Abstract

West Nile virus (WNV) has become established in the northeastern United States, where mosquitoes are inactive during winter. There have been no documented studies to explain how this virus survives winter and reinitiates infection in spring. We report that WNV was vertically transmitted to 2 F1 female Culex pipiens from a naturally infected female collected in Stratford, Connecticut. One vertically infected F1 female, which was 168 days old, fed on a hamster that died 8 days later of West Nile disease. This suggests that WNV survives winter in unfed, vertically infected C. pipiens with amplification initiated in spring by horizontal transmission

West Nile virus (WNV) has survived and annually caused human disease in the northeastern United States since 1999, even though mosquitoes are inactive during winter. Mosquitoes transmit the virus horizontally to vertebrate hosts during summer and fall [1], but the mechanisms for maintenance of WNV during periods of mosquito inactivity in winter and amplification of infection in spring are unknown. Vertical (passage of virus from female parent directly to offspring) followed by horizontal transmission enables many Bunyaviridae, including LaCrosse virus, to survive winter and to initiate infection in spring [2]. WNV, a Flaviviridae, has been vertically transmitted in laboratory studies in which Culex adult females were infected by intrathoracic inoculation and F1 adults were tested within 8 days after emergence [3–5 ]. The “long-term survival” of several months, which is needed during winter, and infection of vertebrate animals in spring have not been demonstrated. The isolations of WNV from male [6, 7] and nulliparous (i.e., those that have not produced eggs) female [7] Culex adults in the field during summer and from a pool of Culex females in winter [8] suggest that vertical transmission may be occurring naturally. We report the isolation of WNV from >180-day-old F1 adult females of a naturally infected C. pipiens and the infection of a hamster on which a single vertically infected F1 female fed after termination of diapause (i.e., hibernation)

Materials and methodsAdult C. pipiens mosquitoes were collected live on Water Pollution Control Authority land in Stratford, Connecticut (41°10′41″N, 73°07′34″W) during September 2005. Weekly minimum infection rates of C. pipiens infected with WNV were relatively high in Stratford during September, ranging from ∼13 to 43 infected females/1000 specimens [7]. Captured mosquitoes were fed on a guinea pig, and each individual engorged mosquito was transferred into a separate 296-mL, clear, covered plastic cup containing 25 mL of distilled water and 0.5 mL of a 3:2 liver and yeast extract. Mosquitoes were then transferred to a room at 16.5&j0;C±1.5&j0;C and a daily light/dark cycle of 10/14 h. Each female that laid an egg raft was tested for virus in Vero cells [7]. Isolates were identified as WNV by reverse-transcriptase polymerase chain reaction (RT-PCR) [9]

Larvae from each egg mass were reared at 16.5&j0;C±1.5&j0;C and a daily light/dark cycle of 10/14 h to induce diapause in F1 adult females [10]. All larvae from an egg mass were reared as a group in a disposable clear plastic container. Food consisting of a liver/yeast extract was added as needed to each container with larvae. Identification of mosquitoes was based on the characteristics of fourth instar F1 progeny. C. pipiens in the fourth instar are morphologically distinct from all other species of Culex in the northeastern United States. Mortality from the time of egg deposition until adults were tested >200 days later was often 50% or more. This F1 mortality is attributed to handling primarily during the larval stage, but adults also died. Dead mosquitoes were not tested

Adults emerged from pupae that had been placed in a 30.5 cm×30.5cm×30.5 cm mosquito cage. Raisins were provided as a sugar source. Five unfed adult F1 females and all males were tested for virus within 2 weeks of emergence. Males die shortly after mating with females and therefore had to be tested before they died. Previous laboratory studies demonstrating vertical transmission of WNV tested females within days after emergence [3–5 ]. We therefore tested 5 females shortly after emergence, but the remaining females were kept at 16.5&j0;C±1.5&j0;C and a daily light/dark cycle of 10/14 h for ∼140 days, whereupon the daily light/dark cycle was changed to 16/8 h, to initiate termination of diapause. Moist paper or cloth toweling was placed on each cage. Subsequently, mosquitoes were given the opportunity to feed on a hamster. Each engorged female was transferred to a clear plastic cup, and 5–15 days later each was tested in Vero cells for virus. Unfed females were given a chance to feed on a subsequent night

Selected specimens of F1 progeny that tested negative in Vero cell culture were also tested for WNV by RT-PCR [9]. Tissues of brain, spleen, and heart of hamsters were tested for WNV in Vero cells. Surviving hamsters were tested for virus and neutralizing antibody 43 or more days after the mosquitoes had fed. The Connecticut Agricultural Experiment Station Animal Care and Use Committee approved the procedures for handling animals

ResultsThree hundred sixty-one (41.4%) of 872 field-collected C. pipiens that fed on a guinea pig laid eggs. WNV was isolated from 10 of the 361 specimens tested. These 10 infected females were the parents of the F1 progeny

WNV was isolated from F1 adults from 1 of the 10 naturally infected females (table 1). Two F1 females from parent T676 tested positive. One unfed F1 female (this female had been given the opportunity to feed on a hamster) infected with WNV was tested on 15 May 2006, 196 days after it had emerged as an adult on 1 November 2005. The other infected F1 female fed on a hamster on 17 April 2006 and was killed and tested for virus on 3 May 2006, 184 days after it had emerged as an adult. Virus was reisolated from the original suspensions of each positive mosquito, and each of these mosquitoes tested positive by RT-PCR, verifying infection. All F1 male (n=64) and female (n=28) adults from naturally infected parent T676 that tested negative in Vero cell culture also tested negative for WNV by RT-PCR. The filial infection rate of all progeny (male and female) of parent T676 was 1:47 (table 1). The estimated infection rate of all F1 female progeny from the 381 females that laid eggs (assumed average number of 100 adult females/egg mass) was ∼2 infected females/38,100 specimens or 0.5 infected females/1000 females

Table 1

West Nile virus isolation attempts from F1 adult progeny of 10 naturally infected Culex pipiens collected in Stratford, Connecticut, during September 2005

Table 1

West Nile virus isolation attempts from F1 adult progeny of 10 naturally infected Culex pipiens collected in Stratford, Connecticut, during September 2005

The hamster on which parent T676 fed died 8 days after the mosquito bite. Virus was isolated from tissues of spleen, heart, and brain and was identified as WNV. All other hamsters on which F1 females fed were negative for virus and neutralizing antibody

DiscussionWNV was originally isolated in equatorial Africa, where mosquitoes are active throughout the year. Sporadic but not continuous outbreaks of WNV have been noted in humans and horses in Europe, after periodic introductions of the virus by migrating birds [11]. Yet in temperate northeastern United States, where C. pipiens as in Europe, is not active in winter, WNV has persisted and annually caused human disease since 1999. By using field-collected C. pipiens naturally infected with WNV, rearing F1 progeny under diapausing conditions, isolating WNV from a 196-day-old female, and demonstrating vector competency by a 168-day-old F1 female, we have shown how WNV survives winter and amplifies in spring in the northeastern United States

The isolation of WNV from 2 F1 sibling C. pipiens females, which were >180 days old and descendants of a field-collected infected female, demonstrate that vertical transmission is occurring naturally. Studies further show that WNV survives in unfed C. pipiens in diapause from November until mid-April and May of the following year, when increasing day length and warming temperatures terminate diapause and females seek a blood meal [10]. In Connecticut in 2005, C. pipiens mosquitoes were active and seeking hosts in the field during the week of 17 April [7]

We also document that a vertically infected female C. pipiens which had been in diapause and not previously fed on blood, was competent to transmit WNV horizontally to a hamster. A hamster died of WNV infection 8 days after being bitten by a vertically infected F1 female that was 168 days old. These results demonstrate that a vertically infected C. pipiens that entered diapause in November was able to initiate infection the following spring

The filial infection rate of 1:47 for parent T676 is relatively high for WNV [3–5 ], but if our calculation included F1 mosquitoes from all 10 infected parents, the filial infection rate would be 2.8 infected mosquitoes/1000 specimens, which is similar to the filial infection rate of 2.1 infected mosquitoes/1000 mosquitoes reported in laboratory studies for the Kunjin strain of WNV in Aedes albopictus [12] and 3.0 infected mosquitoes/1000 specimens in C. pipiens quinquefasciatus [5]. However, vertical infection rates for WNV and other flaviviruses are significantly lower than rates reported for LaCrosse virus (34% of the F1 mosquitoes were infected) [2]. These differences may be explained by the manner in which F1 progeny become vertically infected. Viruses in the Bunyaviridae family infect oocytes within the ovary of the mosquito vector [13], whereas flaviviruses do not infect the ovary but instead infect the fully formed egg after it has exited the ovary [14]. It is unclear why WNV was vertically transmitted by only 1 of the 10 infected C. pipiens females and why infections were detected in only 2 of the F1 progeny of parent T676. Rosen [14] has suggested that transmission efficacy is dependant on the scale of viral replication in tissues near where eggs are being fertilized. The manner in which the parent female becomes infected (vertical or horizontal) and the number of the ovarian cycle may also influence transmission of the virus to the egg. Even though research is needed to explain how WNV is vertically transmitted, it is clear that this method of transmission is extraordinarily important in the natural history of this virus

The significance of vertical transmission in the natural history of Flaviviridae, including WNV, in tropical environments may not be readily apparent and may be underappreciated, as emphasized by Miller et al. [6]. However, in temperate climates, vertical transmission followed by horizontal transmission is key to continued maintenance of the virus in winter and amplification in spring. The reported WNV infection rate of 2.4 infected females/1000 Culex females collected at Fort Totten, New York, in January and February [8] and our current finding of an estimated infection rate of ∼0.05 infected females/1000 females suggest that transgenerational transmission of WNV is an important means of enabling this virus to persist during winter in temperate climates, where C. pipiens females survive several months of winter without a blood meal. The relatively large numbers of C. pipiens in cities and nearby suburbs in the northeastern United States are also important in enabling the North American WNV strain to persist where transgenerational transmission in the population is relatively low (<1%)

Vertical followed by horizontal transmission is likely also important in contributing to enzootic and epizootic transmission in August and September when minimum infection rates are highest in C. pipiens and the greatest numbers of humans contract West Nile disease. Although many studies have emphasized the importance of maintenance of this virus by the mosquito-bird-mosquito cycle in summer and fall [1], our recent report of isolating WNV from 6 pools of field-collected male C. pipiens with a weekly minimum infection rate as high as 31.1 infected males/1000 specimens and from 6 nulliparous females [7] suggests that females, which likely acquired infections vertically, may also be contributing to avian and human infections by horizontally transmitting the virus during their first and subsequent blood feedings. These findings with WNV in Connecticut may be similar to those reported by Fontenille et al. [15], who suggested that vertical transmission of yellow fever virus (Flaviviridae) in Aedes aegypti contributed significantly to the epidemic of yellow fever in Senegal in 1995. The competency of a previously unfed, but vertically infected, C. pipiens to transmit WNV to a mammalian host suggests that human disease could occur without the mosquito first feeding on an infected avian host

Acknowledgments

We thank Bonnie Hamid, Tanya Petruff, Angela Penna, Michael Vasil, Michael Misencik, Alyson Florek, and Terrill Goodman for technical assistance. We also thank Elaine O’Keefe, Peter Stallings, Bill McCann, and Jeff Ritz for their support on property owned by the Town of Stratford

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Potential conflicts of interest: none reported
Financial support: US Department of Agriculture (specific cooperative agreement 58-6615-1-218); Centers for Disease Control and Prevention (laboratory capacity for infectious diseases cooperative agreement U50/CCU116806-01-1)