Yellow fever virus infectivity for Bolivian Aedes aegypti mosquitoes

Emerging Infectious Diseases, Sept, 2004 by John-Paul Mutebi, Alberto Gianella, Amelia Travassos da Rosa, Robert B. Tesh, Alan D. T. Barrett, Stephen Higgs

The absence of urban yellow fever virus (YFV) in Bolivian cities has been attributed to the lack of competent urban mosquito vectors. Experiments with Aedes aegypti from Santa Cruz, Bolivia, demonstrated infection (100%), dissemination (20%), and transmission of a Bolivian YFV strain (CENETROP-322).

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Yellow fever virus (YFV) may cause severe hemorrhagic fever in humans. The virus is transmitted between susceptible vertebrate hosts by infected mosquitoes ill the genera Aedes, Haemagogus, or Sabethes (1). In the Americas, YFV occurs in two transmission cycles. In the jungle/sylvatic cycle, the virus is transmitted between susceptible monkeys, and possibly other vertebrates, by tree-hole breeding mosquitoes (1). Jungle yellow fever (YF) cases occur when these infected vectors feed on susceptible humans. In the urban cycle, YFV is transmitted to humans by Aedes aegvpti mosquitoes (1). In 2003, a total of 226 cases of jungle YF were reported from South America to the Pan American Health Organization, and as of June 23, ongoing outbreaks in Bolivia, Brazil, Colombia, and Peru during 2004 have thus far resulted in 86 confirmed cases and 41 deaths (2).

An Ae. aegypti eradication campaign initiated by the Pan American Sanitary Bureau in 1947 eliminated this species from most of Central and South America, and urban YF disappeared from the Americas in the 1940s. However, during the past 20 years, many countries abandoned Ac. aegypti control measures, and this urban vector now reoccupies almost the entire area of its distribution preeradication (1). Ac. aegypti was eradicated from Bolivia during the 1960s and 1970s but reappeared in the city of Santa Cruz in 1980, and epidemics of dengue fever occurred during the 1980s and 1990s (3). In 1997 to 1998, six cases of YF were reported among Santa Cruz residents, and some were regarded as urban YF cases (3); despite a population >1 million, low vaccine immunization cover age, and the presence of Ac. aegypti (3), no urban YF outbreak occurred. Based on these observations, researchers have suggested that sylvan strains of YFV circulating in Bolivia may not be infective for Bolivian Ae. aegypti. This study examined that hypothesis and the infectivity of a Bolivian strain of YFV for Bolivian Ae. aegypti.

The Study

All work involving infectious YFV was performed in biosafety level 3 facilities at the University of Texas Medical Branch. Three human isolates of YFV were used: CENETROP-322 (La Paz Department, Bolivia, 1999), Jimenez (Panama, 1974), and Asibi (Ghana, 1927). To facilitate transmission from a viremic vertebrate, viruses were adapted by serial passage through Syrian golden hamsters (Mesocricetus auratus), following the model of Tesh et al. (4,5); CENETROP-322 and Jimenez were passaged 11 times, and the Asibi strain was passaged 10 limes.

The SC strain of Ac. aegypti was started with mosquitoes collected from Santa Cruz, Bolivia, in 2001. Mosquitoes used in this experiment were from laboratory-reared F2-F3 generation. The REX-D strain, an old laboratory colony originally started with mosquitoes collected in Rexville, Puerto Rico and of previously defined susceptibility to YFV infection (6) was used as a control. Mosquitoes were maintained as previously described (7).

Three hamsters were injected intraperitoneally (IP) with 100 [micro]L of clarified liver homogenate, which contained approximately [10.sup.6] [log.sub.10] tissue culture infectious dose 50% (TCI[D.sub.50]/mL) of each YFV strain. Three days after infection, when viremia levels have been shown to peak (4), hamsters were anesthetized (50 mg Pentobarb/kg I P) and simultaneously exposed to 10-day-old Ac. aegypti SC or REX-D mosquitoes for 1 h. Fully engorged mosquitoes in each group were placed in separate cages and incubated for 15 days at 28[degrees]C and 80% relative humidity on a diet of 10% sucrose. Hamster blood samples were collected immediately afterward and stored at -80[degrees]C for viral assay.

Virus Transmission

At day 15 after infection, mosquitoes were allowed to feed on 8-day-old mice. (Mice were used in preference to adult hamsters because they are more susceptible to fatal infection.) After feeding, mosquitoes were assayed for YFV infection and dissemination by whole-body titration and immunofluorescence assay (IFA) of head-squash material, respectively (7). For IFA, a broadly reactive anti-flavivirus monoclonal antibody (813) with biotin-streptavidin amplification was used (8). Suckling mice were observed for illness and death. The brains of two paralyzed mice were tested for viral antigen by culturing on Vero cells. At day 14 after exposure, serum specimens from surviving mice were tested for anti-YFV antibodies by hemagglutination inhibition (Hl) test (9).

All three strains of YFV caused viremia in hamsters on day 3 after infection, with titers of 8.5, 8.7, and 7.3 TCI[D.sub.50][log.sub.l0]/mL (CENETROP-322, Jimenez, and Asibi, respectively), as determined by assay in mosquito cell cultures (4). Determination of mosquito infection and dissemination rates showed that all YFV strains were able to infect both strains of Ac. aegypti, although infection rates varied from 15.l% to 100%. Mean total mosquito YFV titers were relatively low, but the presence of virus after 15 days, evidence of dissemination in the insect, and transmission data are all indicative of replication. At day 15 postinfection, 100% of SC Ae. aegypti were infected with CENETROP-322. Infection rates and mean viral titers for CENETROP-322 were higher in SC Ae. aegypti than in the REX-D strain (Table 1). Infection rates of CENETROP-322 and Jimenez were higher than the rate for Asibi in both mosquito strains.

Virus titers in the mosquitoes varied considerably but were lowest in REX-D strain insects infected with CENETROP-322 (Table 1). These mosquitoes had the highest dissemination rates (80.7%), which indicates little correlation between virus titer and dissemination rates. Dissemination rates were highest in the REX-D strain; but our data demonstrate that both Panamanian and Bolivian strains of YFV disseminated in Santa Cruz Ae. aegypti (Table 1).