Favipiravir treatment prolongs the survival in a lethal mouse model intracerebrally inoculated with Jamestown Canyon virus.

BACKGROUND: Jamestown Canyon virus (JCV) is a mosquito-borne orthobunyavirus that causes acute febrile illness, meningitis, and meningoencephalitis, primarily in North American adults. Currently, there are no available vaccines or specific treatments against JCV infections. METHODOLOGY/PRINCIPAL FINDINGS: The antiviral efficacy of favipiravir (FPV) against JCV infection was evaluated in vitro and in vivo in comparison with that of ribavirin (RBV) and 2'-fluoro-2'-deoxycytidine (2'-FdC). The in vitro inhibitory effect of these drugs on JCV replication was evaluated in Vero and Neuro-2a (N2A) cells. The efficacy of FPV in the treatment of JCV infection in vivo was evaluated in C57BL/6J mice inoculated intracerebrally with JCV, as per the survival, viral titers in the brain, and viral RNA load in the blood. The 90% inhibitory concentrations (IC90) of FPV, RBV, and 2'-FdC were 41.0, 61.8, and 13.6 µM in Vero cells and 20.7, 25.8, and 8.8 µM in N2A cells, respectively. All mice infected with 1.0×104 TCID50 died or were sacrificed within 10 days post-infection (dpi) without treatment. However, mice treated with FPV for 5 days [initiated either 2 days prior to infection (-2 dpi-2 dpi) or on the day of infection (0 dpi-4 dpi)] survived significantly longer than control mice, administered with PBS (p = 0.025 and 0.011, respectively). Moreover, at 1 and 3 dpi, the virus titers in the brain were significantly lower in FPV-treated mice (0 dpi-4 dpi) versus PBS-treated mice (p = 0.002 for both 1 and 3 dpi). CONCLUSIONS/SIGNIFICANCE: Although the intracerebral inoculation route is thought to be a challenging way to evaluate drug efficacy, FPV inhibits the in vitro replication of JCV and prolongs the survival of mice intracerebrally inoculated with JCV. These results will enable the development of a specific antiviral treatment against JCV infections and establishment of an effective animal model.

Venereal transmission of La Crosse (California encephalitis) arbovirus in Aedes triseriatus mosquitoes.

Veneral transmission of La Crosse virus by males of Aedes triseriatus was demonstrated. La Crosse virus was detected in the bursa of females after induced copulation, and disseminated infection was shown to occur occasionally. Since males of Aedes triseriatus have transovarial filial infection rates similar to those of females and can repeatedly mate, veneral transmission may be an important supplement to other natural endemic maintenance mechanisms.

A G1 glycoprotein epitope of La Crosse virus: a determinant of infection of Aedes triseriatus.

Arthropod-borne viruses (arboviruses) have specific vector-vertebrate host cycles in nature. The molecular basis of restriction of virus replication to a very limited number of vector species is unknown, but the present study suggests that viral attachment proteins are important determinants of vector-virus interactions. The principal vector of La Crosse (LAC) virus is the mosquito Aedes triseriatus, and LAC virus efficiently infects the mosquito when ingested. However, a variant (V22) of LAC virus, which was selected by growing the virus in the presence of a monoclonal antibody, was markedly restricted in its ability to infect Ae. triseriatus when it was ingested. Only 15% of the mosquitoes that ingested V22 became infected and 5% of these developed disseminated infections. In contrast, 89% of the mosquitoes that ingested LAC became infected and 74% developed disseminated infections. When V22 was passed three times in mosquitoes by feeding, a revertant virus, V22M3, was obtained that infected 85% of Ae. triseriatus ingesting this virus. In addition, V22M3 regained the antigenic phenotype and fusion capability of the parent LAC virus. These results suggest that the specificity of LAC virus-vector interactions is markedly influenced by the efficiency of the fusion function of the G1 envelope glycoprotein operating at the midgut level in the arthropod vector.

Aedes triseriatus and La Crosse virus: similar venereal infection rates in females given the first bloodmeal immediately before mating or several days after mating.

Venereal infection rates of Aedes triseriatus females mated to males transovarially infected with La Crosse virus were determined in 6 cage-mating trials. In trials 1-4, venereal infection rates averaged 46% and 45% in F2 females bloodfed 6-8 hr before and 7 days after exposure, respectively, to transovarially-infected males. These rates were similar to rates previously reported only in mosquitoes receiving a bloodmeal 6-8 days prior to mating. Lower rates (24%-31%) were obtained using F4 and F7 generation mosquitoes in trials 5B and 6. In most trials, oral and transovarial transmission rates by venereally infected females were less than 25%. In trial 5B, however, the transovarial transmission rates reached 60% and 94% in the second and third ovarian cycles, respectively, with filial infection rates of 46% and 65%, respectively. The oral transmission rate in this trial reached 38% after 32 days. LAC virus was not detected in first ovarian cycle progeny. It is concluded that higher venereal infection rates must be found and/or first ovarian cycle progeny shown to become infected, before venereal transmission can be considered to make more than a modest contribution to offsetting the erosion of virus prevalence during TO transmission.

Higher venereal infection and transmission rates with La Crosse virus in Aedes triseriatus engorged before mating.

Infection of colonized female Aedes triseriatus by La Crosse (LAC) virus occurred more frequently when females were inseminated by infected males after the females engorged blood (49% of 39) than when mating took place before engorgement (4% of 554). Salivary transmission of LAC virus to mice also was more frequent in females venerally infected after engorgement on a normal mouse (35% of 34) than in females mated before engorgement (2% of 49). LAC virus was transovarially transmitted by 40% of 10 females mated by infected males, and in 64% of 279 progeny reared from eggs of second or later ovarian cycles.

Evaluation of the efficiency of transovarial transmission of California encephalitis viral strains in Aedes dorsalis and Aedes melanimon.

California encephalitis (CE) virus was transmitted transovarially by its natural vector, Aedes melanimon. Vertical transmission rates ranged from 13-26% in geographical populations of Ae. melanimon infected with CE virus by intrathoracic inoculation. No consistent pattern of transmission rates was detected for location or time of year mosquito collection. Vertical transmission rates ranged from 9-29% in Aedes dorsalis inoculated with CE viral strains isolated from Ae. melanimon collected in California. The month or year of viral isolation had no effect on the efficiency of vertical transmission. However, a viral strain isolated from the Owens Valley was less efficiently transmitted than strains from the Sacramento Valley, and strains from the San Joaquin Valley were intermediate in efficiency. Filial infection rates were highest in first ovarian cycle progeny and declined with increasing ovarian cycles in both Ae. dorsalis and Ae. melanimon.

Infection rates of Aedes triseriatus following ingestion of La Crosse virus by the larvae.

Infection rates ranged from 0-2.1% in adults of Aedes triseriaus reared from groups of larvae that had ingested La Crosse (LAC) virus (Clifornia encephalitis group) at dosages of 7.0-8.3 log 10 SMICLD50/ml. Females form orally infected larvae transmitted the virus to suckling mice. Larvae that devoured carcasses of transovarially infected larvae containing 3.0 log 10 SMICLD 50/ml failed to become infected. Ingestion by larvae of infected carcasses appears, therefore, to be unimportant as a method of horizontal amplification of LAC virus.

Interference to oral superinfection of Aedes triseriatus infected with La Crosse virus.

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.

Aedes triseriatus and La Crosse virus: lack of infection in eggs of the first ovarian cycle following oral infection of females.

La Crosse (LAC) virus filial infection rates were 0% for 279 first ovarian cycle larvae, 43% for 380 second ovarian cycle larvae, and 58% for 363 third ovarian cycle larvae from orally infected mosquitoes representing 14 Wisconsin populations of Aedes triseriatus. LAC virus was not detected in 72 pools representing 2,250 first ovarian cycle larvae, while 35 pools and 16 pools each containing 30 second and third ovarian cycle larvae, respectively, were all positive for LAC virus. Similar results were obtained when the extrinsic incubation period temperature was 25 degrees C, 27 degrees C, or variable (17, 23, and 29 degrees C for 8 hours each). LAC virus was not detected in 240 second ovarian cycle larvae in which the bloodmeal for the first ovarian cycle was non-infectious. Infection was not detected in 337 first ovarian cycle larvae from female mosquitoes that had been injected intrathoracically with LAC virus concomitantly with receiving a non-infectious bloodmeal. After an extrinsic incubation temperature of 25 degrees C, LAC virus was discovered in dissected mosquito ovarian tissue 7 days postfeeding on an infectious bloodmeal. The epidemiological implications of these findings are discussed.

The location of San Angelo virus in developing ovaries of transovarially infected Aedes albopictus mosquitoes as revealed by fluorescent antibody technique.

Aedes albopictus adult female mosquitoes, transovarially infected with San Angelo (SA) virus, were examined by fluorescent antibody technique during various stages of ovarian development to determine how the virus enters the egg. Upon emergence from the day of adulthood, it was visible in the follicular epithelium, oocytes and nurse cells of the primary follicles. In the 72-hour period between the ingestion of blood and oviposition, there was a marked increase in the amount of viral antigen in the oocyte, indicating rapid virus accumulation. After oviposition, SA viral antigen was also seen in the secondary ovarian follicles. The observed sequence of infection of the mosquito ovariole with SA virus is analogous to that described with certain endosymbionts of insects.

Stabilized infection of California encephalitis virus in Aedes dorsalis, and its implications for viral maintenance in nature.

Three populations of Aedes dorsalis were selected which transmitted California encephalitis (CE) virus vertically to over 90% of their progeny. Infected progeny in these subpopulations transmitted virus at similar rates through five generations; females from the last generation transmitted virus by bite to suckling mice. These high rates of vertical transmission appeared to be due to the development of a stabilized infection with CE virus rather than to genetic selection for a more efficient transmitter or for a mutant strain of virus. In developed nonstabilized infections and transmitted virus vertically to approximately 20% of their progeny. The existence of stabilized infections of CE virus in vector populations and its implications for the natural history of CE virus and on the Fine and LeDuc model are discussed.

Sensitivity to carbon dioxide in mosquitoes infected with California serogroup arboviruses.

Ten species of mosquitoes became sensitive to CO2 following intrathoracic (i.t.) inoculation of California encephalitis (CE) virus. These included field-collected Aedes melanimon, Aedes nigromaculis and Culiseta incidens and laboratory-colonized strains of Aedes dorsalis, Aedes triseriatus, Anopheles freeborni, Culex peus, Culex pipiens pipiens, Culex pipiens quinquefasciatus and Culex tarsalis. Another California serogroup virus, Jerry Slough (= Jamestown Canyon) (JS), also induced CO2 sensitivity in nearly 100% of infected female Ae. dorsalis, Ae. melanimon and Cx. tarsalis. Sensitivity to CO2 developed in Ae. dorsalis and Cx. tarsalis within 3 days after i.t. inoculation of CE virus, remained at a high level through day 10 after inoculation and decreased gradually with time until by 30 days post-inoculation nearly all mosquitoes were nonsensitive. In contrast, Ae. dorsalis infected transovarially with CE virus were not CO2 sensitive. There was homologous but no heterologous interference to the development of CO2 sensitivity when Ae. dorsalis infected transovarially with CE virus were inoculated with CE or JS viruses. Two other bunyaviruses, Main Drain and Turlock, produced CO2 sensitivity in 3-10% and 0%, respectively, of infected Ae. dorsalis, Ae. melanimon and Cx. tarsalis. Two toga-viruses, St. Louis encephalitis and western equine encephalomyelitis, did not induce CO2 sensitivity in Ae. dorsalis and Cx. tarsalis.

Persistence of La Crosse virus (California encephalitis serogroup) in north-central Illinois.

La Crosse (LAC) virus was first isolated in Illinois from a pool of 50 female Aedes triseriatus mosquitoes collected in July 1976, in Peoria Heights. From 1978 through 1981, 27 strains (11 from males and 16 from females) of LAC virus were recovered from 888 pools containing 22,021 adult A. triseriatus mosquitoes from the same study area. These mosquitoes had developed from larvae and pupae collected from 50 individually identified treeholes. Of the 14 trees that yielded LAC virus-positive mosquitoes, one was positive in 3 of 4 years and another was positive in all 4 years. The latter tree had minimum mosquito field infection rates (MFIR) ranging from 3.4 to 12.7/1,000. Eight (57%) of the trees with positive mosquitoes were red oak (Quercus rubra) while 10 (71%) were in the oak genus (Quercus). The four most productive treeholes accounted for 30% of mosquitoes tested and 52% of the LAC isolations. In 1979, 6,729 A. triseriatus mosquitoes were collected in man-baits and tested for virus. From 1,282 tested in 259 pools (mean = 5), 13 LAC isolates were made, resulting in a field infection rate (FIR) of 11.4/1,000. The remaining 5,447 were tested in 218 pools (mean = 25) and 48 strains of LAC were isolated for a FIR of 9.9/1,000. The relationship of these findings to the occurrence of human LAC encephalitis cases in Peoria County, Illinois is discussed. Repeated recovery of virus from this study area reflects a stable, yet dynamic, focus of LAC virus transmission.

Delineation of La Crosse virus in developmental stages of transovarially infected Aedes triseriatus.

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.

Infection rates of Ascocystis-infected Aedes triseriatus following ingestion of La Crosse virus by the larvae.

The La Crosse (LAC) virus infection rate of Aedes triseriatus larvae that ingest LAC virus does not appear to be increased by concomitant infection of larvae by the gregarine parasite, Ascocystis barretti. Infection rates ranged only from 0--2.6% in adult Ae. triseriatus reared from groups of A. barretti-infected larvae that had ingested LAC virus (California encephalitis group) at dosages of 2.0--7.7 log10 SMICLD50/ml. Females resulting from orally infected larvae transmitted LAC virus to suckling mice. Larvae that were infected with A. barretti and devoured carcasses of adult mosquitoes containing 4.7 log10 SMICLD50/ml failed to become infected. A. barretti spores developing in transovarially infected mosquitoes did not harbor LAC virus; thus, A. barretti does not appear to be a mechanism for virus dispersal.

Bloodmeal sources of Aedes triseriatus and Aedes vexans in a southern Wisconsin forest endemic for La Crosse encephalitis virus.

The micro enzyme-linked immunosorbent assay (ELISA) was used to specifically identify bloodmeal sources of Aedes triseriatus Say and Aedes vexans Meigen collected at a site endemic for La Crosse (LAC) encephalitis virus. Deer were the source of 65% of Ae. triseriatus and 94% of Aedes vexans bloodmeals, respectively. Chipmunks and tree squirrels, which are considered to be the major vertebrate amplifying hosts of LAC virus, were the sources of 8% and 16%, respectively, of the bloodmeals of Ae. triseriatus, the vector of LAC virus. The relatively small proportion of vector bloodmeals taken from the amplifying hosts raises further doubts as to the significance of vertebrate amplification in perpetutation of La Crosse virus in nature, i.e. whether vertebrate amplification alone is sufficient to make up for the shortfall of virus infection that occurs during vertical transmission.

Replication and dissemination of La Crosse virus in the competent vector Aedes triseriatus and the incompetent vector Aedes hendersoni and evidence for transovarial transmission by Aedes hendersoni (Diptera: Culicidae).

The time course and pattern of the replication and dissemination of La Crosse virus was studied in orally infected Aedes triseriatus (Say) and Ae. hendersoni Cockerell. Development of La Crosse virus was approximately the same in both species when plaque assay titers of intact mosquitos or dissected tissues were compared. The mosquitoes were equally susceptible to infection; all Ae. hendersoni and 99% of the Ae. triseriatus tested showed detectable midgut infections. Virus was first detected in hemolymph, salivary glands, and ovaries 10-13 d after infection in both species. The pattern of infection suggests virus dissemination beyond the midgut to be via the hemolymph. By 21 d after infection, 100% (10 of 10) of Ae. triseriatus and 70% (7 of 10) of Ae. hendersoni had infected salivary glands, and the geometric mean titer of Ae. hendersoni salivary glands was 10 times higher than the geometric mean titer of those of Ae. triseriatus, However, when tested for transmission 22 d after infection by refeeding on suckling mice, only 9% (2 of 22) of the Ae. hendersoni with disseminated infections transmitted virus versus 71% (12 of 17) of the Ae. triseriatus. A salivary gland escape barrier was shown to be primarily responsible for the failure of Ae. hendersoni to orally transmit La Crosse virus. However, eight parenterally infected Ae. hendersoni females transovarially transmitted the virus to 25% (5 of 20) of their progeny.

Growth of Tahyna virus at low temperatures.

Replication of two Tahyna virus strains in the Aedes albopictus mosquito cell line was studied in the temperature range from 6 to 28 degrees C. The virus grew in this temperature range; its replication rate was related to the temperature of incubation. At lower temperatures the virus titres increased more slowly and did not reach as high maximum values as at higher temperatures. Short increase in incubation temperature resulted in a short increase in the titre of virus previously incubated at 10 degrees C, but not of virus previously incubated at 15 degrees C. At 10 and 15 degrees C, the released virus was demonstrated for more than 300 days past infection (p.i.).

Influence of temperatures corresponding to those of the host animals on Tahyna virus.

The influence of temperature of 36.5 and 39.2 degrees C on uncloned low passage Tahyna virus in chick embryo cell cultures was studied in the course of 10 passages. At a high multiplicity of infection, the virus titres showed a considerable variability, especially at 39.2 degrees C. The cytopathic effect (CPE) apparent at both temperatures started later and was of lower intensity at 39.2 degrees C than at 36.5 degrees C. The temperature of 39.2 degrees C intensified the thermostable character of the virus. Changes in plaque size and virulence were connected mainly with the cultivation substrate of the virus.

Influence of temperature corresponding to that of the vector on Tahyna virus.

The behaviour of uncloned, low-passage Tahyna virus in an Aedes albopictus (AA) cell line at 28 and 20 degrees C was studied in the course of 10 passages. The virus multiplied at both temperatures without any apparent effect on the host cell. At 28 and 20 degrees C reduction of plaque size, decrease of peripheral virulence and weakening of thermostability were observed. Differences between both temperatures were only in the intensity of these changes.