JE en juin (78 lots de C. annu- lus), en aoiut (85 lots) et en septembre (30 lots). L'examen de 288 lots de C. p. fatigans captures entre le 18 octobre 1970 et le 17 avril 1971 a donn6 des r6sultats negatifs. Le processus de l'infection des porcs par le virus JE s'est rev616 remarquablement synchrone dans les deux localit6s etudiees. Lorsqu'un ou deux porcs ( sentinelles*) acqu6raient des anticorps IH contre le virus JE, la plupart des autres porcs, serologiquement negatifs, presentaient une vir6mie. Dans les deux hameaux, tous les porcs ont ete infectes dans 1'espace d'une semaine. Tous etaient porteurs d'anticorps a des titres eleves le 23 juillet. A l'apparition du virus chez les porcs a succ6d6 a breve 6cheance l'infection des moustiques. L'epoque a laquelle on a decele le virus chez les moustiques et la faible teneur en virus de 4 des 6 lots positifs donnent a penser que l'infection des moustiques s'est produite a peu pres au meme moment qu'apparaissait la vir6mie chez les porcs sentinelles. Ces observations corroborent les resultats d'enquetes effectu6es anterieurement dans la province. Elles confir- ment que, durant la saison 6pidemique de l'enc6phalite japonaise, l'infection des moustiques y existe a des taux moins 6leves et pendant moins de temps qu'au Japon ou la circulation du virus persiste habituellement pendant un mois et occasionnellement pendant plus de 2 mois. REFERENCES Buescher, E. L. et al. (1959) Amer. J. trop. Med. Hyg., 8, 651-664 Buescher, E. L. & Scherer, W. F. (1959) Amer J. trop. Med. Hyg., 8, 719-722 Cates, M. D. & Detels, R. (1969) J. med. Ent., 6, 327-328 Cates, M. D. et al. (1969) J. Formosan med. Ass., 68, 663- 665 Clarke, D. H. & Casals, J. (1958) Amer. J. trop. Med. Hyg., 7, 561-573 Detels, R. et al. (1970) Amer. J. trop. Med. Hyg., 19, 716- 723 Gresser, I. et al. (1958a) Amer. J. trop. Med. Hyg., 7, 365- 373 Gresser, I. et al. (1958b) Jap. J. exp. Med., 28, 243-248 Hayashi, K. et al. (1965) End. Dis. Bull. Nagasaki Univ., 7, 155-164 Hayashi, K. et al. (1966) End. Dis. Bull. Nagasaki Univ., 8, 61-73 Hu, S. M. K. (1958) J. med. Ass. Formosa, 67, 911 Hurlbut, H. S. (1964) J. med. Ent., 1, 301-307 Konno, J. et al. (1966) Amer. J. Epidem., 84, 292-300 Lee, H. W., Min, B. W. & Lee, Y. W. (1969) J. Kor. med. Ass., 12, 429-440 Matsuyama, T. et al. (1968) Kitakanto Igaku, 18, 494-500 Mitchell, C. J. & Chen, P. S. (1973) Bull. Wld Hlth Org., 49, 287-292 Mitchell, C. J. et al. (1973) Bull. Wld Hlth Org., 49, 293-299 Muangman, D. et al. (1972) Amer. J. trop. med. Hyg., 21, 482-486 Nakamura, T. et al. (1968) Kitakanto Igaku, 18, 487-493 Okuno, T. et al. (1971) Bull. Wld Hlth Org., 44, 599-604 Shichijo, A. et al. (1968) Trop. Med., 10, 168-180 Takahashi, M. (1967) Shinkei-kenkyu no Shinpo (Progress in Neurology Research), 11, 215-222 Wang, S. P. et al. (1962) Amer. J. trop. Med. Hyg., 11, 141-148 Yamamoto, H. et al. (1966) Report from the Fukuoka Prefectoral Institute of Public Health, 4, 13-25 Yamamoto, H. & Manako, K. (1968) Jap. J. sanit. Zool., 19, 4-14