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.

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.

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: 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.

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.

Interference between bunyaviruses in Aedes triseriatus mosquitoes.

Inhibition of the replication of alternate California serogroup bunyaviruses in Aedes triseriatus mosquitoes has been observed for mosquitoes previously infected with La Crosse (LAC) virus. By contrast, prior infection of mosquitoes with LAC virus did not interfere significantly with the subsequent infection and replication of Guaroa bunyavirus (Bunyamwera serogroup), or heterologous viruses such as West Nile flavivirus, or vesicular stomatitis rhabdovirus.

Experimental model of transovarial transmission of Tahyna virus in Aedes aegypti mosquitoes.

The progeny of 31 viruliferous Aedes aegypti females infected with Tahyna virus by sucking on viraemic newborn mice was investigated for virus presence. Out of 1587 individuals of the F1 generation, 16 suspensions representing the progeny of 7 females were positive in 146 trials. Individuals of the F1 generation failed to transfer the virus by sucking. Electron microscopy revealed the presence of Tahyna virus particles in the cytoplasm of maturing oocytes inevitably confirming the transovarial transfer of the virus by germinal cells of Aedes aegypti mosquitoes.

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.

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.

A membrane glycoprotein that accumulates intracellularly: cellular processing of the large glycoprotein of LaCrosse virus.

The intracellular transport and certain posttranslational modifications of the large glycoprotein (G1) of LaCrosse virus (LAC) in BHK cells have been studied. G1 from released LAC virus was characterized by complex oligosaccharides (endo H-resistant) and covalently attached fatty acid. Only a small fraction of total cellular G1 was present on the baby hamster kidney cell surface. Cell-surface G1 contained complex oligosaccharides, while total G1 in infected cells contained largely unprocessed (endo H-sensitive) oligosaccharides. In addition, cell G1 contained significantly less fatty acid than virion-associated G1. Pulse-chase experiments showed that the oligosaccharides of G1 were processed to the complex from much more slowly than the oligosaccharides of the vesicular stomatitis virus (VSV) glycoprotein (G). In addition, transit of LAC G1 to the cell surface and into extracellular virions was two to three fold slower than the transit of VSV G. Thus LAC G1 accumulates intracellularly and is only slowly processed by intracellular processing enzymes. Treatment with monensin caused accumulation in the cell of a form of G1 with partial sensitivity toward endo H, suggesting that monensin may act to inhibit the glycosylation process directly.

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.

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.

Inhibition of La Crosse virus replication by monensin, monovalent ionophore.

The effect of monensin, a monovalent ionophore, on La Crosse virus particle formation and polypeptide synthesis was examined. Monensin inhibited the release of virus particles (both infectious and non-infectious) from infected BHK-21 cells. Monensin had no detectable effect on the synthesis of polypeptides G1, G2, and N.