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The International Committee on Taxonomy of Viruses (ICTV) has implemented numerous changes to the taxonomic classification of bunyaviruses over the years. Whereas most changes have been justified and necessary because of the need to accommodate newly discovered and unclassified viruses, other changes are a cause of concern, especially the decision to demote scores of formerly recognized species to essentially strains of newly designated species. This practice was first described in the seventh taxonomy report of the ICTV and has continued in all subsequent reports. In some instances, viruses that share less than 75% nucleotide sequence identity across their genomes, produce vastly different clinical presentations, possess distinct vector and host associations, have different biosafety recommendations, and occur in nonoverlapping geographic regions are classified as strains of the same species. Complicating the matter is the fact that virus strains have been completely eliminated from ICTV reports; thus, critically important information on virus identities and their associated biological and epidemiological features cannot be readily related to the ICTV classification. Here, we summarize the current status of bunyavirus taxonomy and discuss the adverse consequences associated with the reclassification and resulting omission of numerous viruses of public health importance from ICTV reports. As members of the American Committee on Arthropod-borne Viruses, we encourage the ICTV Bunyavirus Study Group to reconsider their stance on bunyavirus taxonomy, to revise the criteria currently used for species demarcation, and to list additional strains of public and veterinary importance.

We determined abundance of Aedes aegypti mosquitoes and presence of dengue virus (DENV) in females collected from schools in Mérida, México, during 2008 and 2009. Backpack aspiration from 24 schools produced 468 females of Ae. aegypti and 1,676 females of another human biter, Culex quinquefasciatus. Ae. aegypti females were collected most commonly from classrooms followed by offices and bathrooms. Of these females, 24.7% were freshly fed. Examination of 118 pools of Ae. aegypti females (total of 415 females) for presence of DENV RNA produced 19 positive pools (16.1%). DENV-infected pools were detected from 11 (45.8%) of 24 schools and came from different room types, including classrooms, offices, and bathrooms. The overall rate of DENV infection per 100 Ae. aegypti females was 4.8. We conclude that schools in Mérida present a risk environment for students, teachers, and other personnel to be exposed to mosquitoes and bites of DENV-infected Ae. aegypti females.

We determined abundance of Aedes aegypti mosquitoes and presence of dengue virus (DENV) in females collected from premises of laboratory-confirmed dengue patients over a 12-month period (March 2007 to February 2008) in Merida, Mexico. Backpack aspiration from 880 premises produced 1,836 females and 1,292 males indoors (predominantly from bedrooms) and 102 females and 108 males from patios/backyards. The mean weekly indoor catch rate per home peaked at 7.8 females in late August. Outdoor abundances of larvae or pupae were not predictive of female abundance inside the home. DENV-infected Ae. aegypti females were recovered from 34 premises. Collection of DENV-infected females from homes of dengue patients up to 27 days after the onset of symptoms (median, 14 days) shows the usefulness of indoor insecticide application in homes of suspected dengue patients to prevent their homes from becoming sources for dispersal of DENV by persons visiting and being bitten by infected mosquitoes.

Assessments of spatial risk of exposure to vector-borne pathogens that combine vector and human disease data are needed for areas encompassing large tracts of public land with low population bases. We addressed this need for West Nile virus (WNV) disease in the northern Colorado Front Range by developing not only a spatial model for entomological risk of exposure to Culex tarsalis WNV vectors and an epidemiological risk map for WNV disease but also a novel risk-classification index combining data for these independently derived measures of entomological and epidemiological risk. Risk of vector exposure was high in the densely populated eastern plains portion of the Front Range but low in cooler montane areas to the west that are sparsely populated but used heavily for recreation in the summer. The entomological risk model performed well when applied to the western, mountainous part of Colorado and validated against epidemiologic data.

Surveillance for evidence of West Nile virus (WNV) infection in taxonomically diverse vertebrates was conducted in the Yucatan Peninsula of Mexico in 2003 and 2004. Sera from 144 horses on Cozumel Island, Quintana Roo State, 415 vertebrates (257 birds, 52 mammals, and 106 reptiles) belonging to 61 species from the Merida Zoo, Yucatan State, and 7 farmed crocodiles in Ciudad del Carmen, Campeche State were assayed for antibodies to flaviviruses. Ninety (62%) horses on Cozumel Island had epitope-blocking enzyme-linked immunosorbent assay (ELISA) antibodies to flaviviruses, of which 75 (52%) were seropositive for WNV by plaque reduction neutralization test (PRNT). Blocking ELISA antibodies to flaviviruses also were detected in 13 (3%) animals in the Merida Zoo, including 7 birds and 2 mammals (a jaguar and coyote) seropositive for WNV by PRNT. Six (86%) crocodiles in Campeche State had PRNT-confirmed WNV infections. All animals were healthy at the time of serum collections and none had a history of WNV-like illness.

La Crosse (LAC) virus (family Bunyaviridae, genus Orthobunyavirus) small (S) segment negative-sense RNA genome (vRNA), positive-sense full-length RNA complement (vcRNA), and subgenomic mRNA were assayed in infected cell cultures and female Aedes (Ochlerotatus) triseriatus mosquito tissues using quantitative PCR (Q-PCR). During persistent infection of C6/36 (Aedes albopictus) and MAT (Aedes triseriatus) cultured cells and cytolytic infection of BHK-21 cultured cells, LAC vRNA was the most abundant RNA species, followed by mRNA and vcRNA. RNA copy numbers per cell were quantified and vRNA correlated to virus titer in cell culture medium. The Q-PCR assay proved more sensitive than reverse transcription (RT)-PCR and immunofluorescence assays (IFA) for detecting LAC virus infection of mosquitoes. After infection of female mosquitoes orally, quantities of LAC RNA increased in ovaries for 6 days, and as ovarian biosynthetic activity quiesced, LAC RNA quantities decreased then remained detectable at a low level. After a second, noninfectious blood meal, quantities of LAC RNA in ovaries increased significantly, quantitatively confirming correlation of LAC virus RNA synthesis with vector metabolic activity. Coregulation of viral replication and mosquito ovary metabolic activity may condition efficient transovarial transmission.

A population genetics study of the mosquito Ochlerotatus triseriatus was performed on 36 collections from adjoining regions of Iowa, Minnesota, and Wisconsin covering approximately 120 km 2 . Single nucleotide polymorphism analysis was used to estimate variation in the mitochondrial NADH dehydrogenase subunit 4 (ND4) gene. The heated oligonucleotide ligation assay was used to identify the ND4 haplotype of each mosquito. No evidence of genetic isolation by distance was found, nor did Interstate 90 or the Mississippi River serve as barriers to gene flow. The effective migration rate varied from 18 to 45 reproductive migrants/generation, which is similar to estimates from an earlier study. The collections belong to a single, large, panmictic population. However, within this panmictic population, local genetic drift arises, possibly due to one or a few females ovipositing in larval breeding containers. From generation to generation, there is sufficient gene flow to mix families arising from individual breeding sites and eliminate founder effects due to drift.

Serosurveys were conducted to obtain flavivirus and West Nile virus (WNV) seroprevalence data from mammals. Sera from 513 small- and medium-sized mammals collected during late summer and fall 2003 from Colorado, Louisiana, New York, Ohio, and Pennsylvania were screened for flavivirus-specific antibodies. Sera samples containing antibody to flaviviruses were screened for WNV-specific antibodies by epitope-blocking enzyme-linked immunosorbent assays and confirmed with plaque reduction neutralization tests. Prevalence of WNV antibodies among study sites ranged from 0% to 42.8% among the mammal communities sampled. High prevalence rates for WNV were noted among raccoons (100%, with a very small sample size, N = 2), Virginia opossums (50.0%), fox squirrels (49.1%), and eastern gray squirrels (48.3%). The high WNV antibody prevalence noted for tree squirrels, the peri-domestic tendencies of several of these species, and their ease of observation could make these species useful sentinels for monitoring WNV activity within urban communities.

A dengue (DEN) outbreak occurred in the Yucatan State of Mexico in 2002. Three isolates were obtained from patients presenting with DEN-like symptoms, and examined by partial nucleotide sequencing and phylogenetic analysis. The isolates were identified as DEN-2 viruses of the American-Asian genotype; this is the first report of this genotype in the Yucatan State. The DEN-2 viruses of the American-Asian genotype have been associated with more severe disease outcomes. Thus, its introduction into the Yucatan State presents a serious problem to public health authorities. During this outbreak, DEN virus infection was confirmed in 18% (282 of 1,560) of the patients who presented with DEN-like symptoms. Of these, 87 (31%) patients met the World Health Organization criteria for dengue hemorrhagic fever, including two patients who died. The majority (77%) of the patients experienced secondary infections in this epidemic.

Aedes aegypti from 24 collections in Mexico and the United States were challenged orally with dengue 2 virus JAM1409 (DEN-2 JAM1409). The vector competence (VC) of the populations ranged from 24% to 83%. Mosquito populations from the Yucatan exhibited greater VC than those from other areas of Mexico. The presence or absence of a midgut infection barrier (MIB) and a midgut escape barrier (MEB) was determined for mosquitoes in each population. The percentage of mosquitoes exhibiting an MIB ranged from 14% to 59%, and those exhibiting an MEB ranged from 4% to 43% in the collections. The MIB and MEB were not completely independent as determined by regression analysis. Midgut infection rates were dose dependent.

A population genetic analysis was conducted among 20 Aedes aegypti collections from 19 cities along the south Pacific coast in the Mexican states of Guerrero, Oaxaca, and Chiapas and in Coatepeque, Guatemala. Genetic variation was scored at 131 random amplified polymorphic DNA loci. The amount of genetic differentiation among collections was approximately 3 times as great as detected among collections in an earlier study in northeastern Mexico. Regression analysis of linear or road distances on linearized F(ST) indicated that collections are genetically isolated by distance. Cluster analysis failed to group collections in geographic proximity, and there was as much genetic variation among collections 60 km apart as there was among all collections (approximately 900-km range). The large genetic differentiation in southern Mexico reflects reduced gene flow among mosquitoes arising in a greater diversity of habitats and altitudes than exists among northeastern collections. It is likely that dispersal via human commerce in the northeast confounds patterns of natural gene flow.

The mosquito Aedes aegypti is the major vector of the four serotypes of dengue virus (DENV1-4). Previous studies have shown that Ae. aegypti in Mexico have a high effective migration rate and that gene flow occurs among populations that are up to 150 km apart. Since 2000, pyrethroids have been widely used for suppression of Ae. aegypti in cities in Mexico. In Yucatan State in particular, pyrethroids have been applied in and around dengue case households creating an opportunity for local selection and evolution of resistance. Herein, we test for evidence of local adaptation by comparing patterns of variation among 27 Ae. aegypti collections at 13 single nucleotide polymorphisms (SNPs): two in the voltage-gated sodium channel gene para known to confer knockdown resistance, three in detoxification genes previously associated with pyrethroid resistance, and eight in putatively neutral loci. The SNPs in para varied greatly in frequency among collections, whereas SNPs at the remaining 11 loci showed little variation supporting previous evidence for extensive local gene flow. Among Ae. aegypti in Yucatan State, Mexico, local adaptation to pyrethroids appears to offset the homogenizing effects of gene flow.

A population genetic analysis of gene flow was conducted among 10 Aedes aegypti collections from seven cities along the northeastern coast of Mexico. Four collections were made from Monterrey to examine local patterns of gene flow. Markers included 60 random amplified polymorphic DNA (RAPD) loci amplified by the polymerase chain reaction and single strand conformation polymorphism analysis of variation in a 387-basepair region of the NADH dehydrogenase subunit 4 from the mitochondrial DNA (mtDNA). Seven mitochondrial haplotypes were detected and phylogenetic analysis identified two well-supported clades. Regression analysis of geographic distances and pairwise FST estimated from RAPD markers indicated that populations are isolated by distance and that free gene flow occurs among collections within 90-250 km. Isolation by distance was not detected using mtDNA haplotypes. The Nuevo Laredo collection had unique RAPD and mtDNA haplotype frequencies and reduced heterozygosity suggesting that few mosquitoes established this population.

Single-strand conformation polymorphism (SSCP) analysis was used to characterize genetic polymorphisms among 12 isolates of dengue-2 virus, which were previously genetically characterized by RNase T1 oligonucleotide mapping and by sequencing the viral envelope (E) gene. Specific cDNA fragments from the dengue-2 isolates were amplified by the reverse transcriptase-polymerase chain reaction. The viral E, premembrane (prM), and nonstructural 5 (NS5) gene cDNAs of 291 basepairs (bp), 291 bp, and 201 bp, respectively, were denatured, rapidly chilled to promote intrastrand reassociation, electrophoretically separated on nondenaturing polyacrylamide gels, and SSCP patterns were observed by silver staining. The SSCP analysis revealed polymorphisms among a number of dengue-2 isolates from the same topotype, and these were markedly different between isolates of different topotype (distinct genetic group). Comparison of nucleotide sequence and SSCP analyses of the 291-bp E cDNA demonstrated that virus isolates that produced identical SSCP patterns contained 0-7 nucleotide substitutions, whereas isolates that showed different SSCP patterns contained 4-25 nucleotide substitutions. Positive predictive value and negative predictive value as measures of certainty for predicting identical and different sequences were 26% and 100%, respectively. The SSCP patterns of the 12 dengue-2 isolates suggested greater genetic variation in the prM gene region than in either the E or NS5 gene regions. The SSCP analyses should allow easy, sensitive, and rapid screening of dengue viruses isolates and the assessment of variation at a number of sites in the virus genome. Additionally, SSCP screening of dengue-2 virus for genetic variability may reveal the introduction of new viral genotypes in a given geographic area. These genetic variants of the virus could serve as markers of the epidemic potential of the virus strain.

Low-passage field strains of snowshoe hare (SSH) virus (Bunyaviridae), the prototype SSH virus (originally isolated in Montana), and La Crosse (LAC) virus were compared serologically by plaque-reduction neutralization (PRNT) and molecularly by oligonucleotide fingerprinting (ONF). The PRNT and ONF results confirmed the identity of the field strains, although some differences in the fingerprints were observed. We have examined the RNA genome variability in the two field and three laboratory strains of SSH virus, using direct sequence analysis of selected RNase T1 oligonucleotides. Few changes were observed among three Montana prototype-derived laboratory isolates, although they have different passage histories. In contrast, the field isolates differed greatly from the laboratory strains. In addition, we have located several of the larger T1 oligonucleotides within the known sequence of the small and large RNA genome segments. We then compared the viruses for their ability to replicate in and be transmitted by Aedes triseriatus mosquitoes. The oral infection rates for LAC, the field isolates, and the SSH prototype, as determined by immunofluorescent examination of midgut tissues, were 100%, 82%, and 47%, respectively. All viruses were also transmissible from mosquitoes to mice.

We determined abundance of Aedes aegypti mosquitoes and presence of dengue virus (DENV) in females collected from premises of laboratory-confirmed dengue patients over a 12-month period (March 2007 to February 2008) in Merida, Mexico. Backpack aspiration from 880 premises produced 1,836 females and 1,292 males indoors (predominantly from bedrooms) and 102 females and 108 males from patios/backyards. The mean weekly indoor catch rate per home peaked at 7.8 females in late August. Outdoor abundances of larvae or pupae were not predictive of female abundance inside the home. DENV-infected Ae. aegypti females were recovered from 34 premises. Collection of DENV-infected females from homes of dengue patients up to 27 days after the onset of symptoms (median, 14 days) shows the usefulness of indoor insecticide application in homes of suspected dengue patients to prevent their homes from becoming sources for dispersal of DENV by persons visiting and being bitten by infected mosquitoes.

Assessments of spatial risk of exposure to vector-borne pathogens that combine vector and human disease data are needed for areas encompassing large tracts of public land with low population bases. We addressed this need for West Nile virus (WNV) disease in the northern Colorado Front Range by developing not only a spatial model for entomological risk of exposure to Culex tarsalis WNV vectors and an epidemiological risk map for WNV disease but also a novel risk-classification index combining data for these independently derived measures of entomological and epidemiological risk. Risk of vector exposure was high in the densely populated eastern plains portion of the Front Range but low in cooler montane areas to the west that are sparsely populated but used heavily for recreation in the summer. The entomological risk model performed well when applied to the western, mountainous part of Colorado and validated against epidemiologic data.

DEN-3 parent (strain CH53489) and progeny candidate vaccine (clone 24/28) viruses were compared in their abilities to interact with Aedes aegypti and Ae. albopictus. The parent and progeny virus were equivalent in their ability to infect, to replicate in, and to be transmitted by both species of mosquitoes. The candidate vaccine DEN-3 clone was temperature sensitive and had small plaque morphology. These phenotypic markers remained stable during mosquito passage. Thus, temperature sensitivity and small plaque morphology are not reliable biological markers for attenuation.

Surveillance for evidence of West Nile virus (WNV) infection in taxonomically diverse vertebrates was conducted in the Yucatan Peninsula of Mexico in 2003 and 2004. Sera from 144 horses on Cozumel Island, Quintana Roo State, 415 vertebrates (257 birds, 52 mammals, and 106 reptiles) belonging to 61 species from the Merida Zoo, Yucatan State, and 7 farmed crocodiles in Ciudad del Carmen, Campeche State were assayed for antibodies to flaviviruses. Ninety (62%) horses on Cozumel Island had epitope-blocking enzyme-linked immunosorbent assay (ELISA) antibodies to flaviviruses, of which 75 (52%) were seropositive for WNV by plaque reduction neutralization test (PRNT). Blocking ELISA antibodies to flaviviruses also were detected in 13 (3%) animals in the Merida Zoo, including 7 birds and 2 mammals (a jaguar and coyote) seropositive for WNV by PRNT. Six (86%) crocodiles in Campeche State had PRNT-confirmed WNV infections. All animals were healthy at the time of serum collections and none had a history of WNV-like illness.

La Crosse (LAC) virus (family Bunyaviridae, genus Orthobunyavirus) small (S) segment negative-sense RNA genome (vRNA), positive-sense full-length RNA complement (vcRNA), and subgenomic mRNA were assayed in infected cell cultures and female Aedes (Ochlerotatus) triseriatus mosquito tissues using quantitative PCR (Q-PCR). During persistent infection of C6/36 (Aedes albopictus) and MAT (Aedes triseriatus) cultured cells and cytolytic infection of BHK-21 cultured cells, LAC vRNA was the most abundant RNA species, followed by mRNA and vcRNA. RNA copy numbers per cell were quantified and vRNA correlated to virus titer in cell culture medium. The Q-PCR assay proved more sensitive than reverse transcription (RT)-PCR and immunofluorescence assays (IFA) for detecting LAC virus infection of mosquitoes. After infection of female mosquitoes orally, quantities of LAC RNA increased in ovaries for 6 days, and as ovarian biosynthetic activity quiesced, LAC RNA quantities decreased then remained detectable at a low level. After a second, noninfectious blood meal, quantities of LAC RNA in ovaries increased significantly, quantitatively confirming correlation of LAC virus RNA synthesis with vector metabolic activity. Coregulation of viral replication and mosquito ovary metabolic activity may condition efficient transovarial transmission.

A population genetics study of the mosquito Ochlerotatus triseriatus was performed on 36 collections from adjoining regions of Iowa, Minnesota, and Wisconsin covering approximately 120 km(2). Single nucleotide polymorphism analysis was used to estimate variation in the mitochondrial NADH dehydrogenase subunit 4 (ND4) gene. The heated oligonucleotide ligation assay was used to identify the ND4 haplotype of each mosquito. No evidence of genetic isolation by distance was found, nor did Interstate 90 or the Mississippi River serve as barriers to gene flow. The effective migration rate varied from 18 to 45 reproductive migrants/generation, which is similar to estimates from an earlier study. The collections belong to a single, large, panmictic population. However, within this panmictic population, local genetic drift arises, possibly due to one or a few females ovipositing in larval breeding containers. From generation to generation, there is sufficient gene flow to mix families arising from individual breeding sites and eliminate founder effects due to drift.

Serosurveys were conducted to obtain flavivirus and West Nile virus (WNV) seroprevalence data from mammals. Sera from 513 small- and medium-sized mammals collected during late summer and fall 2003 from Colorado, Louisiana, New York, Ohio, and Pennsylvania were screened for flavivirus-specific antibodies. Sera samples containing antibody to flaviviruses were screened for WNV-specific antibodies by epitope-blocking enzyme-linked immunosorbent assays and confirmed with plaque reduction neutralization tests. Prevalence of WNV antibodies among study sites ranged from 0% to 42.8% among the mammal communities sampled. High prevalence rates for WNV were noted among raccoons (100%, with a very small sample size, N = 2), Virginia opossums (50.0%), fox squirrels (49.1%), and eastern gray squirrels (48.3%). The high WNV antibody prevalence noted for tree squirrels, the peri-domestic tendencies of several of these species, and their ease of observation could make these species useful sentinels for monitoring WNV activity within urban communities.

A dengue (DEN) outbreak occurred in the Yucatan State of Mexico in 2002. Three isolates were obtained from patients presenting with DEN-like symptoms, and examined by partial nucleotide sequencing and phylogenetic analysis. The isolates were identified as DEN-2 viruses of the American-Asian genotype; this is the first report of this genotype in the Yucatan State. The DEN-2 viruses of the American-Asian genotype have been associated with more severe disease outcomes. Thus, its introduction into the Yucatan State presents a serious problem to public health authorities. During this outbreak, DEN virus infection was confirmed in 18% (282 of 1,560) of the patients who presented with DEN-like symptoms. Of these, 87 (31%) patients met the World Health Organization criteria for dengue hemorrhagic fever, including two patients who died. The majority (77%) of the patients experienced secondary infections in this epidemic.

Single-strand conformation polymorphism (SSCP) and sequence analyses were used to characterize genetic polymorphisms and phylogenetic relationships, respectively, among dengue (DEN) viruses isolated between 1980 and 1997 from Yucatan, Mexico and surrounding states. Amplified cDNAs from the premembrane (prM) coding region of the DEN viruses were characterized by SSCP. There were six distinct haplotypes of DEN-1 viruses, four haplotypes of DEN-2, four haplotypes of DEN-3, and eight haplotypes of DEN-4. The diversity index for DEN-3 isolates was significantly lower than that of the other serotypes, probably reflecting the recent introduction of this viral serotype into Mexico. The SSCP was a sensitive (84.5%) and specific (95.5%) technique for identifying nucleotide substitutions. Sequence analyses provided insight into the phylogenetic relationships of the DEN strains isolated in Yucatan. One DEN-2 isolate from 1996 was demonstrated to cluster with viruses of the Sri Lanka genotype, none of which have been detected before in the Americas.

A population genetic analysis of Aedes aegypti was conducted among 38 collections from throughout coastal regions of Mexico. Multiple collections were made within 5 cities to examine local patterns of gene flow. Single-strand conformation polymorphism analysis was used to screen for variation in a 387-bp region of the Nicotinamide Adenine Dinucleotide Dehydrogenase subunit 4 mitochondrial gene (ND4) and 25 haplotypes were detected. Northeastern Mexico collections were genetically differentiated from and had lower genetic diversity than Yucatan and Pacific coastal collections. Yucatan and Pacific collections were genetically homogeneous. Regression analysis of geographic distances and F(ST) values indicated that collections were genetically isolated by distance in the Pacific and the Yucatan, but not among collections in the northeast. Free gene flow occurred among all collections within 130 km of one another in the northeast and within 180 km in the Yucatan. F(ST) values were never large among Pacific collections, suggesting extensive gene flow along the Pacific coast.

The co-circulation of all 4 dengue virus serotypes in the same community, common since the 1950s in Southeast Asia, has now become a frequent occurrence in many Caribbean Islands, Mexico, and Central and South America in the past 20 years. As a consequence, the frequency of concurrent infections would be expected to increase in these areas. To assess this, using state of the art technology, we screened viremic serum samples and mosquitoes inoculated with serum samples collected during epidemics involving multiple dengue virus serotypes in Indonesia, Mexico, and Puerto Rico for virus isolation. Of 292 samples tested, 16 (5.5%) were found to contain 2 or more dengue viruses by an indirect immunofluorescence test and/or the reverse transcriptase-polymerase chain reaction.

A quantitative genetic study of the ability of Aedes aegypti to propagate dengue-2 (DEN-2) virus in the midgut and in a disseminated infection in the head was conducted with a standard half-sib breeding design. Aedes aegypti aegypti and A. aegypti formosus differ markedly in oral susceptibility to DEN-2 virus. Mosquitoes were orally infected and, after an extrinsic incubation period of 14 days, virus titer (by tissue culture infectious dose, 50% endpoint) was determined in the midgut (MT) and head (HT). Body size as measured by wing length was not significantly different between infected and uninfected mosquitoes and was not correlated with MT or HT The heritability for MT in both subspecies was 0.41 and was 0.39 for HT in A. aegypti formosus. In A. aegypti aegypti, HT appeared to be controlled by dominant alleles. The MT was not correlated with HT nor did MT determine whether virus disseminated out of the midgut. These results suggest that it is the barriers to infection and dissemination, independent of virus titer, that determine vector competence for DEN-2 virus.

La Crosse (LAC) virus is transmitted horizontally to vertebrates and vertically to progeny by Aedes triseriatus mosquitoes, and in northern midwestern states, this virus overwinters in diapausing eggs of the vector. In Florida, the vector remains active throughout the year and does not diapause. To determine if there is an association between diapause and vertical transmission efficiency of LAC virus, transovarial transmission (TOT), and filial infection (FI) rates were determined for geographic strains of Ae. triseriatus. The TOT rates were not significantly different for Ae. triseriatus originating from Florida (78%) and those from Wisconsin (85%). The FI rates did differ significantly between the two groups (33% and 45%, respectively, for the Florida and Wisconsin mosquitoes). Furthermore, a line of mosquitoes was selected from a Wisconsin colony that had a reduced diapause phenotype (the AD- strain). While this strain displayed TOT rates that were the same as the other Wisconsin mosquitoes (85%), the FI rates were significantly lower (34%), indicating a reduction in TOT efficiency. The role of vertical transmission capacity in LAC virus endemicity remains to be determined.

Artificial selection on strains of Aedes aegypti showing susceptibility and refractoriness to oral infection with yellow fever virus (YFV) suggests that there is a significant genetic component to this trait. Using a population with an average susceptibility of 15%, inbreeding of isofemale lines followed by individual selection produced susceptible (29% infected) and refractory (11% infected) lines. The difference between lines was largely apparent before individual selection, which failed to increase/decrease susceptibility significantly. The findings suggest that very few loci with a major bearing on the trait segregated genetic variation in the original population sample, and that non-genetic factors also play a major role in determining whether or not Ae. aegypti females become infected with YFV.

The Japanese encephalitis vaccine virus (JE 2-8) and its parent virus (JE SA 14) strains were compared for their ability to infect, to replicate in, and to be transmitted by Culex tritaeniorhynchus mosquitoes. Both viruses replicated in the mosquitoes after intrathoracic infection. The JE 2-8 vaccine was transmitted to 1/36 (3%) of suckling mice bitten by intrathoracically infected mosquitoes; however, the parent JE SA 14 strain was transmitted to 46/46 (100%) of the mice bitten. In oral infection trials, only 4/36 (11%) of the Cx. tritaeniorhynchus mosquitoes ingesting the vaccine JE 2-8 strain became infected, whereas 19/19 (100%) of those ingesting the parent JE SA 14 strain became infected. The vaccine JE 2-8 strain did not revert to virulence during passage in mosquitoes.

The timing and sequence of eastern equine encephalitis (EEE) virus replication was studied in the organs from a colony strain of orally infected Culiseta melanura. Three methods of virus assay were used: fluorescent antibody (FA) staining of dissected organs; virus titration in cell culture of whole mosquitoes, dissected organs, hemolymph, and egg rafts; and transmission electron microscopy (TEM) of infected hindguts. EEE virus replicated rapidly in Cs. melanura, first in the posterior midgut, after which it disseminated into the hemocoel where hemolymph transported virus to other organs causing a systemic infection that eventually involved all organs examined, except ovarioles. No initial decrease in virus titer of whole mosquitoes or dissected organs was observed when mosquitoes were collected at daily intervals. Muscle tissue contained the greatest amount of specific fluorescence and the largest aggregates of virus that were visible by TEM. Dissemination of virus occurred rapidly, in some mosquitoes after less than or equal to 17 hours of extrinsic incubation (EI). All infected mosquitoes had disseminated infections after 3 days of EI. Maximum amounts of virus were obtained from whole mosquitoes on the 7th day of EI. FA staining of hindguts was determined to be a rapid and reliable method for detection of EEE virus dissemination and replication in Cs. melanura.

Two colonies of Aedes aegypti were established from two independent col lections from Vero Beach, Florida. Eleven sequential generations of the first colony were tested for variation in oral susceptibility to infection with yellow fever virus (YFV). Each generation was also assayed for genetic variability at seven enzyme loci using elec- trophoretic techniques. Significant differences in infection rates were detected between some generations. These differences were significantly correlated with genetic variation at the malate dehydrogenase locus. Seven generations from the second colony were examined simultaneously for variation in susceptibility to YFV. Significant differences were also detected between some of these generations. The results suggest that colonization may have an effect on the genetic and phenotypic variation in a mosquito strain, and that genetically based variation for susceptibility to infection with YFV occurs in populations of Ae. aegypti.

The frequency and distribution of La Crosse (LAC) virus overwintering in Aedes triseriatus diapause eggs were studied during 1974 by following 64 tree-hole oviposition sites in four enzootic hardwood forest areas. A direct fluorescent antibody technique, adapted for detection of LAC virus in individual mosquitoes, proved to be a rapid, reliable and economical tool for ascertaining true field infection rates. Virus was found in larvae from each of the four study areas before the seasonal emergence of adults and detected in 10 (0.6%) of 1,698 individually processed adults reared from collected larvae. In one of these study areas, all 12 located tree-holes were enclosed with screen before the seasonal emergence of adults, to ensure that all collected from these sites originated from overwintered eggs. Of 1,280 Aedes triseriatus processed from this area throughout the season, 16 (1.2%) contained virus. Isolates were found in overwintered Aedes triseriatus throughout the summer months, demonstrating the role of these tree-hole sites as foci for both overwintering and continuing summer season source of LAC virus in these forested areas.

A technique was developed for microinjection and transformation of Aedes triseriatus mosquitoes. Mosquito ova can be readily microinjected with approximately 0.9 nl of inoculum if desiccated and inoculated before hardening of the chorion. Ova were injected with molecular constructs that conferred antibiotic (G418) resistance to larvae. Insertion of the antibiotic resistance construct into the mosquito genome was demonstrated by Southern blot analysis.

Twenty-eight populations representing a worldwide distribution of Aedes aegypti were tested for their ability to become orally infected with yellow fever virus (YFV). Populations had been analyzed for genetic variations at 11 isozyme loci and assigned to one of 8 genetic geographic groups of Ae. aegypti. Infection rates suggest that populations showing isozyme genetic relatedness also demonstrate similarity to oral infection rates with YFV. The findings support the hypothesis that genetic variation exists for oral susceptibility to YFV in Ae. aegypti.

A molecular hybridization technique was developed to detect bunyavirus RNA in cells. Complementary DNAs (cDNAs) to the small (S) RNA segment of La Crosse (LAC) virus and to a portion of the middle (M) RNA segment of snowshoe hare (SSH) virus were used as probes to detect LAC or SSH viral RNA by in situ hybridization. Protocols were developed and standardized using radiolabeled DNA probes, and adapted for use with biotin labeled probes. The in situ hybridization procedure detected an estimated 3,600 copies of viral S RNA/cell at 24 hr postinfection. In growth curve studies, LAC nucleocapsid antigen was detectable slightly before S RNA. LAC S RNA synthesis was first seen about the nucleus. By 12 hr postinfection, hybridization signal was detected throughout the cytoplasm of the cell. The LAC S RNA probe was group-specific and cross-hybridized to 5 other California group viruses. The SSH M RNA probe was type-specific.

Transovarial transmission of two strains of yellow fever virus was demonstrated in three colonized geographical strains of Aedes aegypti following infection by intrathoracic inoculation. Infected progeny were detected in F1 offspring from only the first three ovarian cycles (OVC). The overall minimum filial infection rate for the first three OVC was 1:596; rates were loser in the second and third OVC. Virus survived in eggs for longer than 4 months and was recovered from progeny of three immersions of first OVC eggs. Infected progeny averaged a longer time to pupation than noninfected siblings. Transovarial transmission of virus was also demonstrated in first OVC progeny of Aedes mascarensis.

Enzyme immunoassays (EIAs) producing either chromogenic or fluorogenic end products were developed and evaluated for detection of eastern equine encephalomyelitis (EEE) and Highlands J (HJ) viruses in pools of Aedes triseriatus mosquitoes. Overnight incubation of the mosquito samples in the EIA significantly enhanced the sensitivity of the test. Both the EEE and HJ EIAs were sensitive, readily detecting one infected mosquito in a pool with 99 noninfected, and specific, distinguishing homologous from the alternate alphavirus and other arboviruses. By 3 days post-infection after intrathoracic inoculation, EEE virus was isolated from 100% (30/30) of the mosquitoes examined. Concurrently, EEE virus antigen was detectable by EIA in 100% (30/30) of examined mosquitoes and by indirect fluorescent antibody (IFA) technique in 77% (23/30) of the examined mosquito head squash preparations.

Aedes triseriatus orally infected with a temperature-sensitive mutant of La Crosse virus were, at predetermined times post-infection, orally challenged with wild type La Crosse or Tahyna virus. Most mosquitoes challenged with wild type La Crosse virus within 24 hr of ingestion of the temperature-sensitive virus became superinfected. In contrast, the majority of mosquitoes challenged at 72 hr were resistant to superinfection. Mosquitoes challenged at 7 days or thereafter were refractory to superinfection with La Crosse or Tahyna virus. The onset of interference was correlated with virus titer and antigen expression in midgut cells.

The dengue-2 vaccine virus (S-1), and its parent virus (PR-159), were compared for their ability to infect orally, to replicate in, and subsequently to be transmitted by Aedes aegypti mosquitoes. The vaccine virus was markedly less efficient in its ability to infect mosquitoes orally. After ingesting infectious bloodmeals containing 3, 7 to 8.2 log10MID50/ml of the respective viruses, 56% (220/396) of the mosquitoes became orally infected with the parent virus contrasted with 16% (66/397) for the vaccine virus. None of the 16 infected mosquitoes transmitted the vaccine virus, while 14% (3/22) of the mosquitoes transmitted the parent virus. The vaccine virus remained temperature-sensitive (restrictive temperature 39 degrees C) after orally infecting and replicating in Ae. aegypti mosquitoes.

The tropisms and development of La Crosse (LAC) virus in stages of transovarially infected Aedes triseriatus were studied with fluorescent antibody (FA) stained dissected organs and titrations of individual arthropods in suckling mice. Viral antigen was detected by FA in 95 of 387 dissected larvae, pupae, and adults. In larvae highest levels of fluorescence were detected in the alimentary tract, followed by ganglia, malpighian tubule, muscle, and other tissues. No specific organs or germ layer-derived tissues appeared to be the sole source of viral replication. Most tissues and organs of A. triseriatus are capable of maintaining LAC virus. Antigen was detected in the identifiable organs immediately upon emergence from the egg. In pupae and adults antigen was detected at high levels in foregut, gonadal and associated tissues, and in salivary glands, which would indicate females could be infective upon emergence. Virus was isolated from all arthropod stages, in 32 of 130 individuals inoculated into suckling mice. Titrations ranged from less than 1.0 log10 SMICLD50 per 0.02 ml for eggs and 1st instar larvae to 3.0 log10 SMICLD50 for 4th instar larvae. Adults and pupae averaged between 2.0 and 3.0 log10 SMICLD50. Increases in titer during maturation were mainly related to increases in size of the organism rather than in titer per unit volume.

A cell culture-adapted variant of the rabies-related Mokola virus was demonstrated to replicate in inoculated Aedes aegypti mosquitoes. Replication was slow compared to many arboviruses in their vectors. Maximum titers were not obtained until after approximately 6 weeks of extrinsic incubation. Mokola virus underwent nine mosquito-mosquito passages at approximately monthly intervals and was thus maintained in insects for 340 days before terminating the study. Virus antigen was detected by immunofluorescence in a variety of mosquito tissues and organs, including salivary glands, but primarily in nervous tissue. Irrefutable virus transmission by bite could not be demonstrated because of equivocal results. Transovarial passage of virus was observed in the mosquito. Viremia in baby mice was demonstrable. Ornithodoros moubata nymphal ticks were exposed to viremic mice but failed to become infected.

Female Aedes aegypti mosquitoes infected with yellow fever virus by intra-thoracic inoculation transmitted the virus to a small percentage of their F1 progeny. Infected offspring were obtained from surface-sterilized as well as from untreated eggs, indicating that the virus was transovarially transmitted. Vertical transmission of yellow fever virus in mosquitoes may be an alternative mechanism for biological survival of the virus during adverse periods or in the absence of susceptible vertebrate hosts.