J.
Meld. Entomol. Vol. 14,
DO.
10 February 1978
5: 523-526
ORUNGO VIRUS: TRANSMISSION STUDIES WITH AEDES ALBOPICTUS AND AEDES AEGYPTI (DIPTERA: CULICIDAE)} By Oyewale Tornori2 and Thornas H. G. Aitken3 Abstract: Adult female Aedes albopictlls and Aedes aegfPti mosquitoes were exposed to Orungo, an orbivirus, by intrathoracic inoculation and oral ingestion. After 6 days, virus was demonstrated in I orally exposed mosquito, but only inoculated mosquitoes transmitted the virus to blood droplets after an extrinsic incubation of 6-10 days. Transmission attempts utilizing infant mice wert' negative.
MATERIALS
AND METHODS
Virus. The IbAr 52302 strain of Orungo virus isolated from a pool of Aedes dentatus in Nigeria was used for these studies. The strain was at the 7th mouse brain and 2nd BHK-21 tissue culture passage level. The virus was stored at -60°C in Eagle's Basal Medium (EBM) supplemented with 2.5% fetal calf serum (FCS). Virus titrations were done in 2-day-old mice inoculated by the intracerebral (ic) route. Virus titers were calculated by the method -
--
'The studies and observations on which this paper is based were eonducted at the Yale Arbovirus Research Unit, New Haven, Connecticut 06510, U.S.A., where the benior author was on a study leave under a staff Development Grant from the University of Ibadan, Nigeria. The research was supported in part under U,S. Army Contract DADA 17-72-C-2170,National Institute of Health Grant AI 10984and a grant from the World Health Organization. 'Virus Research Laboratory, University of Ibadan, Ibadan, ~igeria, to which reprint requests should be addressed. 'Yal~ Arbovirus Research Unit, New Haven, Connecticut OG51O, U,S.A.
Mosquitoes. Two species, Aedes albopictus and Aedes aegypti, were used. They represented longestablished laboratory colonies. The former originated in the Virus Research Centre, Poona, India, and came to Yale Univeristy via Fort Detrick, Maryland; the latter is the Amphur strain from Thailand (12-20 F generations) obtained from Dr George Craig, Notre Dame University. Infection of mosquitoes was attempted either by permitting mosquitoes to engorge on a virus-blood suspension or by intrathoracic inoculation with virus suspension. Starved females (4-5 days old), in net-covered O.236-liter ~arton cages, were allowed to feed on cotton pledgets soaked in a virus-defibrinated blood suspension or else through the air sac membrane of chicken egg feeding chambers (Wallis 1962). Each feeding chamber contained 1 ml of the virus-blood suspension, which was prepared as follows: 2 ml of undiluted virus stock, 7.5 ml of inactivated and defibrinated chick blood and 0.5 ml of antibiotic solution (500 units of penicillin and 500 [Jog of streptomycin). Virus titer in this suspension was 4.3 dex LD50/ml. A fully engorged Ae. aegyPli takes up about 0.003 ml of blood; thus, these females acquired about 1.8 dex LD50 of Orungo virus. Intrathoracic inoculations of adult female mosquitoes (2-5 days old) were accomplished by means of an inoculating needle made with glass capillary tubing (l-mm diam.) drawn into a fine point. The tubing was marked off in I-mm graduations with an indelible ink wick pen, each unit representing the amount of inoculum per mosquito. The inoculation site was the membranous area below the paratergite between the meso thoracic spiracle and the wing base. Each mosquito received :1: 0.0006 ml of undiluted virus stock suspension, approximately 1.8 dex LD50 of virus. All manipulations were carried out on mosquitoes lightly anesthetized with carbon dioxide and kept immobilized in a petri dish resting on wet icc. Inoculated and blood-engorged mosquitoes were held in net-covered cages provided with wicks
Downloaded from http://jme.oxfordjournals.org/ by guest on June 8, 2016
Orungo virus was first isolated from Anopheles funes/us mosquitoes in Uganda and later in Nigeria ii'om man and Aedes denla/us mosquitoes (Tomori & Fabiyi 1977a). It has been included in the 01'bivirus taxonomic group (Tomori et al. 1976) established by Borden et al. (1971) and Murphy et al. (1971), based on physicochemical, morphological and morphogenetical similarities. Experimental arthropod transmission of bluetongue (BLU), African horse sickness (AHS), Colorado tick fever (CTF), and epizootic hemorrhagic disease of deer (EHD) viruses, other members of the orbivirus taxon, has been reported by various workers (du Toit 1944, Ozawa & Nakata 1965, Ozawa et al. 1966, Florio et al. 1950, Burgdorfer 1960, Boorman & Gibbs 1973). The present report describes attempts to transmit Orungo virus following experimental infection of Aedes albopictus and Aedes aegypti.
of Reed & Muench (1938) and expressed in dex (loglO) (Haldane 1960). Stock virus titer was 5.0 dex mouse LD50/ml.
J. Med.
524
soaked in 10% dextrose. Mosquitoes were held for 6 to 11 days at 26.7°C and 68-78% RH.
Each blood meal (droplet or capillary) sampled by feeding mosquitoes subsequently was mixed with a drop (about 0.03 ml) of 2.5% FCS and inoculated intrathoracically into 10-15 fresh adult Ae. albopictus Or Ae. aegypti females in order to facilitate the TABLE 1.
Vol. 14, no. 5
multiplication of any virus which might have been transmitted in the feeding process. These mosquitoes were then held for 7-10 days at 26.7 °C, 68-78% RH and maintained on 10% dextrose. At the end of the incubation period, all surviving mosquitoes from each transmission experiment were pooled and tested for virus; mosquito pools were triturated in suspensions of 0.5-1.0 ml of 2.5% FCS, centrifuged, and the supernatants then inoculated ic into 2-day-old mice. Brains from mice showing typical infection were harvested and tested for virus, which if present was identified as Orungo by CF test, using the technique of Casals (1967) and specific hyperimmune, reference-mouse ascitic fluid. The infected mosquitoes used for the virus transmission feedings and then stored at -60°C were likewise tested for virus by suspending each in 1 ml of FCS and inoculating the centrifuged specimen into a group of 2-day-old mice. Brains of sick or dying mice were tested for the presence of Grungo virus as described previously. All mice surviving feedings by infected mosquitoes or surviving ic inoculations of infected mosquito material were exsanguinated 28 days later and their sera tested for specific Grungo virus neutralizing (N) antibody in baby mice. Neutralization tests were done in mice by standard methods using the constant-serumvarying virus technique. Sera were inactivated at 56°C for 30 min. before use. RESULTS
The results of the transmission experiments in
TABLE
1.
Aedes alboPictus Following parenteral inoculations of 1.8 dex LD50 of Orungo virus per mosquito, artificial
Infection and transmission rates in Aedes albopictus and Aedes aeg)pti after parenteral and oral infection with Orungo virus.
MOSQUITO
ROUTE OF INFECTION
At. alboPictus
Parenteral
Oral
EXTRINSIC INCUBATION (DAYS)
Mouse
Blood droplet
6 7
1/50/2
3/6 1/9
10
2/7 3/14
INFECTION RATE
1/4
0/7 0/14 0/3 0/2
1/4 4/19
7
5/19 0/5 0/8 0/7 0/20 1/3
7
0/2
9
0/3 0/5
1/3
0/2 1/5
Parenteral Oral
Blood droplet 3/6 0/9
11
Ae. aeg)'jiti
TRANSMISSION RATE
Mouse 0/5 0/2
6 7
with
Ae. albopictus and Ae. aegypti mosquitoes are presented
0/5
-No. mosquitoes transmitting or virus positive/no. tested feeding on mouse or blood.
0/5 0/8 0/7 0/20 1/3 0/2 0/3 0/5
Downloaded from http://jme.oxfordjournals.org/ by guest on June 8, 2016
Transmission attempts. At intervals, following exposure to virus infection, single mosquitoes were allowed to bite individual 1- to 2-day-old mice. Engorged mosquitoes were stored at -60°C until tested for virus by ic inoculation of baby mice. Bitten mice were watched for signs of illness, and virus isolation attempts were made from brains where indicated. Brain suspensions (10% in bovine albumen diluent) of suspiciously sick mice were passaged ic to groups of fresh mice. In addition, 2 other methods were employed to demonstrate possible transmission of Orungo virus by feeding mosquitoes. In the 1st method (for details, see Gubler & Rosen 1976), infected mosquitoes were allowed to engorge on a drop (0.03 ml) of blood composed of equal parts of 2.5% FCS, 10% dextrose, and inactivated, defibrinated chick blood. One mosquito was confined in a cotton-plugged tube covered at the other end with fine-mesh synthetic netting on which was placed a drop of the blood meal. The 2nd method allowed feeding mosquitoes to engorge on blood in a capillary tube similar to that used for inoculating mosquitoes. The technique, fully described elsewhere (Aitken 1977), calls for inserting the infected mosquito's proboscis into the blood-charged tip of the capillary tube following which feeding usually begins. Using either technique, feeding mosquitoes were removed from the meal before completing engorgement and stored at -60°C.
Entomol.
Tomori & Aitken:
IS78
525
Transmission of Orungo virus by Aedes
transmission, by allowing mosquitoes to feed on blood droplets, was achieved on extrinsic incubation days 6 and 10, but not on day 7. The details are as follows; Del.)! 6: Three of the 6 females transmitted virus; however, only 1 had a titer of 2.3 dex LD50, while the others titered below 1.8 dex LD50, the initial inoculum. On the same day, none of 5 mosquitoes feeding on normal mice transmitted virus, as all exposed mice survived the experimental period and all failed to develop specific N antibody; only 1 of these 5 mosquitoes had demonstrable virus which titered 2.8 dex LD50'
Da.v 10: One of 4 females, containing virus titering 3.0 dex LI\Ol transmitted virus to a drop of blood; the remaining individuals were negative. Among the 7 mosquitoes feeding on mice, 2 were infected (virus titers of 2.4 and 2.7 dex LDsO) , but neither transmitted virus. Of the orally infected mosquitoes, only I had a virus titer of 1.9 dex LDso; however, it failed to transmit virus to a mouse after 6 days of extrinsic incubation. Other mosquitoes were negative when tested at 7 and 11 days. Aedes aegypti
One of 3 inoculated females transmitted virus to a blood droplet after 7 days of incubation; it contained virus titering 2.5 dex LDso- The remaining 2 mosquitoes as well as 10 others, all orally infected, were negative for virus. DISCUSSION
Orungo virus has been isolated on 3 occasions from mosquitoes in Uganda and Nigeria (Tomori & Fabiyi 1977a) and the Central African Republic (Y. Robin & P. Sureau, pel's. commun., 1975), as well as from the blood of humans during episodes of febrile diseases in Nigeria. Neutralizing antibodies have been demonstrated in the human, bovine and ovine populations in Nigeria (Tomori & Fabiyi 1976). The prevalence of Orungo virus antibodies in the human population suggests the possible role of mosquitoes as biological vectors of Grungo viruses in Nigeria. Demonstration of experimental mosquito transmission of the virus between animals was hampered by failure to find a suitable laboratory host. Mice, hamsters and chickens are not infected by subcutaneous inoculations of Orungo virus, but the
LITERATURE
CITED
Aitken, T. H. G. 1977. An in vitro feeding technique for artificially demon~trating virus transmission by mosquitoes. Mosg. News 37: 130-33.
Downloaded from http://jme.oxfordjournals.org/ by guest on June 8, 2016
Da..v 7: One of 9 females contained demonstrable virus, titering 2.2 dex LD50; however, it failed to transmit virus to the blood droplet. Of2 mosquitoes feeding on mice, neither had virus.
first 2 do produce a low-grade viremia following ic inoculation (Tomori & Fabiyi 1977b). In the present studies, Orungo virus was transmitted artificially by allowing intrathoracically inoculated mosquitoes to feed on drops of blood; moreover, there was low-grade replication of virus in these infected mosquitoes (between 0.5-1.2 dex LDso increase). None of the orally infected mosquitoes transmitted Orungo virus, although virus was recovered after 6 days from 1 Ae. albopictus. Various factors might have been responsible for transmission failure by orally infected mosquitoes. The low level (about 4.0 dex) of Or un go virus in the blood meal might account for failure of the virus to establish itself in the mosquito. Schaeffer & Arnold (1954) suggested a viremia level of4.5 dex and above as a high mosquito-infecting potential for Eastern equine encephalomyelitis virus. Even when the virus level is high, the ingested virus must survive anatomic, physiologic and biochemical barriers in several organs of the mosquito before progeny virus is delivered to the salivary secretions for possible transmission (Murphy 1975). These barriers referred to as "gut barrier" must be overcome before virus replication takes place in the mosquito, and it is not known if virus is actively destroyed by means unknown or dies off gradually in the absence of favorable conditions (Chamberlain & Sudia 1961). The duration of incubation required to attain maximal virus growth and transmitting efficiency is also important. The II-day extrinsic incubation period may not have been sufficiently long to attain enough virus for transmission by mosquitoes orally exposed to virus, as compared to those inoculated intrathoracically and, therefore, not requiring gutwall passage. Infection rates of inoculated mosquitoes were likewise inexplicably low, but they, too, might be related to the short incubation period. Unfortunately, experimental time limits were dictated by extraneous factors. The multiplication of Orungo virus in and its artificial transmission (albeit sporadic) by Aedes mosquitoes, coupled with the detection of viremia and development of N antibody in man and domestic animals, suggests categorization of Orungo as an arbovirus. However, a suitable animal host that will circulate virus and develop antibody following exposure to an infected arthropod, as well as further transmission experiments, are required for confirmation.
526
J.
Moo. Entomol.
infection. In: Pathology of invertebrate vectors of disease. Ann. N. Y. Acad. Sci. 266: 197-203. Murphy, F. A., E. C. Borden, R. E. Shope & A. Harrison. 1971. Physicochemical and morphological relationships of some arthropod-borne viruses to bluetongue virus-A new taxonomic group. Electron microscopic studies. j. Cm. Viral. 13: 273--88. Ozawa, Y. & G. Nakata. 1965. Experimental transmission of African horse-sicknessby means of mosquitoes. Am. J. Vet. Res. 26: 744-48. Ozawa, Y., G. Nakata, F. Shad-Del & S. Naval. 1966. Transmission of Mrican horse-sickness by a species of mosquitoes Aedes aegypti Linnaeu~. Am. J. Vet. Res. 27: 695-97. Reed, L. J. & H. Muench. 1938. A simple method of estimating fiftyper cent endpoints. Am. j. Hyg. 27: 493-97. Schaeffer, M. & E. H. Arnold. 1954. Studies on the North American arthropod-borne encephalitides. I. Introduction. Contributions of newer field-laboratory approaches. Am. J. Hyg. 60: 231-35. TODlori, O. & A. Fabiyi. 1976. Neutralizing antibodies to Orungo virus in man and animals in Nigeria. Trop. Ceogr. Med. 28: 233-38. 1977a. Orungo virus, a new agent from mosquitoes and man in Uganda and Nigeria. Niger. Med. j. 7: 5--8. 1977b. Susceptibility of laboratory and domestic animals to experimental infection with Orungo virus. Acta Viral. 21: 133-38. Tomori, 0., A. Fabiyi & F. A. Murphy. 1976. Characterization of Orungo virus, an orbivirus from Uganda and Nigeria. Arch. Viral. 51: 285-98. Wallis, R. C. 1962. Chicken egg chorio-allantoic membrane lor mosqUito feeding. Mosq. News 22: 305--06.
Downloaded from http://jme.oxfordjournals.org/ by guest on June 8, 2016
Boorman, J. & E. P. J. Gibbs. 1973. Multiplication of the virus of epizootic haemorrhagic disease of deer in Culicoides species (Diptera, Ceratopogonidae). Arch. Cesamte Vin/sforsch. 41: 259-66. Borden, E. C., R. E. Shope & F. A. Murphy. 1971. Physicochemical and morphological relationships of some arthropod-borne viruses to bluetongue virus-A new taxonomic group. Physicochemical and serological studies. j. Cm. Virol. 13: 261-71. Burgdorfer, W. 1960. Colorado tick fever. II. The behaviour of Colorado tick fever virus in rodents. j. Infict. Dis. 107: 384-88. Casals, J. 1967. Immunological techniques for animal viruses. p. 113-98. I,I: Maramorosch, K. & H. Koprowski, eds., Methods in virology. Vol. 3. Academic Press, New York & London. ChaDlberlain, R. W. & W. D. Sudia. 1961. Mechanism of transmissionof viruses by mosquitoes. Amlu. Rev. Entomol. 6: 371-90. du Toit, R. M. 1944. The transmission of bluetongue and horse sickness by Culicoides. Onderstepoort J. Vet. Sci. Anim. Ind. 19: 7-16. Florio, L., M. S. Miller & E. R. Mugrage. 1950. Colorado tick fever. Isolation of the virus from Dermacentor andersoni in nature and a laboratory study of the transmissionof the virus in the tick. J. Immtlnol. 64: 257--63. Gubler, D. J. & L. Rosen. 1976. A simple technique for demonstrating transmission of dengue virus by mosquitoes without the use of vertebrate hosts. Am. J. Trap. Med. Hyg. 25: 146--50. Haldane, J. B. S. 1960. "Dex" or "Order of Magnitude"? Nahm, London 187: 879. Murphy, F. A. 1975. Cellular resistance to arbovirus
Vol. 14, no. 5
Meld. Entomol. Vol. 14,
DO.
10 February 1978
5: 523-526
ORUNGO VIRUS: TRANSMISSION STUDIES WITH AEDES ALBOPICTUS AND AEDES AEGYPTI (DIPTERA: CULICIDAE)} By Oyewale Tornori2 and Thornas H. G. Aitken3 Abstract: Adult female Aedes albopictlls and Aedes aegfPti mosquitoes were exposed to Orungo, an orbivirus, by intrathoracic inoculation and oral ingestion. After 6 days, virus was demonstrated in I orally exposed mosquito, but only inoculated mosquitoes transmitted the virus to blood droplets after an extrinsic incubation of 6-10 days. Transmission attempts utilizing infant mice wert' negative.
MATERIALS
AND METHODS
Virus. The IbAr 52302 strain of Orungo virus isolated from a pool of Aedes dentatus in Nigeria was used for these studies. The strain was at the 7th mouse brain and 2nd BHK-21 tissue culture passage level. The virus was stored at -60°C in Eagle's Basal Medium (EBM) supplemented with 2.5% fetal calf serum (FCS). Virus titrations were done in 2-day-old mice inoculated by the intracerebral (ic) route. Virus titers were calculated by the method -
--
'The studies and observations on which this paper is based were eonducted at the Yale Arbovirus Research Unit, New Haven, Connecticut 06510, U.S.A., where the benior author was on a study leave under a staff Development Grant from the University of Ibadan, Nigeria. The research was supported in part under U,S. Army Contract DADA 17-72-C-2170,National Institute of Health Grant AI 10984and a grant from the World Health Organization. 'Virus Research Laboratory, University of Ibadan, Ibadan, ~igeria, to which reprint requests should be addressed. 'Yal~ Arbovirus Research Unit, New Haven, Connecticut OG51O, U,S.A.
Mosquitoes. Two species, Aedes albopictus and Aedes aegypti, were used. They represented longestablished laboratory colonies. The former originated in the Virus Research Centre, Poona, India, and came to Yale Univeristy via Fort Detrick, Maryland; the latter is the Amphur strain from Thailand (12-20 F generations) obtained from Dr George Craig, Notre Dame University. Infection of mosquitoes was attempted either by permitting mosquitoes to engorge on a virus-blood suspension or by intrathoracic inoculation with virus suspension. Starved females (4-5 days old), in net-covered O.236-liter ~arton cages, were allowed to feed on cotton pledgets soaked in a virus-defibrinated blood suspension or else through the air sac membrane of chicken egg feeding chambers (Wallis 1962). Each feeding chamber contained 1 ml of the virus-blood suspension, which was prepared as follows: 2 ml of undiluted virus stock, 7.5 ml of inactivated and defibrinated chick blood and 0.5 ml of antibiotic solution (500 units of penicillin and 500 [Jog of streptomycin). Virus titer in this suspension was 4.3 dex LD50/ml. A fully engorged Ae. aegyPli takes up about 0.003 ml of blood; thus, these females acquired about 1.8 dex LD50 of Orungo virus. Intrathoracic inoculations of adult female mosquitoes (2-5 days old) were accomplished by means of an inoculating needle made with glass capillary tubing (l-mm diam.) drawn into a fine point. The tubing was marked off in I-mm graduations with an indelible ink wick pen, each unit representing the amount of inoculum per mosquito. The inoculation site was the membranous area below the paratergite between the meso thoracic spiracle and the wing base. Each mosquito received :1: 0.0006 ml of undiluted virus stock suspension, approximately 1.8 dex LD50 of virus. All manipulations were carried out on mosquitoes lightly anesthetized with carbon dioxide and kept immobilized in a petri dish resting on wet icc. Inoculated and blood-engorged mosquitoes were held in net-covered cages provided with wicks
Downloaded from http://jme.oxfordjournals.org/ by guest on June 8, 2016
Orungo virus was first isolated from Anopheles funes/us mosquitoes in Uganda and later in Nigeria ii'om man and Aedes denla/us mosquitoes (Tomori & Fabiyi 1977a). It has been included in the 01'bivirus taxonomic group (Tomori et al. 1976) established by Borden et al. (1971) and Murphy et al. (1971), based on physicochemical, morphological and morphogenetical similarities. Experimental arthropod transmission of bluetongue (BLU), African horse sickness (AHS), Colorado tick fever (CTF), and epizootic hemorrhagic disease of deer (EHD) viruses, other members of the orbivirus taxon, has been reported by various workers (du Toit 1944, Ozawa & Nakata 1965, Ozawa et al. 1966, Florio et al. 1950, Burgdorfer 1960, Boorman & Gibbs 1973). The present report describes attempts to transmit Orungo virus following experimental infection of Aedes albopictus and Aedes aegypti.
of Reed & Muench (1938) and expressed in dex (loglO) (Haldane 1960). Stock virus titer was 5.0 dex mouse LD50/ml.
J. Med.
524
soaked in 10% dextrose. Mosquitoes were held for 6 to 11 days at 26.7°C and 68-78% RH.
Each blood meal (droplet or capillary) sampled by feeding mosquitoes subsequently was mixed with a drop (about 0.03 ml) of 2.5% FCS and inoculated intrathoracically into 10-15 fresh adult Ae. albopictus Or Ae. aegypti females in order to facilitate the TABLE 1.
Vol. 14, no. 5
multiplication of any virus which might have been transmitted in the feeding process. These mosquitoes were then held for 7-10 days at 26.7 °C, 68-78% RH and maintained on 10% dextrose. At the end of the incubation period, all surviving mosquitoes from each transmission experiment were pooled and tested for virus; mosquito pools were triturated in suspensions of 0.5-1.0 ml of 2.5% FCS, centrifuged, and the supernatants then inoculated ic into 2-day-old mice. Brains from mice showing typical infection were harvested and tested for virus, which if present was identified as Orungo by CF test, using the technique of Casals (1967) and specific hyperimmune, reference-mouse ascitic fluid. The infected mosquitoes used for the virus transmission feedings and then stored at -60°C were likewise tested for virus by suspending each in 1 ml of FCS and inoculating the centrifuged specimen into a group of 2-day-old mice. Brains of sick or dying mice were tested for the presence of Grungo virus as described previously. All mice surviving feedings by infected mosquitoes or surviving ic inoculations of infected mosquito material were exsanguinated 28 days later and their sera tested for specific Grungo virus neutralizing (N) antibody in baby mice. Neutralization tests were done in mice by standard methods using the constant-serumvarying virus technique. Sera were inactivated at 56°C for 30 min. before use. RESULTS
The results of the transmission experiments in
TABLE
1.
Aedes alboPictus Following parenteral inoculations of 1.8 dex LD50 of Orungo virus per mosquito, artificial
Infection and transmission rates in Aedes albopictus and Aedes aeg)pti after parenteral and oral infection with Orungo virus.
MOSQUITO
ROUTE OF INFECTION
At. alboPictus
Parenteral
Oral
EXTRINSIC INCUBATION (DAYS)
Mouse
Blood droplet
6 7
1/50/2
3/6 1/9
10
2/7 3/14
INFECTION RATE
1/4
0/7 0/14 0/3 0/2
1/4 4/19
7
5/19 0/5 0/8 0/7 0/20 1/3
7
0/2
9
0/3 0/5
1/3
0/2 1/5
Parenteral Oral
Blood droplet 3/6 0/9
11
Ae. aeg)'jiti
TRANSMISSION RATE
Mouse 0/5 0/2
6 7
with
Ae. albopictus and Ae. aegypti mosquitoes are presented
0/5
-No. mosquitoes transmitting or virus positive/no. tested feeding on mouse or blood.
0/5 0/8 0/7 0/20 1/3 0/2 0/3 0/5
Downloaded from http://jme.oxfordjournals.org/ by guest on June 8, 2016
Transmission attempts. At intervals, following exposure to virus infection, single mosquitoes were allowed to bite individual 1- to 2-day-old mice. Engorged mosquitoes were stored at -60°C until tested for virus by ic inoculation of baby mice. Bitten mice were watched for signs of illness, and virus isolation attempts were made from brains where indicated. Brain suspensions (10% in bovine albumen diluent) of suspiciously sick mice were passaged ic to groups of fresh mice. In addition, 2 other methods were employed to demonstrate possible transmission of Orungo virus by feeding mosquitoes. In the 1st method (for details, see Gubler & Rosen 1976), infected mosquitoes were allowed to engorge on a drop (0.03 ml) of blood composed of equal parts of 2.5% FCS, 10% dextrose, and inactivated, defibrinated chick blood. One mosquito was confined in a cotton-plugged tube covered at the other end with fine-mesh synthetic netting on which was placed a drop of the blood meal. The 2nd method allowed feeding mosquitoes to engorge on blood in a capillary tube similar to that used for inoculating mosquitoes. The technique, fully described elsewhere (Aitken 1977), calls for inserting the infected mosquito's proboscis into the blood-charged tip of the capillary tube following which feeding usually begins. Using either technique, feeding mosquitoes were removed from the meal before completing engorgement and stored at -60°C.
Entomol.
Tomori & Aitken:
IS78
525
Transmission of Orungo virus by Aedes
transmission, by allowing mosquitoes to feed on blood droplets, was achieved on extrinsic incubation days 6 and 10, but not on day 7. The details are as follows; Del.)! 6: Three of the 6 females transmitted virus; however, only 1 had a titer of 2.3 dex LD50, while the others titered below 1.8 dex LD50, the initial inoculum. On the same day, none of 5 mosquitoes feeding on normal mice transmitted virus, as all exposed mice survived the experimental period and all failed to develop specific N antibody; only 1 of these 5 mosquitoes had demonstrable virus which titered 2.8 dex LD50'
Da.v 10: One of 4 females, containing virus titering 3.0 dex LI\Ol transmitted virus to a drop of blood; the remaining individuals were negative. Among the 7 mosquitoes feeding on mice, 2 were infected (virus titers of 2.4 and 2.7 dex LDsO) , but neither transmitted virus. Of the orally infected mosquitoes, only I had a virus titer of 1.9 dex LDso; however, it failed to transmit virus to a mouse after 6 days of extrinsic incubation. Other mosquitoes were negative when tested at 7 and 11 days. Aedes aegypti
One of 3 inoculated females transmitted virus to a blood droplet after 7 days of incubation; it contained virus titering 2.5 dex LDso- The remaining 2 mosquitoes as well as 10 others, all orally infected, were negative for virus. DISCUSSION
Orungo virus has been isolated on 3 occasions from mosquitoes in Uganda and Nigeria (Tomori & Fabiyi 1977a) and the Central African Republic (Y. Robin & P. Sureau, pel's. commun., 1975), as well as from the blood of humans during episodes of febrile diseases in Nigeria. Neutralizing antibodies have been demonstrated in the human, bovine and ovine populations in Nigeria (Tomori & Fabiyi 1976). The prevalence of Orungo virus antibodies in the human population suggests the possible role of mosquitoes as biological vectors of Grungo viruses in Nigeria. Demonstration of experimental mosquito transmission of the virus between animals was hampered by failure to find a suitable laboratory host. Mice, hamsters and chickens are not infected by subcutaneous inoculations of Orungo virus, but the
LITERATURE
CITED
Aitken, T. H. G. 1977. An in vitro feeding technique for artificially demon~trating virus transmission by mosquitoes. Mosg. News 37: 130-33.
Downloaded from http://jme.oxfordjournals.org/ by guest on June 8, 2016
Da..v 7: One of 9 females contained demonstrable virus, titering 2.2 dex LD50; however, it failed to transmit virus to the blood droplet. Of2 mosquitoes feeding on mice, neither had virus.
first 2 do produce a low-grade viremia following ic inoculation (Tomori & Fabiyi 1977b). In the present studies, Orungo virus was transmitted artificially by allowing intrathoracically inoculated mosquitoes to feed on drops of blood; moreover, there was low-grade replication of virus in these infected mosquitoes (between 0.5-1.2 dex LDso increase). None of the orally infected mosquitoes transmitted Orungo virus, although virus was recovered after 6 days from 1 Ae. albopictus. Various factors might have been responsible for transmission failure by orally infected mosquitoes. The low level (about 4.0 dex) of Or un go virus in the blood meal might account for failure of the virus to establish itself in the mosquito. Schaeffer & Arnold (1954) suggested a viremia level of4.5 dex and above as a high mosquito-infecting potential for Eastern equine encephalomyelitis virus. Even when the virus level is high, the ingested virus must survive anatomic, physiologic and biochemical barriers in several organs of the mosquito before progeny virus is delivered to the salivary secretions for possible transmission (Murphy 1975). These barriers referred to as "gut barrier" must be overcome before virus replication takes place in the mosquito, and it is not known if virus is actively destroyed by means unknown or dies off gradually in the absence of favorable conditions (Chamberlain & Sudia 1961). The duration of incubation required to attain maximal virus growth and transmitting efficiency is also important. The II-day extrinsic incubation period may not have been sufficiently long to attain enough virus for transmission by mosquitoes orally exposed to virus, as compared to those inoculated intrathoracically and, therefore, not requiring gutwall passage. Infection rates of inoculated mosquitoes were likewise inexplicably low, but they, too, might be related to the short incubation period. Unfortunately, experimental time limits were dictated by extraneous factors. The multiplication of Orungo virus in and its artificial transmission (albeit sporadic) by Aedes mosquitoes, coupled with the detection of viremia and development of N antibody in man and domestic animals, suggests categorization of Orungo as an arbovirus. However, a suitable animal host that will circulate virus and develop antibody following exposure to an infected arthropod, as well as further transmission experiments, are required for confirmation.
526
J.
Moo. Entomol.
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