Diptera: Culicidae) in the Laboratory Sara Marie Erickson Iowa State University

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1-1-2005 Infection and transmission of West Nile virus by Ochlerotatus triseriatus (Diptera: Culicidae) in the laboratory Sara Marie Erickson Iowa State University

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Recommended Citation Erickson, Sara Marie, "Infection and transmission of West Nile virus by Ochlerotatus triseriatus (Diptera: Culicidae) in the laboratory" (2005). Retrospective Theses and Dissertations. 18773. https://lib.dr.iastate.edu/rtd/18773

This Thesis is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Infection and transmission of West Nile virus by Ochlerotatus triseriatus (Diptera: Culicidae) in the laboratory

Sara Marie Erickson

A thesis submitted to the graduate faculty

in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

Major: Entomology

Program of Study Committee: Wayne A. Rowley, Major Professor Russell A. Jurenka Kenneth B. Platt Marlin E. Rice

Iowa State University Ames, Iowa 2005

Graduate College Iowa State University

This is to certify that the master's thesis of

Sara Marie Erickson

has met the thesis requirements of Iowa State University

Signatures have been redacted for privacy lll

TABLE OF CONTENTS

LIST OF TABLES lV

ABSTRACT v

CHAPTER 1. GENERAL INTRODUCTION

Thesis organization 1

Literature review 1

References 18

CHAPTER 2. INFECTION AND TRANSMISSION OF WEST NILE VIRUS

BY OCHLEROTATUS TRISERIATUS (DIPTERA: CULICIDAE) IN THE

LABORATORY

Abstract 28

Introduction 29

Materials and methods 30

Results 34

Discussion 35

Acknowledgements 37

References 38

CHAPTER 3. GENERAL CONCLUSIONS 47

ACKNOWLEDGMENTS 48 lV

LIST OF TABLES

Table 1.1. Ochlerotatus triseriatus blood meal identification 7

Table 1.2. West Nile virus isolations from field-collected mosquitoes 12

Table 1.3. Vector competence of mosquitoes orally infected with 15

West Nile virus and tested 12-14 days after exposure

Table 2.1. Blood feeding success of Ochlerotatus triseriatus and 42

Culex p. pipiens on 2-5-day-old chickens

Table 2.2. Infection of Ochlerotatus triseriatus and Culex p. pipiens with 43

West Nile virus (IA- 02) 14 days after blood feeding on

viremic chickens

Table 2.3. Blood meal titers of West Nile virus predicted by regression 44

models to infect Ochlerotatus triseriatus and Cul exp. pipiens at

specific rates

Table 2.4. Estimated coefficients used to construct logistic regression models 45

and areas under the receiver operating characteristic curves

Table 2.5. West Nile virus (IA- 02) transmission to 2-4-day-old 46

chickens by the bite of infected Ochlerotatus triseriatus v

ABSTRACT

West Nile virus (WNV) is a newly recognized arbovirus in North America. The

WNV transmission cycle involves mosquitoes in the genus Culex, and avian species as the amplification hosts. Mosquitoes that feed on both birds and mammals are known as bridge vectors, which transmit WNV from avian amplification hosts to other vertebrates susceptible to WNV infection. These secondary hosts do not develop viremias high enough to continue the infection cycle, and are known as dead end hosts.

The primary objective of this study was to compare the susceptibility of Ochlerotatus triseriatus (Say) to Culex p. pipiens (L.), a primary amplification vector of WNV.

Ochlerotatus triseriatus is the primary vector of La Crosse encephalitis virus (LAC). It feeds primarily on mammalian hosts, and is a possible bridge vector of WNV.

West Nile virus infection rates of Oc. triseriatus and Cx. p. pipiens were determined following WNV viremic blood meals ranging from 104·0 to 108·2 PFU/ml. The minimum blood meal titer to infect Oc. triseriatus was 104·3 PFU/ml, and it was 104·0 PFU/ml for Cx. p. pipiens. West Nile virus infection rates in both species significantly increased as the viremic blood meal titers increased. Maximum infection of 98 and 100% was observed at 10~ 8 · 0 <85 and 10~7 - 0 < 7 · 5 PFU/ml for Oc. triseriatus and Cx. p. pipiens, respectively. West Nile virus titers predicted to infect Cx. p. pipiens at ID 10, ID50, and ID90 are 103·2, 1042, and 106·5

PFU/ml. Predicted titers to infect Oc. triseriatus at ID10, ID50, and ID90 are 104.7, 106·5, and

108.3 PFU/ml. Observed and predicted infection rates suggest that Oc. triseriatus is significantly less susceptible than Cx. p. pipiens to WNV.

Ochlerotatus triseriatus orally infected from viremic chicken blood meals with a

WNV titer> 108·0 PFU/ml were able to transmit virus by bite to susceptible chickens on days

14 and 18 post infection. The infection, dissemination, and transmission rates of Oc. triseriatus were 94, 91, and 60 percent respectively. Vl

Ochlerotatus triseriatus is less susceptible to WNV infection then Cx. p. pipiens. It is a competent vector with a 55% estimated transmission rate among individuals infected at high titer WNV viremic blood meals. Ochlerotatus triseriatus is a potential bridge vector of

WNV and may play a role in transmission among susceptible mammalian hosts.

Characterization of WNV infection within primary hosts of Oc. triseriatus, i.e. squirrels, chipmunks, rabbits, and deer, is needed to assess the possibility of a mammalian WNV transmission cycle. 1

CHAPTER 1. GENERAL INTRODUCTION

THESIS ORGANIZATION

This thesis consists of three chapters. Chapter 1 is a general introduction consisting of an explanation of thesis organization and literature review. Chapter 2 is a research article to be submitted to the Journal of Vector-borne and Zoonotic Diseases, and Chapter 3 is general conclusions of the thesis. References cited are listed at the end of each chapter. All results and conclusions from this study were inteq~reted and written into manuscript form by the author.

LITERATURE REVIEW

Classification and distribution of Ochlerotatus triseriatus

Ochlerotatus triseriatus (Say) was originally described within the genus Culex (Say

1823). Since 1823 there have been several systematic descriptions (WRAIR 2001) of this species, commonly known as the Eastern treehole mosquito. The current classification is as follows; Diptera: Culicidae: Aedini: Ochlerotatus (Protomacleaya) triseriatus (Say) (Reinert

2000). This species was formerly in the genus Aedes. Reinert (2000) elevated Ochlerotatus to generic rank, formerly a subgenus of Aedes, based on morphological characteristics of female and male genitalia, pupae, and 4th _stage larvae. This publication initiated discussions on culicid systematics by many in medical entomology. Published scientific opinions have both disputed (Savage and Strickman 2004) and supported (Black 2004) the generic elevation of Ochlerotatus.

Ochlerotatus triseriatus is a member of the Triseriatus group of the subgenus

Protomacleaya. This group is composed of four closely related mosquitoes that share morphological characteristics, but differ in biological attributes. It includes Ochlerotatus 2

hendersoni (Cockerell), Ochlerotatus brelandi (Zavortink), Ochlerotatus zoosophus (Dyar and Knab), and Ochlerotatus triseriatus (Say) (Craig 1983).

Sibling species Ochlerotatus triseriatus and Ochlerotatus hendersoni are difficult to identify by morphological characteristics alone (Reno et al. 2000). They are sympatric in the

Eastern United States, east of the Great Plains (Craig 1983), where hybridization can occur

(Truman and Craig 1968). Ochlerotatus hendersoni was first described as a subspecies of

Oc. triseriatus (Cockerell 1918), and was elevated to specific status by Breland (1960).

Identification of sibling adults can be almost impossible with rubbed specimens (Craig

1983). Molecular techniques insure proper species differentiation, such as electrophoresis

(Saul et al. 1977) and restriction fragment length polymorphisms (Reno et al. 2000).

Oviposition

Gravid Ochlerotatus triseriatus are attracted to tree holes by decomposing organic matter, such as oak leaf infusion (Trexler et al. 1998), and engage in multiple ovipositional events within one gonotrophic cycle. Ovipositional traps in woodlots measured 20-60 eggs

(Kitron et al. 1989), and 31±9.8 eggs (Beehler and DeFoliart 1990) per oviposition event.

Although Oc. triseriatus are diurnal, oviposition occurs in all hours of the day, including night, with an increased percentage of eggs laid during the evening crepuscular period in the laboratory (Trexler et al. 1997).

Ochlerotatus triseriatus lay eggs in basal tree holes, from ground level to 1.2 meters, more often than in holes > 1.2 meters and <3 .2 meters (Aziz and Hayes 1987). A greater number of Oc. triseriatus eggs are found in tree holes located in mesic habitats in contrast to those found within border trees (Ballard et al. 1987). The presence of mosquito eggs on the surface of an ovipositional site negatively affects the number of eggs laid at that location.

Field studies by Kitron et al. (1989) found a nonrandom ovipositional pattern among 300 3 traps located within a woodlot, in which the number of eggs in each trap appeared to be laid in an overly dispersed manner. Tree holes can be a limited resource within woods. This ovipositional practice may contribute in the spread of Oc. triseriatus progeny within wooded habitats and therefore increasing survival probabilities due to larval habitat crowding.

Ochlerotatus triseriatus is a container-breeding mosquito that is not restricted to woodlot habitats. Mather and DeFoliart (1984b) studied the dispersion of Oc. triseriatus from woodlots by placing ovipositional traps in nearby open terrain fields. Gravid females are able to locate small water containers outside of wooded areas. Dispersal from woodlots to open terrain increases potential for Oc. triseriatus-human contact.

Seasonal survival of Oc. triseriatus occurs in the egg or larval stage in temperate regions. Induction of both types of diapause is the effect of short day length on eggs or larvae. Photoperiod treatment on adult Oc. triseriatus has no effect on induction of diapause of their eggs, but the induction of diapause increases when eggs are directly exposed to reduced daylength (Kappus and Venard 1967). Love and Whelchel (1955) demonstrated larval diapause of Oc. triseriatus in which 4th instars delayed pupation when treated with a decreased photoperiod. Egg diapause terminates with prolonged exposure to cold temperatures (Shroyer and Craig 1983).

Larval habitats and behavior

Larval competition occurs when mosquito populations compete for ovipositional sites in a given habitat. Adults emerging from a crowded container habitat may be smaller than adults that did not have competition for food resources as larvae. Crowding within larval habitats also increases the developmental time of both male and female Oc. triseriatus

(Mahmood et al. 1997). Third and forth instars have a longer developmental time than younger instars. High larval densities may also result in significantly increased mortality. 4

Walker and Merritt ( 1991) constructed larval habitats to resemble natural tree holes to study the larval behavior of Oc. triseriatus. Of the behaviors described and observed, Oc. triseriatus larvae spent 90.8% of its time feeding. The flexible feeding behaviors of Oc. triseriatus larvae enabled the authors to define the entire larval habitat as the 'feeding zone' .

This contrasts with other larval feeding habits of mosquitoes in the tribe Aedini, which are known as brushers or bottom feeders.

Adults

Mark and recapture studies by Beier et al. (1982) found a 0.80 daily survivorship for female Oc. triseriatus, and 0.81 for males. Female and male recapture percentages were 5.9 and 4.7, respectively. The majority of captured mosquitoes were aggregated in a small area of the study woodlot, which contained dense ground cover. Haramis and Foster (1983) measured daily survivorship, distribution, dispersal, and population densities of female Oc. triseriatus within a woodlot through mark and recapture techniques. These authors reported a daily survivorship ranging from 0.93 to 0.97 for females, with 41 and 12 percent recapture rates during the two years of the study. Ochlerotatus triseriatus are found in low densities, and dispersal rates to a nearby lot were low in comparison to other mosquito species

(Haramis and Foster 1983). Walker et al. (1987) found a survival probability range of 0.917-

0.960 for the sympatric sibling species, Oc. triseriatus and Oc. hendersoni. Field populations of Oc. triseriatus were studied over two mosquito seasons to determine adult body size in a

LAC endemic region in Wisconsin, USA. There were significant variations in adult female size, but there was not a strong relationship between increased size and survival advantages

(Laundry et al. 1988).

Ochlerotatus triseriatus is physiologically able to take a blood meal several days before they are receptive to mating (Porter et al. 1986). The median time from emergence to 5 blood feeding on suckling mice in the laboratory was 2.6 days (38 h) (Porter et al. 1986).

Ochlerotatus triseriatus reared in captivity are not receptive to mating until the seventh day of adult life. However they become receptive immediately following blood feeding (Mather and DeFoliart 1984b). Field observations by Loor and DeFoliart (1970) reported Oc. triseriatus forming swarms during evening crepuscular periods in close proximity to human and rabbit hosts. In some cases, mating occurred in the swarms above the host, and on the hosts before or after female engorgement.

Blood Meal Source

Ochlerotatus triseriatus is considered to be diurnal (Scholl et al. 1979) with peak activity observed one hour before lights off in the laboratory (Clarke 1988). Mosquito activity is often reflective of gonotrophic status, i.e. insemination, blood-feeding, and oviposition. A 50% decrease in flight activity was observed between non-inseminated and inseminated Oc. triseriatus, and relatively no activity in Oc. triseriatus for 48 hr post blood feeding (Clarke 1988). Field studies by Aziz and Hayes (1987) report late-afternoon biting of Oc. triseriatus with activity lasting into the crepuscular period. In contrast, Clark et al.

(1985) did not observe a marked increase or decrease in Oc. triseriatus biting activity during the daylight hours in the field. In natural habitats, host seeking Oc. triseriatus are found at ground level during all daylight hours, but at lower frequencies during early morning and late afternoon hours (Scholl et al. 1979).

Ochlerotatus triseriatus blood feed primarily on mammals, as summarized in Table

1.1. Ritchie and Rowley ( 1981) found Oc. triseriatus fed exclusively on mammalian hosts collected from urban sites throughout the state of Iowa. In northern Indiana, an area with low

La Crosse encephalitis virus (LAC) activity, Oc. triseriatus fed predominantly on chipmunks and deer (Nasci 1982). The majority of blood meal events were from chipmunks in the urban 6 locations, and from deer in suburban and rural areas. Blood meal hosts of Oc. triseriatus 'in a

LAC enzootic and LAC nonenzootic area were predominately tree squirrels and eastern chipmunks respectively (Nasci 1985). To a lesser degree Oc. triseriatus in these two areas also blood fed on human, rabbit, white tail deer, cats, and avian hosts. Within a Wisconsin forest endemic for LAC, Oc. triseriatus fed on deer (65%), grey squirrels (16%), chipmunks

(8%), and a small percentage on humans and rabbits (Burkot and DeFoliart 1982).

Ochlerotatus triseriatus collected in North Carolina blood fed predominantly on reptiles; with 75% feeding on turtles (Irby and Apperson 1988). Twenty-two percent fed on mammals, including deer, rabbit, and dog/fox hosts. Few blood meals originated from birds

(3%). Ochlerotatus triseriatus collected following the introduction of West Nile virus fed on mammals and birds in New Jersey, and in New York City they fed on deer (89%), equines, and humans (Apperson et al. 2004).

Fecundity

Many factors are involved in the quality and quantity of eggs produced by female mosquitoes, including adult size, age, insemination, volume of blood meal, and blood source. > Mather and DeFoliart (1983) measured the effect of the blood meal source on the reproductive capacity of Oc. triseriatus. Laboratory reared females were monitored for two gonotrophic cycles, with an individual mosquito feeding on the same host on each event.

Membrane feeders were presented to mosquitoes containing the blood from white tail deer, suckling mice, grey squirrel, human, and chipmunk hosts. The highest mean number of eggs laid were from mosquitoes that blood fed on chipmunk blood, with squirrels the second highest. These small mammals are often the preferred hosts of Oc. triseriatus in wooded areas, as previously described. Ochlerotatus triseriatus also feed on deer. Mosquitoes engorged on deer blood, required the shortest time (four fewer days) for the completion of Table 1.1 Ochlerotatus triseriatus blood meal identification

Blood meal source

Mosq. Mammalian Avian Reptilian

origins Raccoon Unknown Cat Chipmunk Deer Dog/Fox Horse Human Rabbit /Skunk Squirrel mammal Turtle (Statet

IA1 + + + + +

IL & IN4 + + + + + + + INJ + +

NC5 + + + 3% 75%

Nf 50% 50%

NY6 89% + +

WI2 8% 65% + + 16%

aAdapted from: 1Ritchie and Rowley (1981), 2Burkot et al. (1982), 3Nasci (1982), 4Nasci (1985), 5Irby et al. (1988), and 6Apperson et al. (2004).

-.J 8 two gonotrophic cycles, and the shortest time (1.7 and 2.0 days) needed to oviposit during the first and second gonotrophic cycles respectively.

Surveillance and Control

Some aspects of the Oc. triseriatus life cycle exclude it from many surveillance and control measures. Mosquito populations are generally monitored by attracting mosquitoes with light to traps that are operated at night. This strategy collects Culex, Aedes, and

Ochlerotatus mosquitoes, but is not effective in collecting diurnal mosquitoes such as Oc. triseriatus. Carbon-dioxide baited traps can be used to a limited extent to capture host seeking females. However, male mosquitoes are not targeted with these traps, and low collections are expected among mosquito species that occur in low adult populations such as

Oc. triseriatus. Identification and control of larval developmental sites in some containers, i.e. tree holes, may be impossible in remote localities. Ochlerotatus triseriatus have long been a problem/concern for public health workers due to difficulties in surveillance and control of the species (Craig 1983).

History of West Nile virus

West Nile virus (WNV) is a positive, single-stranded RNA virus in the family

Flaviviridae, and genus Flavivirus. It is a member of the Japanese encephalitis complex, which also includes St. Louis encephalitis virus (SLE), Murray Valley encephalitis virus

(MVE), and Kunjin virus (KUN). It was originally isolated from a febrile woman in the

West Nile district of Uganda in 1937 (Smithburn et al. 1940).

Outbreaks of WNV have occurred in several European countries, Africa, the Middle

East, and India. West Nile virus was not considered a public health problem, even with epidemics in France (1962) and South Africa (1974) (Dauphin et al. 2004). Since 1996, 9

WNV became a major public health concern with outbreaks in Italy (1998), France (2000,

2003 ), Romania ( 1996), Russia ( 1999), Algeria ( 1994 ), Morocco ( 1999, 2003 ), Tunisia

(1997), Israel (1998-2000), and the United States (1999-2004) (Dauphin et al. 2004).

In late August 1999, an outbreak of arboviral encephalitis occurred in New York City

(CDC 1999). Initial serological tests reported St. Louis encephalitis virus (SLE) activity in the area, but viral-specific tests of human, avian, and mosquito samples confirmed that a

West Nile-like virus was responsible for the outbreak. In the New York City area local health officials documented increased bird fatalities, especially crows, which is not typical of previous SLE or WNV epidemics. High mortality rates in crows and other birds have occurred in WNV viral introduction to a naive bird population (CDC 1999).

The mechanism of WNV introduction to the United States is not known, nor is the period of time the virus was present in the United States before detection (Lanciotti 1999).

Migratory birds have long been suspected as the primary introduction hosts of WNV into new regions in Europe, Asia, Africa, and the Middle East (Rappole et al. 2000). Other possibilities for WNV introduction also include international travel of infected humans, and the importation of infected birds or other wildlife (CDC 1999).

During the WNV epidemic (1999-2004), surveillance programs in the United States reported isolation of WNV from 60 species of mosquitoes representing 11 genera (Table 1.2

(CDC 2005a)). The virus has been associated with avian mortalities in 284 native and exotic bird species (CDC 2005b).

The West Nile virus strain isolated in New York (WN-NY99) is closely related to a

West Nile virus isolate from a dead goose in Israel in 1998 (Lanciotti 1999). Based on the E glycoprotein ofWNV, Lanciotti (1999) assembled a phylogenetic tree of two lineages.

Lineage I contains WN-NY99 and West Nile strains recently isolated from North Africa,

Romania, Kenya, Italy, and the Middle East. Lineage II contains West Nile strains found 10 exclusively on the African continent that are maintained in enzootic cycles without apparent human or equine outbreaks. Investigation of the genetic variances in West Nile virus strains has potentially identified differences in epidemic and enzootic viral strains. Studies of viral mutational change add to the understanding of viral evolution. Genetic recombination occurs in all three genera of Flaviviridae, but has not been observed in West Nile virus, or Yellow fever virus (Twiddy and Holmes 2003).

The WNV transmission cycle involves mosquitoes in the genus Culex, and avian species as the amplification hosts. Mosquitoes that feed on both birds and mammals are known as bridge vectors, which transmit WNV from avian amplification hosts to other vertebrates susceptible to WNV infection. These secondary hosts do not develop viremias high enough to continue the infection cycle, and are known as dead end hosts.

Twenty-five North American bird species are known to become infected with WNV infection. Species in the orders Passeriformes and Charadriiformes on average develop the highest magnitude and longest duration of WNV viremias (Komar et al. 2003). The top five reservoir competent North American birds are passerines: blue jay, common grackle, house finch, American crow, and house sparrow. Characterizations of WNV infection in other susceptible vertebrates have yet to be published.

Laboratory studies of North American mosquitoes have evaluated vector potential by determining infection, dissemination, and transmission rates which are summarized in Table

1.3. Mosquito species are categorized in three groups: Relatively refractory to WNV infection (Ae. vexans, Ae. aegypti, and Ae. taeniorhynchus); moderately susceptible (Cx. p. pipiens and Cx. sollicitans); highly susceptible (Ae. albopictus, Ae. atropalpus, and Ae. japonicus) (Turell et al. 2001a). These categories are supported by Sardelis et al. (2001), who include breeding habitats to separate the Aedes and Ochlerotatus mosquitoes into laboratory vector competence categories: Inefficient vectors - floodwater-breeding Aedes 11 and Ochlerotatus species; Moderately efficient vectors - Culex species; Highly efficient - container-breeding Aedes and Ochlerotatus species. Laboratory studies of several North

American mosquito species have yet to discover a mosquito refractive to West Nile virus infection.

Vector competence

There are a number of biological and ecological elements necessary for a mosquito species to be an efficient viral vector. These include: (1) viral replication within the mosquito midgut; (2) dissemination of the virus from the midgut and infection of other organs (i.e. salivary glands and reproduction organs); (3) transmission of infectious virus in subsequent blood feedings, or vertically transmitted to progeny; ( 4) mosquito has a natural relationship with a vertebrate viral reservoir; and (5) field evidence of mosquito infection.

Midgut Infection

Upon ingestion of a blood meal, mosquito midgut epithelial cells excrete digestive enzymes. The insect peritrophic membrane (PM), or peritrophic matrix, is formed of chitin and protein excreted by midgut epithelial cells. The porous PM compartmentalizes the blood bolus within the midgut, aiding in nutrient absorption by epithelial cells. The PM may also provide the first protective barrier of infection to ingested pathogens such as bacteria, microfilaria, malarial zygotes, or arboviruses. The average pore size of the mosquito PM is

20-30nm, which is small enough to prohibit arboviral passage (Hardy et al. 1983). However, the time of PM formation varies among mosquito species. Ochlerotatus triseriatus forms an initial peritrophic membrane structure 20-50 min after blood meal ingestion (Richardson and

Romoser 1972). Arboviral infection of the midgut epithelial cells is therefore possible between the time of ingestion and PM formation. 12

Table 1.2 West Nile virus isolations from field-collected mosquitoes

Genus Species Aedes Aedes aegypti Aedes cinereus Aedes albopictus Aedes vexans

Anopheles Anopheles atropos Anopheles hermsi Anopheles barberi Anopheles punctipennis Anopheles crucians/bradleyi Anopheles quadrimaculatus Anopheles franciscanus Anopheles walkeri Anopheles freeborni

Coquillettidia Coquilettidia perturbans

Cul ex Culex coronator Culex restuans Culex erraticus Culex salinarius Culex erythrothorax Culex stigmatosoma Culex nigripalpus Culex tarsalis Culex p . pipiens Culex territans Culex p . quinquefasciatus Culex thriambus

Culiseta Culiseta impatiens Culiseta melanura Culiseta inornata Culiseta morsitans

Deinocerites Deinocerites cancer

Mansonia Mansonia tittilans

Ochlerotatus Ochlerotatus atlanticus/tormentor Ochlerotatus japonicus Ochlerotatus atropalpus Ochlerotatus melanimon Ochlerotatus canadensis Ochlerotatus nigromaculis Ochlerotatus cantator Ochlerotatus provocans Ochlerotatus condolescens Ochlerotatus sollicitans Ochlerotatus dorsalis Ochlerotatus squamiger Ochlerotatus dupreei Ochlerotatus sticticus Ochlerotatus fitchii Ochlerotatus stimulans Ochlerotatus fulvus pallens Ochlerotatus taeniorhynchus Ochlerotatus grossbecki Ochlerotatus triseriatus Ochlerotatus infirmatus Ochlerotatus trivittatus

Orthopodomyia Orthopodomyia signifera

Psorophora Psorophora ciliata Psorophora ferox Psorophora columbiae Psorophora howardii

Uranotaenia Uranotaenia sapphirina 13

Initial West Nile virus replication occurs in the singular layer of epithelial cells within the mosquito midgut. The columnar midgut epithelial cells border neighboring epithelial cells on two sides, have a microvillar membrane towards the midgut, and are separated from the body cavity by the basal lamina. The microvillar membrane provides increased surface area for nutrient absorption, and also contains cellular receptors that facilitate virion entry.

Girard et al. (2004) demonstrated cell-to-cell spread of WNV within midgut epithelial cells of Culex. p. quinquefasciatus. Replicated virus must next exit the midgut epithelial cells to subsequently infect other tissues, including the salivary glands and reproductive organs.

Dissemination Barrier

Ingested blood first passes through the mosquito foregut. Insect foregut and hindgut cells are covered with a chitinous barrier that is impermeable to water. This barrier protects cells from arboviral infection due to the absence of cellular membrane receptors. A cardiac sphincter muscle divides the foregut and midgut sections. The intussuscepted foregut consists of foregut cells that line the sphincter junction and are present in the anterior midgut.

The foregut/midgut region is a possible location for arbovirus dissemination in the mosquito.

Rift Valley fever virus (RVF) was detected in foregut tissues before it was present in the hemolymph in 85% of Culexp. pipiens examined (Romoser et al. 1987). Recently, another possible arboviral dissemination conduit involving tracheae and visceral muscles has been described. The midgut basal lamina is penetrated by tracheae and potentially connects infected midgut epithelial cells with the rest of the tracheal system. Ochlerotatus taeniorhynchus midgut tracheal cells became infected with Venezuelan equine encephalitis virus (VEE) after infection of tracheae and muscle tissues in intrathoracically inoculated mosquitoes (Romoser et al. 2004).

Dengue 3 virus (DEN-3) infection is primarily detected in intussuscepted foregut, salivary glands, and nervous tissue in Aedes aegypti (Linthicum et al. 1996). Nervous tissue 14 is the principal site of amplification of DEN-3 in the mosquito, but was not detected in muscles, tracheae, malphigian tubules, and the posterior midgut. Tissue tropisms of West

Nile virus in Cx. p. quinquefasciatus were the midgut, salivary glands, nervous system, and fat body (Girard et al. 2004). The primary site of amplification for WNV is fat body within the abdomin, thorax, and head of Cx. p. quinquefasciatus. Dissemination of WNV in Cx. p. quinquefasciatus escapes the posterior midgut and following this it appears in fat body; anterior midgut and muscles; abdominal ganglia and salivary glands, in that order. Culex p. quinquefasciatus tracheal cells were not replication sites for WNV (Girard et al. 2004).

Transmission Barrier

Viral transmission depends on the ability of infected mosquitoes to either infect vertebrate hosts through blood feeding, or vertically transmit the virus to their offspring. The extrinsic incubation (EI) period is the time between infection of the vector (viremic blood meal) and its ability to transmit the virus. The EI time has epidemiological importance considering the relatively short mosquito lifespan. The minimum extrinsic incubation of

Culex tritaeniorhynchus with West Nile virus was seven days at 28°C (Hayes et al. 1980), with 100% of mosquitoes capable of transmission 12 days post infection. Salivary glands of

Culex p. quinquefasciatus were infected with WNV eight days following oral infection

(Girard et al. 2004). Environmental temperatures have a direct effect on arboviral vector competency. Culex p. pipiens held at temperatures ranging from 18 to 30°C following a

WNV viremic blood meal have markedly different dissemination rates (Dohm et al. 2002b ).

Low incubation temperatures resulted in decreased dissemination rates compared to higher temperatures >90%, WNV dissemination. 15

Table 1.3 Vector competence of mosquitoes orally infected with West Nile virus and tested 12-14 days after exposure WNV No. Infect. Dissem. Trans. Est. trans. Speciesa sourceb tested ratec rated ratee rater Ae. aegypti5 7.2 ± 0.3 19 16 16 2:16 Ae. albopictus4 4.5 21 0 0 0 0 Ae. albopictus4 5.9 31 6.5 6.5 0 0 Ae. albopictus5 7.2 ± 0.3 61 90 85 73 Ae. albopictus4 7.5 30 93.3 86.7 60 52 Ae. albopictus4 8.7 10 90 90 90 81 Ae. atropalpus5 7.2 ± 0.3 12 92 92 92 Ae. vexans5 5.2 ± 0.2 3 0 0 0 Ae. vexans3 7.1±0.1 22 32 23 Ae. vexans5 7.2 ± 0.3 13 46 8 8 Coquillettidea perturbans2 6.6 ± 0.3 11 18 9 2 Culiseta inornata3 7.1±0.1 28 75 21 Culiseta melanura6 7.1±0.4 19 26 11 0 n.a. Cx. erythrothorax3 4.9 ± 0.1 20 65 30 Cx. erythrothorax3 7.1±0.1 25 100 64 Cx. nigripalpus2 4.6 7 29 0 0 Cx. nigripalpus2 5.7 ± 0.5 132 78 8 7 Cx. nigripalpus2 6.8 ± 0.4 127 84 12 10 Cx. p. pipiens4 4.0 25 4 0 0 0 Cx. p. pipiens3 4.9 ± 0.1 35 23 60 Cx. p. pipiens5 5.2 ± 0.2 46 17 2 2 Cx. p. pipiens4 5.9 13 76.9 30.8 7.7 23.7 Cx. p. pipiens1 6.0 ± 0.5 17 82 23 20 Cx. p. pipiens1 7.0 ± 0.4 78 79 24 21 aAdapted from: 1Sardalis and Turell (2001 ), 2Sardalis et al. (2001 ), 3Goddard et al. (2002), 4Tiawsirisup (2003), 5Turell et al. (2001 b), and 6Turell et al. (2005). bTiter expressed as PFU ± MSE /ml cvirus detected in mosquito body dVirus detected in legs eVirus detected in saliva by in vitro or in vivo assay rEstimated transmission rate = the percentage of mosquitoes with a disseminated infection multiplied by the transmission rate. 16

Table 1.3 (continued)

WNV No. Infect. Dissem. Trans. Est. trans. Speciesa sourceb tested ratec rated ratee rater Cx. p. pipiens3 7.1±0.1 31 IOO 71 Cx. p. pipiens5 7.2 ± 0.3 95 81 23 20 Cx. p. pipiens4 7.5 24 100 70.8 54.2 38.4 Cx. p. pipiens4 8.7 19 100 79 47.4 37.4 Cx. p. quinquefasciatus3 4.9 ± 0.1 55 0 0 Cx. p. quinquefasciatus3 4.9 ± 0.1 50 IO 0 Cx. p. quinquefasciatus2 5.0 13 46 0 0 Cx. p. quinquefasciatus2 5.5 16 50 6 6 Cx. p. quinquefasciatus2 6.3 17 94 12 ~ 13 Cx. p. quinquefasciatus2 7.0 ± 0.5 78 91 22 20 Cx. p. quinquefasciatus3 7.1±0.1 50 58 52 Cx. p. quinquefasciatus3 7.1±0.1 58 28 19 Cx. p. quinquefasciatus3 7.1±0.1 50 66 36 Cx. restuans2 6.6 ± 0.3 11 100 55 55 Cx. salinarius2 6.6 ± 0.3 20 95 60 34 Cx. stigmatosoma3 4.9 ± 0.1 29 69 34 Cx. stigmatosoma3 7.1±0.l 48 77 19 Cx. tarsalis3 4.9 ± 0.1 11 36 82 Cx. tarsalis3 4.9 ± 0.1 45 7 0 Cx. tarsalis3 7.1±0.1 35 74 60 Cx. tarsalis3 7.1±0.1 55 85 62 Cx. tarsalis3 4.9 ± 0.1 IO 0 0 Oc. canadensis6 6.3 ± 0.3 24 13 0 n.a. n.a. Oc. canadensis6 7.1±0.4 73 44 19 11 18 aAdapted from: 1Sardalis and Turell (2001), 2Sardalis et al. (2001), 3Goddard et al. (2002), 4Tiawsirisup (2003), 5Turell et al. (2001 b) , and 6Turell et al. (2005). bTiter expressed as PFU ± MSE /ml cvirus detected in mosquito body dVirus detected in legs evirus detected in saliva by in vitro or in vivo assay rEstimated transmission rate = the percentage of mosquitoes with a disseminated infection multiplied by the transmission rate. 17

Table 1.3 (continued)

WNV No. Infect. Dissem. Trans. Est. trans. Speciesa sourceb tested ratec rated ratee rater Oc. canator6 6.3 ± 0.3 51 22 18 0 0 Oc. canator6 7.1±0.4 8 50 13 100 11 Oc. dorsalis3 4.9 ± 0.1 25 4 4 Oc. dorsalis3 7.1±0.1 29 41 34 Oc. j. japonicus1 6.0 ± 0.5 92 57 56 54 Oc. j. japonicus5 7.2 ± 0.3 36 69 64 64 Oc. j. japonicus1 7.0 ± 0.4 83 80 77 75 Oc. melanimon3 4.9 ± 0.1 60 3 2 Oc. melanimon3 7.1 ± 0.1 60 48 20 Oc. taeniorhynchus5 5.2 ± 0.2 45 2 0 0 Oc. taeniorhynchus5 7.2 ± 0.3 75 12 3 3 Oc. sierrensis3 4.9 ± 0.1 30 0 0 Oc. sierrensis3 7.1±0.1 50 14 6 Oc. sollicitans5 5.2 ± 0.2 9 11 11 7 Oc. sollicitans5 7.2 ± 0.3 50 70 16 11 Oc. triseriatus6 7.1±0.4 29 31 17 12 10 Oc. trivittatus4 4.0 16 12.5 0 0 0 Oc. trivittatus4 5.9 26 61.5 42.3 15.4 6.5 Oc. trivittatus4 7.5 29 96.6 86.2 51.7 44.6 Oc. trivittatus4 8.7 10 100 100 40 40 Psorophoraferox6 6.3 ± 0.3 17 29 12 0 13 Psorophoraferox6 7.1±0.4 24 33 0 0 0 aAdapted from: 1Sardalis and Turell (2001), 2Sardalis et al. (2001), 3Goddard et al. (2002), 4Tiawsirisup (2003), 5Turell et al. (2001 b), and 6Turell et al. (2005). bTiter expressed as PFU ± MSE /ml cvirus detected in mosquito body dVirus detected in legs evirus detected in saliva by in vitro or in vivo assay fEstimated transmission rate = the percentage of mosquitoes with a disseminated infection multiplied by the transmission rate. 18

Vertical transmission of WNV has been detected in the offspring of infected Cx. p. pipiens1'2, Ae. albopictus2·3, Ae. aegypti3, Cx. tritaeniorhynchus3, Cx. tarsalis4, and Culexp . quinquefasciatus4 (1Turell et al. 2001 b, 2Dohm et al. 2002a, 3Baqar et al. 1993, 4Goddard et al. 2003). The minimal filial infection rates (the number infected per 1,000 progeny) ranged from <0.1 to 6.9 in Ae. albopictus and Cx. tarsalis respectively. Few studies have documented the incidence of vertical transmission of arboviruses in nature. West Nile virus was detected in wild caught male Cx. univittatus in Kenya, which is the primary amplification vector in the area. (Miller et al. 2000).

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CHAPTER 2. INFECTION AND TRANSMISSION OF WEST NILE VIRUS BY OCHLEROTATUS TRISERIATUS (DIPTERA: CULICIDAE) IN THE LABO RATORY

A paper to be submitted to the Journal of Vector-Borne and Zoonotic Diseases

SARA M. ERICKSON', KENNETH B. PLATT2, WAYNE A. ROWLEY1,2,

AND BRAD J. TUCKER'

1Department of Entomology, College of Agriculture,

Iowa State University, Ames, IA 50011

2Department of Veterinary Microbiology and Preventative Medicine,

College of Veterinary Science, Iowa State University, Ames, IA 50011

ABSTRACT The susceptibility of Ochlerotatus triseriatus (Say) to West Nile virus (WNV) was studied by comparing it to the susceptibility of Culex p. pipiens (L.) a known amplification species. Dissemination and transmission rates were also determined for Oc. triseriatus orally infected with WNV to assess vector competency. Ochlerotatus triseriatus, the primary vector of La Crosse encephalitis virus, fed readily on 2-4 day-old chickens with WNV serum titers ranging from 104·0 to 108·2 PFU/ml. The lowest infective titer for Oc. triseriatus and

Cx. p. pipiens was 104.3 and <104·0 PFU/ml respectively. Infection rates varied from 8 to

98% of Oc. triseriatus and 25 to 100% of Cx. p. pipiens that fed on chickens with viremias of

10~4 · 0 < 4 .s and 10~ 8 · 0 < 8 · 5 PFU/ml, respectively. The predicted infection dose 50 (ID50) for Oc. 29

triseriatus was 106·5 PFU/ml. Culex p. pipiens is considerably more susceptible to WNV infection than Oc. triseriatus suggested by statistically different infection rates and by a significantly lower ID50 of 104·6 PFU/ml. Ochlerotatus triseriatus orally infected by viremic chickens with titers > 108 0 PFU/ml had infection, dissemination, and transmission rates of 94,

91, and 60%. Although Oc. triseriatus may be less susceptible than a primary amplification vector, it has the potential to be a bridge vector of WNV and a possible maintenance vector among susceptible mammals.

Key words: West Nile virus - Vector competence - Ochlerotatus triseriatus - Culex p. pipiens.

INTRODUCTION

West Nile virus (WNV) was isolated in the West Nile district of Uganda in 1937

(Smithbum et al. 1940). This single-stranded, positive sense RNA virus (Flaviviridae:

Flavivirus) was involved in sporadic outbreaks, and a few epidemics until the mid 1990' s.

Increased West Nile virus human incidence worldwide, including recent establishment in

North America (CDC 1999, O'Leary 2004), has elevated WNV to a level of public health concern (Dauphin et al. 2004). Predictions of WNV activity in North America is nearly impossible due to incomplete knowledge of the complex ecology of the virus (Petersen and

Roehrig 2001).

The epidemic ofWNV (1999-2004) in North America involved most of the contiguous United States of.America, and some areas in Canada, Mexico, the Caribbean, and parts of South America. Surveillance programs detected WNV in 60 wild mosquito species in 11 genera (CDC 2005a), and avian mortality due to WNV in 284 North American bird species (CDC 2005b). West Nile virus viremias in 25 North American bird species, from 11 30 orders, have been characterized (Komar et al. 2003), but little is known about potential mammalian reservoirs.

Several studies have evaluated the vector competency of North American mosquitoes for West Nile virus (Sardalis and Turell 2001, Sardalis et al. 2001, Goddard et al. 2002,

Turell et al. 2001, Turell et al. 2005), but there have been few investigations at infection titers

<105·0 PFU/ml (Tiawsirisup et al. 2004). Container-breeding Ochlerotatus and Aedes mosquitoes are competent laboratory vectors of WNV (Sardalis et al. 2001), and several are potential bridge vectors in WNV transmission (Turell et al. 2005).

Ochlerotatus triseriatus (Say), commonly known as the Eastern treehole mosquito, is distributed throughout the eastern half of the United States (Craig 1983). It is the principle vector of La Crosse encephalitis virus (LAC), and blood feeds primarily on mammals (i.e. chipmunks, squirrels, deer, and rabbits) including humans (Burket et al. 1982, Nasci 1982 and 1985), Apperson et al. 2004). West Nile virus was identified in field collected Oc. triseriatus from 2000-2004 (CDC 2005a).

This study compared the WNV susceptibility of a potential bridge vector

Ochlerotatus triseriatus (Say) to that of Culex p. pipiens (L.), an amplifying vector.

Mosquitoes were orally exposed to WNV infection at ranges of virus titers that occur in viremic vertebrate hosts. Regression modeling was used to predict infection rates at low, medium, and high titers. The ability of Oc. triseriatus to transmit WNV by bite to susceptible vertebrate hosts was also evaluated.

MATERIALS AND METHODS

All studies were conducted within Biosafety level 3 (BL3) facilities at Iowa State

University, in the Department of Veterinary Microbiology and Preventative Medicine, Ames,

Iowa. 31

Experimental design - infection

Female Ochlerotatus triseriatus and Culex p. pipiens were allowed to feed on 2- to 5- day-old chickens that had been inoculated 1 to 3 days earlier with West Nile virus. Infectious blood meal titers were determined by viral plaque assay of chicken sera collected promptly following mosquito feeding. Engorged mosquitoes were sorted from non-blood fed mosquitoes, and maintained for 14 days at 27 ± 1°C and 80 ± 5% RH. Surviving females were killed by freezing and tested for WNV on Vero cell monolayers. West Nile virus infection rates were determined for Oc. triseriatus and Cx. p . pipiens that fed on viremic chicken with different titers. Omnibus chi-square tests were used to determine the overall effect of chicken viremias on the infection rate of each species. Observed differences were considered significant at p <0.05, and as approaching statistical significance at 0.1

When statistically significant, post-hoc pairwise Fischer's exact tests were used to analyze differences in infection rates of mosquitoes that fed on a series of low to high WNV viremic chickens. Similarly, infection rates of Oc. triseriatus and Cx. p. pipiens were compared at specified WNV titer levels. JMP version 5.1 software was used for statistical analysis (SAS

Institute Inc., Cary, NC).

Experimental design - transmission

Ochlerotatus triseriatus females were blood fed on WNV-inoculated 2- to 5-day-old chickens, allowed to oviposit, and then individually introduced to a susceptible chicken for a second blood meal at 14 and 18 days post viral exposure. Mosquitoes were processed immediately following the second blood meal, with the legs removed from the body and triturated separately. Chicken sera were collected 36-48 hours post mosquito exposure, and stored at -70°C until viral assay. 32

Mosquitoes

Ochlerotatus triseriatus were collected in Scott County, Iowa in 2002, and colonized by the

Iowa State University (ISU) Medical Entomology Laboratory, Ames, Iowa. Culex p. pipiens originated from Ames, Iowa and were colonized in 2003 by the ISU Medical Entomology

Laboratory. Mosquitoes were maintained at 27 ± 1°C and 80% ± 5%. RH with a 16:8 photoperiod (light: dark). Adults were provided access to 0.3M sucrose ad libitum except 48 h before to 24 h post blood feeding. During this time a water pledget was provided.

Chickens

Biosafety level 3 (BL3) facilities were used to house 1- to 2-day-old chickens obtained from a commercial hatchery (Hoover's hatchery, Inc., Rudd, IA). These chickens were WNV antibody-free broilers (Ross x Ross).

Cells and Media

African green monkey kidney cells (Vero-76) were cultured for virus propagation and assay.

Cell culture growth media, maintenance media, and mosquito grinding media were previously described by Tiawsirisup et al. (2004).

Virus

West Nile virus strain IA-02 was isolated from a crow that died in Iowa in 2002. The virus has been passed once in Vero cells and two times in 2- to 3-day-old chickens.

Virus assay

Blood samples were taken from chickens following mosquito blood feeding, placed in a vacutainer (Becton Dickinson), and centrifuged to separate the blood serum. Sera were 33 stored at -70°C until assayed. West Nile virus titers are expressed as plaque forming units

(PFU)/ml.

Virus isolation

Mosquitoes were killed by freezing, and placed individually into a l .5ml microcentrifuge tube containing 300µ1 cold grinding media. Each sample was triturated with a sterilized grinding pestle, and stored at -70°C until assayed. Each 300µ1 sample was first extended to

2.0ml with cold maintenance media and passed through a 450nm filter directly into a 25cm2 cell culture flask containing a confluent Vero 76 cell monolayer. The flask was incubated for

1 hour after which 5 ml of maintenance media were added. Observations of cytopathic effect

(CPE) were made for up to 8 days. Reverse transcriptase-polymerase chain reaction (RT

PCR) was used to verify West Nile virus infection in 10% of the positive and negative CPE results.

Reverse transcription-polymerase chain reaction (RT-PCR)

RNA extraction was performed using QIAamp®Viral RNA Mini Kit (Qiagen,

Valencia, CA). Spin-column procedures were conducted according to product protocols for cell culture medium samples. Extracted RNA samples were stored at -70°C until amplification. Superscript™ One-Step RT-PCR with Platinum™ Taq (Invitrogen, Carlsbad,

CA) was used for amplification. WN233 and WN640c primers produced a 408 bp product

(Lanciotti et al. 2000), which was electrophoresed through a 0.8% agarose gel (Nusieve®,

FMC Bioproducts) prepared with IX Tris-Acetate-EDTA buffer (Fischer Scientific) containing 0.3mg ethidium bromide per 100 ml gel (Sigma-Aldrich Co.). 34

RESULTS

Infection

The incidence of blood feeding on 2- to 5-day-old chickens by Oc. triseriatus and Cx. p. pipiens is presented in Table 2.1. In five laboratory experiments, an average of 61.6 ±

6.6% and 22.7 ± 8.1 % of Oc. triseriatus and Cx. p. pipiens blood fed on chickens respectively. The mean number of mosquitoes that blood fed to repletion from chickens was statistically significant (t=-3. 73, p<0.003). Mosquitoes ranged in age (time post emergence) at time of blood feeding. Ochlerotatus triseriatus and Cx. p. pipiens were exposed to a WNV viremic chicken at 4-14 and 1-9 days post emergence respectively.

Ochlerotatus triseriatus and Cx. p. pipiens became infected with West Nile virus infection after feeding on viremic chickens. Mosquitoes ( Oc. triseriatus or Cx. p. pipiens), and the viral exposure level (viremic blood meal) had overall effects on WNV infection rates

(p<0.0001). Table 2.2 summarizes infection rates among viremic chicken blood meals starting at 104·0 PFU/ml and increased by 1o 0·5 PFU/ml increments.

Infection rates in Oc. triseriatus and Cx. p. pipiens were 8% and 25% respectively following a blood meal from chickens with WNV titers 10:::4.0<4·5 PFU/ml. The highest infection rate for Oc. triseriatus was 97% when fed on chickens with WNV titers 10:::8·0<8·5

PFU/ml, and 100% for Cx. p. pipiens orally infected at titers 10:::7.0<7·5 PFU/ml.

West Nile virus infection rates of Oc. triseriatus increased significantly as chicken serum titers increased from 10<:4-5<5.o PFU/ml to llf6·5<7.o PFU/ml. At titers ~ 10 6 · 5 PFU/ml

>50% of the Oc. triseriatus became infected with WNV. Culex p. pipiens that fed on chickens with WNV titers ~ 105 · 5 PFU/ml had infection rates >75%. Culex p. pipiens infection rates steadily increased from 77% to 100% when fed on chickens with titers

102:5.5<6 0 PFU/ml to 102:7.0<7.5 PFU/ml respectively, but the differences were not significant 35

(p>0.05). Infection rates of Oc. triseriatus and Cx. p. pipiens were significantly different within each titer range tested.

Chicken titers and 95% confidence intervals predicted to infect Oc. triseriatus and

Cx. p. pipiens at specific rates are presented in Table 2.3. The infection dose 50s (ID50) were

1065 and 104·9 PFU/ml for Oc. triseriatus and Cx. p. pipiens respectively. Coefficients used to construct the regression models are listed in Table 2.4. The area under the receiver operating characteristic (ROC) curves were 0.80 and 0.81 for the regression models, indicating a good fit between observed and predicted values.

Transmission

Infection, dissemination, and transmission rates of Oc. triseriatus orally infected at

WNV titer levels of 108·1-8·2 PFU/ml are presented in Table 2.5. There were no significant differences among mosquitoes studied on 14 or 18 days post exposure (PE). Ninety-four percent of mosquitoes tested were infected with WNV on days 14 and 18 PE. Dissemination and transmission rates were 91 and 60% respectively.

DISCUSSION

Ochlerotatus triseriatus and Cx. p. pipiens are active at different times of the day.

Ochlerotatus triseriatus are diurnal, and Cx. p. pipiens are crepuscular/night feeders (Turell et al. 2005). In this study blood feeding events were performed 1-4 hours after lights on, at which time both species were concurrently exposed to chickens. The observed difference in blood feeding percentages among Oc. triseriatus and Cx. p. pipiens on chickens may reflect the time of day feeding was permitted. Age of mosquitoes (days post emergence) also appears to have had an effect on the feeding rates of Cx. p. pipiens. One of the primary physiological needs of mosquitoes after emergence is the buildup of lipids in fat body 36

(Briegel 2003). Culex p. pipiens in experiment 5 were <2 days of age at the time of blood feeding, and may not have had time to sugar feed and develop sufficient fat body.

Ochlerotatus triseriatus and Cx. p. pipiens were exposed to WNV by feeding on viremic chickens with titers ranging from 104·0 PFU/ml to 108·2 PFU/ml, a range of titers representing WNV viremias in vertebrates. Each blood meal from viremic chickens was placed within a half-titer range to analyze infection trends in Oc. triseriatus and Cx. p. pipiens as the WNV exposure level changed. Culex p. pipiens had higher infection rates than

Oc. triseriatus at each titer level investigated. Therefore, Oc. triseriatus is less susceptible to

WNV infection than Cx. p. pipiens, a known primary WNV amplification vector.

Studies involving a range of viral exposures have identified infection, dissemination, and transmission barriers to arboviruses (Hardy et al. 1983). The WNV infection rate in Oc. triseriatus that fed on viremic chickens with titers ~10 8 · 0 PFU/ml was significantly higher than those that had a viremic blood meal with a titer <108·0 PFU/ml. This difference suggests that there might be a midgut infection barrier in Oc. triseriatus that was surpassed when mosquitoes fed at infection levels ~10 8 · 0 PFU/ml. Additional studies at lower WNV titers are necessary to evaluate the presence of a midgut infection barrier.

Ochlerotatus triseriatus that blood fed from chickens with WNV serum titers > 108·0

PFU/ml transmitted WNV by bite to susceptible vertebrate hosts. The estimated transmission rate is 55% at this infection level. The possibility of Oc. triseriatus feeding on vertebrate hosts with WNV viremic titers > 108 0 PFU/ml is not known. This study establishes that Oc. triseriatus is a competent WNV vector.

In this study WNV viremic blood meal titers of 106·5 (6.4-6·7) and 104·9 C4·6-5·11 ) PFU/ml are the titers and 95% CI predicted to infect 50% of Oc. triseriatus and Cx. p. pipiens respectively (Table 2.3). Previously, a principle WNV viremic titer ~10 5 · 0 PFU/ml was used as a guideline to define avian reservoir potential (Komar et al. 2003). This study indicates a 37 greater susceptibility of Cx. p. pipiens to WNV than previously described. At blood meal titers of 1032 <25-3·6) and 104·2 <3·8-4·5) PFU/ml, ten and thirty percent of Cx. p. pipiens become infected with WNV. Similarly, at titers of 104·7 <4·34-9) and 105·8 <5·6-6·0) PFU/ml, ten and thirty percent of Oc. triseriatus become infected with WNV, respectively.

Unpublished studies in our laboratory demonstrated WNV viremias in cottontail rabbits (Sylvilagus floridanus) that exceed the predicted ranges to infect Oc. triseriatus, and

Cx. p. pipiens. Additional studies have demonstrated Oc. triseriatus infection from blood feeding on WNV viremic rabbits, and subsequent WNV transmission to susceptible vertebrate hosts by bite. Investigations of WNV infection in potential mammalian reservoirs are required to better define the role of Oc. triseriatus in the transmission of WNV in the western hemisphere.

ACKNOWLEDGEMENTS

The research reported in this publication was supported by the Centers for Disease

Control and Prevention (CDC) Cooperative Agreement for Applied Research in Emerging

Infections; Investigations of West Nile virus. The contents are the sole responsibility of the authors and do not necessarily represent the official views of the CDC. This study represents a portion of a thesis submitted by the senior author to the Graduate College of Iowa State

University in partial fulfillment of the requirements for the M.S. degree. · 38

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Table 2.1. Blood feeding success3 of Ochlerotatus triseriatus and Culex p. pipiens on 2-5- day-old chickens

Ochlerotatus triseriatus Culex p. pipiens

Experiment Ageb % Blood fed Age % Blood fed

1 7-14 42 (84/200t 7-9 6 (11/200)

2 5-8 66 ( 186/280) 4-5 43 (104/240)

3 6-9 59 (210/357) 6-8 27 (25/94)

4 5-7 58 (238/410) 4-5 36 (140/393)

5 4-5 83 (242/293) 1-2 3 (8/300)

TOTAL (960/1540) (288/1227)

MEANd ± SEe 61.61 ± 6.6 22.72 ± 8.1 aBlood feeding events occurred 1-4 hours after lights on in the laboratory bAge of mosquito = days post emergence cPercent blood fed (No. engorged/No. exposed) dStatistically different percentages of Oc. triseriatus and Cx. p. pipiens blood fed on 2- to 5- day-old chickens in five experiments. (p<0.05) eSE= Standard Error 43

Table 2.2. Infection of Ochlerotatus triseriatus and Cu/exp. pipiens with West Nile virus

(IA-02) 14 days after blood feeding on viremic chickens

Ochlerotatus triseriatus Culex p. pipiens

Blood No. of No. of Infection No. of No. of Infection

meal titer infected mosq. rate infected mosq. rate

levela chickens tested (%) chickens tested (%)

4.0Sx<4.5c 6 127 85,b 5 63 253

4.5Sx<5.0c 5 85 115 3 42 452,3

5.5Sx<6.0c 6 108 234 2 13 7?1 ,2

6.0Sx<6.5c 6 113 423 4 62 841

6.5Sx<7.0c 10 170 552 4 45 91 1

7.0Sx<7.5c 3 43 652 1 11 1001

8.0Sx<8.5 4 48 981 NDd ND ND

aBlood meal virus titer expressed as log10 PFU/ml serum. Each infectious blood meal was

placed into titer ranges of 10°5 increments. blnfection rates within species that do not share the same superscript are significantly

different (p<0.05) clnfection rates within a blood meal titer range are significantly different between mosquito

species. (p<0.05) dND= Not Determined 44

Table 2.3. Blood meal titers of West Nile virus predicted by regression models to infect

Ochlerotatus triseriatus and Culex p. pipiens at specific rates

Predicted virus titer

log 1o PFU/ml serum (95% confidence interval)

Infection rate (%)b Ochlerotatus triseriatus Cu/exp. pipiens

10 4.66 (4.30-4.93) 3.19 (2.48-3.64)

20 5.34 (5.09-5.53) 3.81 (3.29-4.15)

30 5.79 (5.61-5.95) 4.22 (3.81-4.51)

50 6.50 (6.35-6.67) 4.87 ( 4.60-5.11)

80 7.66 (7.41-7.99) 5.93 (5.64-6.33)

90 8.34 (8.00-8.80) 6.54 (6.17-7.12) astatistically different virus titer is predicted to infect Oc. triseriatus and Cx. p. pipiens at all rates of infection. 45

Table 2.4. Estimated coefficients used to construct logistic regression models and areas under the receiver operating characteristic curves

Model Area under ROC curved

Ochlerotatus triseriatus -7.76 ± 0.65 1.19±0.10 0.80

Culex p. pipiens -6.38 ± 0.89 1.31 ± 0.17 0.81 a130 =intercept bl31 =slope cSE =standard error ct Area under the receiver operating characteristic (ROC) curve indicates the goodness of fit

between observed and predicted values. Values approaching 1 indicate a high degree of

fit. 46

Table 2.5. West Nile virus (IA-02) transmission to 2-4-day-old chickens by the bite of infected3 Ochlerotatus triseriatus.

Mosquito Disseminated Transmission

infectionb infectionc rate ct

14 days PEe 100 (16/16)f 94 (15/16) 50 (6/12)

18 days PE 88 (14/16) 88 (14116) 75 (6/8)

TOTAL 94 (30/32) 91 (29/32) 60 (12/20)

3Mosquitoes infected by blood feeding on WNV viremic chickens with titers between 1081-82

PFU/ml. blnfection rate =Number of mosquitoes with virus isolated from torso/ Number of

mosquitoes that blood fed on viremic chickens cDissemination rate =Number of mosquitoes with virus isolated from legs/ Number of

mosquitoes that blood fed on viremic chickens ctTransmission rate = Number of infected chickens/ Number of mosquitoes that blood fed on

individual chickens ePE = post exposure to West Nile virus fPercentage (No. detected/ No. tested) 47

CHAPTER 3. GENERAL CONCLUSIONS

Four main conclusions can be made in completion of this thesis project:

1. Culex p. pipiens and Ochlerotatus triseriatus become infected after feeding on 2- to

5-day-old chickens with WNV titers :Sl 045 PFU/ml.

2. Ochlerotatus triseriatus is a competent WNV laboratory vector.

3. Ochlerotatus triseriatus is less susceptible to WNV infection than Culex p. pipiens.

4. Ochlerotatus triseriatus primarily feeds on mammalian vertebrate hosts in the wild,

and is therefore a potential bridge vector for WNV.

The role of mammals in the transmission cycle of West Nile virus is largely unknown. To better understand the role of potential bridge vector mosquitoes, including Ochlerotatus triseriatus, studies characterizing WNV infection in mammalian species are required. 48

ACKNOWLEDGMENTS

I thank my committee members Drs. Wayne A. Rowley, Kenneth B. Platt, Russell A.

Jurenka, and Marlin E. Rice. It was my pleasure to work with you in the laboratory, and to be your student in the classroom. I am grateful for your support before, during, and in conclusion of my thesis project.

I am especially grateful for the guidance of Dr. Wayne Rowley. I admire your dedication to research and teaching. Thank you for introducing me to the field of medical entomology and providing numerous opportunities within it.

Working in a BL3 laboratory requires continuous attention to detail and in turn a tremendous amount of energy. No one knows this better than Dr. Ken Platt, Dr. Flor

Fabiosa, and Mr. Brad Tucker. Thank you for your assistance on this and other WNV projects.

Thanks Mom and Dad! Your support has helped me live my dreams. Whether it's a trip across the globe, or a busy week at school, you have always provided advice and love.