An antiviral substance from Penicillium funiculosum. IV. Inquiry into the mechanism by which helenine exerts its antiviral effect.

1. Helenine injected intraperitoneally 24 hr prior to a regularly fatal dose of Semliki Forest virus saves most of the mice to which it is administered. 2. Mice saved by helenine develop no viral immunity and regularly succumb when rechallenged 2 wk later with the same dose of virus from which they were originally saved. 3. The time during which helenine is optimally effective in protecting mice from death by Semliki Forest virus covers a period of approximately 36 hr beginning after about 12 hr and extending to 48 hr before virus infection. When periods of less than 12 hr, or more than 48 hr, elapse between the time of helenine administration and virus inoculation, its protective effectiveness diminishes progressively. 4. Repeated injections of helenine at 2- or 3-day intervals, if continued long enough, exhaust the capacity of a host to respond favorably to helenine administered 24 hr before virus inoculation. 5. Helenine injections at intervals of 4, 3, and 2 wk before its administration 24 hr prior to infection do not decrease the effectiveness of this final dose in protecting mice from fatal infection by the virus. The experimental results here reported indicate that, as suggested by the findings of earlier work, helenine does not act directly as an antiviral substance, but instead exerts its effect through some substance that it induces the host to elaborate. The nature of this induced antiviral substance is as yet unknown though, to judge from the failure of spared mice to acquire viral immunity, it appears to act at a stage in viral replication prior to that at which antigenic viral protein is produced. The findings with helenine and those thus far reported for interferon afford no factual basis for judging the relationship of the two, if any.

An antiviral substance from Penicillium funiculosum. VII. An attempt to determine whether the material responsible for the antipassive immunity effect exhibited by mice injected with helenine is an interferon.

Helenine, NDV, and statolon, all known inducers of interferon in mice, all exerted a marked antiviral effect against Semliki Forest virus. This AV effect was, so far as can be demonstrated, mediated through, the induced interferon. The same three materials also exerted a marked antipassive immunity effect. All the evidence that can be brought to bear indicates that this API effect like the AV effect is mediated through interferon known to be induced by the three materials. If the API effect does indeed have interferon as its basis, this represents a new and totally unsuspected action of interferon.

An antiviral substance from Penicillium funiculosum. VI. Prevention of the establishment of passive immunity to Semliki Forest virus infection in mice by Helenine.

1. Helenine prevents the establishment in mice of passive viral immunity by anti-Semliki serum of swine, rabbit, or guinea pig origin. 2. A period of 12 days must elapse, between the antiviral serum administration and challenge with virus, for prevention of the establishment of passive immunity to become apparent. This period is believed to correspond to that in which injected antibody persists in circulation in the injected host. 3. Helenine is effective in preventing the establishment of passive viral immunity by heterologous antiviral sera when it is administered any time during a period of 6 days, extending from 4 days before to 2 days after injection of the antiviral serum. 4. Helenine does not prevent the establishment of passive viral immunity by antiviral sera of mouse origin (homologous). 5. Evidence is presented to indicate that the phenomenon of the prevention of the establishment of passive viral immunity by heterologous antiviral sera is not effected directly, but rather is mediated through some substance that helenine induces the injected host to elaborate. 6. The capacity to prevent the establishment of passive viral immunity could not be exhausted by repeated preceding injections of helenine at 2- or 3-day intervals. 7. Evidence is presented to indicate that the helenine-induced material does not act upon antiviral antibody per se but rather on heterologous foreign protein that happens to be labeled as Semliki Forest virus antibody. This helenine-induced material, whatever its nature, appears to enhance the capacity of the injected host to recognize and dispose of foreign protein. 8. Statolon, a material that like helenine is a known inducer of interferon, is, like helenine, also capable of preventing the establishment of passive viral immunity by heterologous antiviral sera. 9. Experiments designed to determine whether the induced material responsible for the antipassive immunity effect of helenine is interferon have yielded inconclusive answers thus far.

The presence in Penicillium funiculosum of an inhibitor to the antiviral agent helenine.

The present findings indicate that the wide fluctuation observed in the antiviral activity of various crude helenine preparations may be attributable to the presence of varying amounts of inhibitor. Antiviral activity could be enhanced by removal of the inhibitor. A meaningful value for helenine titer in a preparation clearly should be determined in the inhibitor-free zone.

An antiviral substance from Penicillium funiculosum. V. Induction of interferon by helenine.

Helenine, a substance obtained from broth cultures of Penicillium funiculosum and known to exert a protective effect in vivo in experimental animals against several unrelated viruses, has been shown to elicit the formation in cell cultures and in intact mice of an inhibitor of viral plaque formation. Because the biological and chemical characteristics of the viral inhibitor induced by helenine are similar to those of interferon, it is suggested that the antiviral effect of helenine may be mediated through the formation of interferon.

Molecular basis of bunyavirus transmission by mosquitoes: role of the middle-sized RNA segment.

In an examination of the molecular basis of oral transmission of bunyaviruses by mosquitoes., La Crosse (LAC), snowshoe hare (SSH), and LAC-SSH reassortant viruses were compared in their ability to be transmitted to laboratory mice by the natural mosquito vector of LAC virus, Aedes triseriatus. Both LAC virus and the reassortment viruses containing the middle-sized (M) segment from the LAC parent were efficiently transmitted. In contrast, SSH virus and reassortment viruses containing the M RNA from the SSH parent were inefficiently transmitted. Thus, the M RNA segment, which codes for the virion glycoproteins, may be a major determinant of oral transmission of bunyaviruses by mosquitoes.

Antibody response to arboviruses. Absence of increased response in amyotrophic lateral sclerosis and multiple sclerosis.

Complement fixation and hemagglutination imhibition tests were conducted on the serums of patients with amyotrophic lateral sclerosis and multiple sclerosis using a variety of arboviral antigens. Seventy-eight complement fixation and 15 hemagglutination-inhibition viral antigens were used representing togaviruses, orbiviruses, rhadoviruses, bunyaviruses, arenaviruses, and several ungrouped agents. The serological results did not indicate any relationship between these viruses and either amyotrophic lateral sclerosis or multiple sclerosis.

Jatobal virus is a reassortant containing the small RNA of Oropouche virus.

Jatobal (JAT) virus was isolated in 1985 from a carnivore (Nasua nasua) in Tucuruí, Pará state, Brazil and was classified as a distinct member of the Simbu serogroup of the Bunyavirus genus, family Bunyaviridae on the basis of neutralization tests. On the basis of nucleotide sequencing, we have found that the small (S) RNA of JAT virus is very similar (>95% identity) to that of Oropouche (ORO) virus, in particular, the Peruvian genotype of ORO virus. In comparison, limited nucleotide sequencing of the G2 protein gene, encoded by the middle (M) RNA, of JAT and ORO viruses, revealed relatively little identity (<66%) between these two viruses. Neutralization tests confirmed the lack of cross-reactivity between the viruses. These results suggest that JAT virus is a reassortant containing the S RNA of ORO virus. JAT virus was attenuated in hamsters compared to ORO virus suggesting that the S RNA of ORO virus is not directly involved in hamster virulence.

Single inoculation immune hamster sera for typing California group arboviruses by the complement-fixation test.

Eight reference California group viruses of North America were typed using sera of immune hamsters bled 21 days after a single inoculation. The complement-fixation test reactions were relatively specific, although only 2-fold differences were observed reciprocally with the closely-related La Crosse and snowshoe hare viruses. Hamster serum taken 10 days post inoculation was more specific than a 21-day serum. Sepcificity after second inoculation was lost with some antigens. Jamestown Canyon and South River viruses were identical by complement-fixation test and showed minor differences in the plaque reduction neutralization test.

Description of Guanarito virus (Arenaviridae: Arenavirus), the etiologic agent of Venezuelan hemorrhagic fever.

This paper characterizes Guanarito virus, the etiologic agent of Venezuelan hemorrhagic fever. Based on its morphology and antigenic properties, Guanarito virus appears to be a new member of the Tacaribe complex of the genus Arenavirus, family Arenaviridae. Complement fixation and indirect fluorescent antibody tests showed that Guanarito virus and its antiserum are broadly cross-reactive with other members of the Tacaribe complex, but it can be differentiated from other members of the complex by neutralization test. Guanarito virus causes mortality in suckling mice and adult guinea pigs, but not in adult mice. Inoculated rhesus monkeys developed viremia and became ill; however, they subsequently recovered and responded with production of antibody. To date, all isolates of Guanarito virus have come from sick persons or wild rodents living within a single geographic focus in the central plains of Venezuela.

Aedes aegypti strain fitness for yellow fever virus transmission.

Three geographical strains of Aedes aegypti from Thailand (Amphur), East Africa (Kampala), and the West Indies (Santo Domingo) were compared for susceptibility to infection with low-passage yellow fever virus (French viscerotropic) as well as for ability to transmit virus by bite at varying extrinsic incubation periods. Santo Domingo strain appeared the most competent and Kampala the least when mosquitoes were exposed to a low level virus-infecting blood meal; at higher virus levels, a similar trend was noted but differences were less evident and in no case were the differences statistically significant. All three strains were infected with and transmitted yellow fever virus.

Flavivirus type-specific antigens produced from fusions of a portion of the E protein gene with the Escherichia coli trpE gene.

Based on previous data showing that JEV and DEN-1 fusion proteins can be used as antigens, we engineered analogous fusion proteins for the same regions of the E proteins of DEN-2, DEN-3, and DEN-4, using the polymerase chain reaction. The resulting fusion proteins were tested in a modified ELISA for their reactivity with mouse immune ascitic fluids (MIAF) generated against 10 different flaviviruses. The results showed that these recombinant antigens could be used as type-specific immunological reagents, and suggest that these antigens could serve as the basis for rapid, safe, and inexpensive flavivirus serosurveys.

Seroepidemiology of California and Bunyamwera serogroup (Bunyaviridae) virus infections in native populations of Alaska.

This study investigated the geographic distribution and prevalence of antibodies to California and Bunyamwera serogroup viruses in Native populations of Alaska, and demographic and ecologic risk factors associated with exposure. Sera (n = 1,635) from 18 communities were screened using an ELISA. All age groups were tested for antibodies to Jamestown Canyon (JC), Inkoo (INK), snowshoe hare (SSH), and Northway (NOR) viruses; persons > or = 45 years old (n = 90) from six communities were additionally tested for antibodies to Tahyna (TAH), Batai (BAT), Cache Valley (CV), and Sindbis (SIN) viruses. Thirty free-ranging mammals were tested by a plaque reduction neutralization test (PRNT) for antibodies to all eight viruses and to Getah (GET) virus. In Natives, overall antibody prevalence was 24.9% (JC = 17.6%, monotypic JC = 6.5%, INK = 11.1%, monotypic INK = 0.6%, SSH = 6.8%, monotypic SSH = 3.5%, and NOR = 6.2%). Five TAH, CV, and BAT virus exposures may be serologic cross-reactions, and no SIN virus antibodies were detected. Sindbis-like virus antibodies were found in 30% of the mammals. Most mammals had antibodies to NOR (83.3%) and California serogroup (70.0%) viruses; no GET virus exposures were found. Significant risk factors for human bunyavirus exposures were age group, ethnic-linguistic group, biotic province, climate zone, terrestrial vegetation, and presence of some ungulates and small mammals in communities. Sex was not a significant risk factor.

A rapid diagnostic assay for eastern equine encephalomyelitis viral RNA.

Eastern equine encephalomyelitis virus (EEEV) has been a low-frequency, but serious human and veterinary health problem. Increased frequency of this mosquito-borne virus is anticipated as wetlands are maintained and re-established. Control of EEEV has depended on mosquito abatement in response to increasing frequency of EEEV in the environment. A coupled reverse transcription/polymerase chain reaction assay was designed to rapidly, sensitively, and specifically detect EEEV RNA. The assay successfully detected the viral RNA in a single-blind study of a set of field samples composed of either pooled mosquitoes or bird tissue. These results suggest that it would be practical to use this assay for deciding when and where to implement mosquito abatement.

The effect of chloroquine prophylaxis on yellow fever vaccine antibody response: comparison of plaque reduction neutralization test and enzyme-linked immunosorbent assay.

Weekly oral chloroquine prophylaxis for malaria has been associated with impaired antibody response to intradermal rabies vaccination. Experimental data indicate that chloroquine may inhibit yellow fever virus in vitro, yet there has been no clinical evidence to suggest that antibody response to yellow fever vaccine is impaired by concomitant oral administration of chloroquine. A prospective trial was undertaken to evaluate the antibody response to yellow fever 17D vaccine (Connaught Laboratories) of volunteers who were randomized to taking either chloroquine or no drug. Of fifty subjects, 28 were randomized to taking chloroquine, 22 were randomized to taking no drug. Yellow fever 17D vaccine was administered on day 0 and blood sampled on days 0, 14, 35 and 210. Chloroquine was administered weekly for four weeks. There was no significant difference in peak antibody titer by plaque reduction neutralization testing (PRNT) between the group that took chloroquine (mean log peak of reciprocal titer 1.43 +/- SD 0.60) with vaccine subcutaneously compared to vaccine-only group (mean log peak of reciprocal titer = 1.21 +/- 0.55). All fifty subjects seroconverted to yellow fever vaccine by day 210. ELISA testing was also performed on all subjects. The two tests showed good correlation (Spearman r = 0.675), although ELISA readings were positive by day 14 in significantly more subjects (p = .01). We conclude that routine anti-malarial doses of chloroquine do not affect antibody response to yellow fever 17D vaccine. ELISA testing, a less complex and less time-consuming test, correlates well with PRNT and is proposed for additional trials to measure yellow fever 17D vaccine response in flavivirus non-immune subjects.

Pacui virus, phlebotomine flies, and small mammals in Brazil: an epidemiological study.

Pacui virus, originally obtained from forest rodents, was isolated 100 times from 61,437 specimens (658 pools) of the phlebotomine fly Lutzomyia flaviscutellata, collected from rodent-baited traps in the forests of Belem, Para, Brazil in the period October 1968 through September 1970. Isolations were made from engorged and unengorged females and from males (3 strains), and occurred in all 24 months. Pacui virus also was isolated from the blood of two wild rodents (Oryzomys), but not from 424 L. infraspinosa, 12,000 mosquitoes, or sentinel mice. Pacui virus neutralizing antibodies were detected in serum of six bait animals after exposure to biting flies in the forest, in 30% of wild rodents surveyed (including two from Amapa Territory), and in 10% of marsupials, but were absent in human survey sera and in bats. Low-passage Pacui virus produced viremia in and was lethal to infant mice by the subcutaneous route. L. flaviscutellata was most abundant in the dry season, in which period Pacui virus isolations increased. This fly is strongly attracted to rodents close to the ground. L. flaviscutellata also yielded single strains of Guama, Icoaraci, and BeAr 177325 viruses.

Antigenic relationships among phlebotomus fever group arboviruses and their implication for the epidemiology of sandfly fever.

The antigenic relationships of 21 known or presumed Phlebotomus fever group serotypes and of 2 ungrouped, solvent sensitive, sandfly-associated arboviruses (Pacui and Charleville) were studied by complement fixation, plaque neutralization, and hemagglutination-inhibition methods. Results of complement fixation and neutralization tests were specific, allowing clear separation of the various serotypes, while those of the hemagglutination-inhibition test showed broader crossing and lack of specificity. Pacui virus was shown to be a member of the Phlebotomus fever serogroup. Six new Phlebotomus fever group serotypes are also described, increasing the known members of the group to 22. The implications of these and other recent data about the epidemiology of sandfly fever are discussed.

Effect of human gamma interferon on yellow fever virus infection.

We studied yellow fever virus infection in two species of monkey: Saimiri sciureus (squirrel monkeys) and Macaca mulatta (rhesus monkeys). Human gamma interferon was administered intravenously in five equal doses, one was given 24 hr before infection followed by four doses 24 hr apart. Interferon reduced the levels and duration of viremia and the severity of hepatitis in squirrel monkeys. Interferon prolonged survival time and delayed the appearance of viremia and hepatitis in infected rhesus monkeys, but it did not change overall mortality.

Arthropod studies with rabies-related Mokola virus.

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.

Biological and antigenic characterization of Netivot virus, an unusual new Orbivirus recovered from mosquitoes in Israel.

The antigenic and biological characteristics of a new Orbivirus, designated Netivot virus, are described. This agent was originally recovered in cultures of the C6/36 clone of Aedes albopictus cells from a pool of Culex pipiens captured in Israel. Netivot virus is not pathogenic for newborn mice, nor did it initially produce detectable cytopathic effect (CPE) in Vero cells. It is closely related antigenically to Umatilla and Llano Seco viruses; these 3 agents appear to constitute a new serogroup within the genus Orbivirus. Netivot virus is also more distantly related to a number of other orbiviruses in the blue-tongue, epizootic hemorrhagic disease of deer, and Eubenangee serogroups. Netivot virus replicated to high titer and produced CPE in a variety of mosquito cell cultures, but it did not grow in 2 sand fly cell lines. Inoculation of Ae. aegypti and Ae. albopictus with Netivot virus resulted in almost 100% mortality in both species within 15 days after infection. The recovery of this and a number of other yet unidentified viral agents from field-collected mosquitoes in cultures of C6/36 cells, but not in the conventional vertebrate assay systems, suggests the existence in nature of many yet unrecognized mosquito-associated viruses. It also demonstrates the value of using new isolation methods in arbovirus studies.