[Experimental and clinicolaboratory evaluation of complex therapy efficacy in arboviral infections].

Search for drugs efficient in prophylaxis and treatment of dangerous infections (especially arboviral ones) is rather actual, since no specific therapy is available. Many-year investigations of interferon inductors showed that they had immunomodulating, antiviral and antiinflammatory effects and were low toxic. The present study demonstrated that the protective effect was the following: Venezuelan equine encephalitis (VEE)--cycloferon > amixin = ridostin, Rift Valley fever (RVF)--cycloferon > amixin > ridostin, predator pox (PP)--cycloferon > amixin = ridostin, that was obvious that cycloferon was the most active agent in the treatment of VEE, RVF and PP, thus making it possible to acknowledge its priority in prophylaxis and therapy of dangerous viral infections (DVI). Ribavirin in combination with cycloferon solution or cycloferon tablets provided shorter periods of the fever, minimized the intoxication syndrome, promoted earlier resolution of hemorrhagic eruption and lowered the frequency of complications, which was in favour of the disease prognosis.

Infection and dissemination of Venezuelan equine encephalitis virus in the epidemic mosquito vector, Aedes taeniorhynchus.

The mosquito Aedes taeniorhynchus is an important epidemic vector of Venezuelan equine encephalitis virus (VEEV), but detailed studies of its infection are lacking. We compared infection by an epidemic VEEV strain to that by an enzootic strain using virus titrations, immunohistochemistry, and a virus expressing the green fluorescent protein. Ae. taeniorhynchus was more susceptible to the epidemic strain, which initially infected the posterior midgut and occasionally the anterior midgut and cardia. Once dissemination beyond the midgut occurred, virus was present in nearly all tissues. Transmission of the epidemic strain to mice was first detected 4 days after infection. In contrast, the enzootic strain did not efficiently infect midgut cells but replicated in muscles and nervous tissue on dissemination. Because VEEV emergence can depend on adaptation to epidemic vectors, these results show that epidemic/enzootic strain comparisons not only comprise a useful model system to study alphavirus transmission by mosquitoes, but also have important public health implications.

Envelope glycoprotein mutations mediate equine amplification and virulence of epizootic venezuelan equine encephalitis virus.

Epidemics of Venezuelan equine encephalitis (VEE) result from high-titer equine viremia of IAB and IC subtype viruses that mediate increased mosquito transmission and spillover to humans. Previous genetic studies suggest that mutations in the E2 envelope glycoprotein allow relatively viremia-incompetent, enzootic subtype ID strains to adapt for equine replication, leading to VEE emergence. To test this hypothesis directly, chimeric VEEV strains containing the genetic backbone of enzootic subtype ID strains and the partial envelope glycoprotein genes of epizootic subtype IC and IAB strains, as well as reciprocal chimeras, were used for experimental infections of horses. Insertion of envelope genes from two different, closely related enzootic subtype ID strains into the epizootic backbones resulted in attenuation, demonstrating that the epizootic envelope genes are necessary for the equine-virulent and viremia-competent phenotypes. The partial epizootic envelope genes introduced into an enzootic ID backbone were sufficient to generate the virulent, viremia-competent equine phenotype. These results indicate that a small number of envelope gene mutations can generate an equine amplification-competent, epizootic VEEV from an enzootic progenitor and underscore the limitations of small animal models for evaluating and predicting the epizootic phenotype.

Transmission cycles, host range, evolution and emergence of arboviral disease.

Many pandemics have been attributed to the ability of some RNA viruses to change their host range to include humans. Here, we review the mechanisms of disease emergence that are related to the host-range specificity of selected mosquito-borne alphaviruses and flaviviruses. We discuss viruses of medical importance, including Venezuelan equine and Japanese encephalitis viruses, dengue viruses and West Nile viruses.

Aerosol infection of cynomolgus macaques with enzootic strains of venezuelan equine encephalitis viruses.

Because Venezuelan equine encephalitis viruses (VEEVs) are infectious by aerosol, they are considered to be a biological-weapons threat. Nonhuman-primate models are needed to evaluate the efficacy of candidate vaccines. In the present study, cynomolgus macaques, after aerosol exposure to either VEEV-IE or VEEV-IIIA, developed fever, viremia, and lymphopenia; the severity of the fever response, viremia, and lymphopenia correlated with the inhaled dose of VEEV. Of the 10 macaques in our study, 7 developed clinical signs indicative of encephalitis, including loss of balance and hypothermia. In the macaque, the enzootic strains used are infectious by aerosol and lead to disease, including clinical encephalitis.

Venezuelan equine encephalomyelitis.

The emergence of epidemic VEE viruses has been reported ever since the virus was first described; this phenomenon is likely to continue to occur because of the high mutation rate of these RNA viruses. A vaccine that was first developed by the US Military for human use has proved helpful in curtailing the spread of VEE virus during epizootics of the disease in equids but not during human epidemics. It has not, however, eliminated the source of these highly pathogenic and transmissible viruses. Occurrences of VEE in equids in Mexico in recent years suggest that the present vaccine is not effective in interrupting transmission of new epizootic viruses arising from what were previously known as avirulent enzootic cycles. Future vaccines against VEE should be based on immunogens derived from enzootic viruses to interrupt VEE virus transmission at the source itself rather than waiting for virulent phenotypes of VEE virus to emerge.

Humane endpoints are an objective measure of morbidity in Venezuelan encephalomyelitis virus infection of mice.

The clinical signs are described of Venezuelan encephalomyelitis virus (VEEV) infection in mice after both airborne and subcutaneous (s.c.) challenge. Group clinical scores reflected the known pathogenesis of infection by both s.c. and airborne challenge, and with epizootic and enzootic strains of VEEV. This observation confirms the specific relationship of the observed clinical signs to VEEV infection. Within an experiment, those who are assessing the animals for clinical signs must have a common understanding of their appearance, including severity, and should be unaware of the allocation of treatments. If these conditions are met, the progress of clinical signs may be used to determine objectively the time of culling for humane endpoints.

Apoptotic cell death is an important cause of neuronal injury in experimental Venezuelan equine encephalitis virus infection of mice.

Mice develop a fatal encephalomyelitis after infection with the Trinidad donkey strain of Venezuelan equine encephalitis (VEE) virus. Adult mice were inoculated intraperitoneally with VEE virus and the brains were examined at different time points. Morphological changes were assessed by histological staining. VEE virus antigen was detected with immunoperoxidase staining, and DNA fragmentation was evaluated in situ using the terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) method. VEE antigen was found in many areas of the brain and it was prominent in neurons. There were mild associated inflammatory changes. DNA fragmentation was demonstrated in many of these areas using TUNEL. In areas with TUNEL staining, morphological neuronal changes ranged from nuclear chromatin condensations to nuclear and cellular fragmentation, which are characteristic of apoptosis. There is strong morphological and biochemical evidence of apoptotic cell death in this experimental model of VEE virus infection.

Venezuelan equine encephalitis virus vaccines induce mucosal IgA responses and protection from airborne infection in BALB/c, but not C3H/HeN mice.

Immunization with either a live-attenuated (TC-83) or formalin-inactivated (C-84) vaccine for Venezuelan equine encephalitis (VEE) virus protected BALB/c mice from lethal VEE infection acquired subcutaneously or by aerosol. While vaccinated C3H/HeN mice were also protected from parenteral infection, neither vaccine protected these mice from an aerosol infection. The apparent vaccine failures in C3H/HeN mice could not be attributed to deficiencies in virus-neutralizing antibodies in serum, as these responses were typically of equal or higher titer than those observed in protected BALB/c mice before challenge. IgG subclass analysis offered no facile explanation: profiles of IgG2 alpha dominance were observed in C3H/HeN mice given either vaccine and in BALB/c mice given the live-attenuated vaccine, whereas BALB/c antibody responses shifted toward IgGl dominance after immunization with the killed C-84 vaccine. Data from immunized congenic mice showed that the H-2 genes from the C3H/He mice were not singularly responsible for the inability of these mice to resist aerosol infection with VEE virus. VEE virus-specific IgA responses were detected more frequently in respiratory and vaginal secretions obtained from the protected BALB/c mice.

A molecular genetic approach to the study of Venezuelan equine encephalitis virus pathogenesis.

Viral pathogenesis can be described as a series of steps, analogous to a biochemical pathway, whose endpoint is disease of the infected host. Distinct viral functions may be critical at each required step. Our genetic approach is to use Venezuelan equine encephalitis virus (VEE) mutants blocked at different steps to delineate the process of pathogenesis. A full-length cDNA clone of a virulent strain of VEE was used as a template for in vitro mutagenesis to produce attenuated single-site mutants. The spread of molecularly cloned parent or mutant viruses in the mouse was monitored by infectivity, immunocytochemistry, in situ hybridization and histopathology. Virulent VEE spread through the lymphatic system, produced viremia and replicated in several visceral organs. As virus was being cleared from these sites, it began to appear in the brain, frequently beginning in the olfactory tracts. A single-site mutant in the E2 glycoprotein appeared to block pathogenesis at a very early step, and required a reversion mutation to spread beyond the site of inoculation. The feasibility of combining attenuating mutations to produce a stable VEE vaccine strain has been demonstrated using three E2 mutations.

[Presence of antibodies against Venezuelan equine encephalitis virus subtype VI in patients with acute febrile illness]. / Presencia de anticuerpos contra el virus de la encefalitis equina venezolana subtipo VI en pacientes con enfermedad aguda febril.

In Argentina, there is no record of human cases produced by Dengue virus (Flavivirus), but Paraguay and Brasil (neighbouring countries) have notified human outbreaks of Dengue Haemorrhagic Fever. In this report, we inform the serological results of a limited human outbreak of a Dengue-like acute illness that occurred in General Belgrano Island, Formosa, Argentina in April 1989. This island is 35 km far from Clorinda city of Paraguay river, with a human population of 150 inhabitants. The weather of this area is humid with abundant rainfall, favouring mosquitoes proliferation. Two samples of serum from 28 human notified cases were studied using hemagglutination inhibition test (HI), complement fixation (CF), and plaque reduction neutralization (NT) test in Vero cell cultures. All tested sera were negative to Dengue, St. Louis encephalitis, Yellow Fever, Bussuquara, Rocio, Eastern and Western Equine Encephalitis arboviruses as well as Influenza and Rubella viruses. By contrast, infection with Venezuelan equine encephalitis virus (VEE), subtype VI-AG80-663 strain was demonstrated (34.5% positive by HI, 39.1% by CF and 51.6% by NT). Seroconversion was detected by NT in six cases and only five were positive by CF. The 26.8% of the sera reacted also with VEE subtype I AB by NT. Considering that no cross reaction were detected in NT with these two subtypes, our results suggest that both viruses are concomitantly circulating in the studied area. Furthermore, the seroconversions detected with AG80-663 strain firmly indicate that during the outbreak this virus subtype was circulating in the island, although we could not assure that it was the causal agent of the acute disease.

Review of accidents caused by incomplete inactivation of viruses.

Inactivation of viruses and bacterial toxins with formaldehyde for the preparation of vaccines has been a favourite method for most of this century. The Cutter incident in 1955 with poliovaccine focussed attention on the problems accompanying the procedure for inactivating viruses although it had been known since the 1930s that the method was not without its dangers. It had also been known since about the same time that foot-and-mouth disease vaccines prepared in this way could carry residual infectivity. The molecular methods of analysis introduced in the 1970s proved without any doubt that the outbreaks in France in 1981 and in other countries of Western Europe in the 1980s were caused by improperly inactivated vaccines. Recent molecular evidence has now shown that formaldehyde-inactivated Venezuelan equine encephalitis vaccines were the probable cause of the outbreaks of the disease during the 1969-1972 pandemic in Central America. In the author's opinion it is remarkable that formaldehyde is still used for the preparation of inactivated vaccines, particularly since it is known that the procedure also affects the immunogenic epitopes of the viruses.

[The protective activity of the attenuated strain 15 of the Venezuelan equine encephalomyelitis virus in rodent infection via the respiratory tract]. / Protektivnaia aktivnost' attenirovannogo shtamma 15 virusa venesuél'skogo éntsefalomielita loshadei pri infitsirovanii gryzunov cherez dykhatel'nye puti.

Administration to rodents (Syrian hamsters, mice, guinea pigs, rabbits) of minimal doses of attenuated strain 15 VEE (up to 20 ImD50) provided protection against respiratory challenge with a highly virulent strain of Venezuelan equine encephalomyelitis virus. The protection is observed in a wide range of doses (up to LD50 hundreds and even thousands).

Identification of a new Venezuelan equine encephalitis virus from Brazil.

Two strains of recently isolated Venezuelan equine encephalitis (VEE) complex virus from southern Brazil, avirulent for 6- to 8-week-old mice and short-haired guinea pigs, were characterized by biologic, serologic, and biochemical means. They were shown serologically to represent a single, newly recognized variant of subtype I. Two-dimensional polyacrylamide gel electrophoresis (PAGE) of ribonuclease T1 digests of viral ribonucleic acid showed considerable homology between the genomes of the new variant prototype and variant IA. Three structural proteins were visualized by discontinuous sodium dodecyl sulfate-PAGE (SDS-PAGE). Although the smallest protein of both recent isolates migrates with the capsid proteins of other subtype I viruses, the larger structural proteins of the new variants differ in molecular weight from the E1 and E2 envelope glycoproteins of the other subtype I variants. The new isolates produced peptide fragment patterns that were identical to each other, but different from the patterns of other subtype I viruses, following SDS-PaGE of dissociated virions digested with Staphylococcus aureus V8 protease. Since these two isolates were from Culex (Melanoconion) species mosquitoes and from a bat (Carollia perspicillata), were postulated that this is an enzootic VEE virus variant for which the classification IF is suggested.