Modification of Venezuelan equine encephalomyelitis virus infection in mice by X radiation

A highly virulent strain of Venezuelan equine encephalomyelitis (VEE) virus produced less severe histopathologic changes in brain tissues of mice previously exposed to sublethal total-body x-irradiation than it caused in nonirradiated mice. Prior exposure to 600 R of x-irradiation virtually eliminated the lesions of vasculitis and encephalitis that were found in the infected nonirradiated control mice. Mean peak brain lesion scores generally decreased as radiation exposure dose was increased. Irradiation of mice before inoculation often decreased median time to death, whereas the severity of pathologic changes in brain tissues from inoculated irradiated mice was often reduced, without significantly altering ultimate host survival. The inflammatory response did not appear to have a significant role in clearance of this virus from the brain. There was no evidence that participation of the immune response contributed to total mortality from VEE virus encephalitis, as indicated by the failure of radiation immunosuppression to reduce mortality. Death apparently was caused by the direct cytocidal effects of VEE virus replication.     

Re-emergence of epidemic Venezuelan equine encephalomyelitis in South America. VEE Study Group

BACKGROUND: Venezuelan equine encephalomyelitis (VEE) virus has caused periodic epidemics among human beings and equines in Latin America from the 1920s to the early 1970s. The first major outbreak since 1973 occurred in Venezuela and Colombia during 1995, and involved an estimated 75,000 to 100,000 people. We report an epidemiological and virological investigation of this epidemic. METHODS: Virus isolates were made in cell culture from human serum, human throat swabs, and brain tissue from aborted and stillborn human fetuses, as well as from horse brain tissue and pooled mosquito collections. Human sera were also tested for VEE-specific antibodies. The serotypes of VEE isolates were identified by antigen assays, and viruses were characterised genetically by sequencing PCR products generated from the E3 and E2 genes. Phylogenetic analyses were done to determine evolutionary relations with respect to previous epidemic/epizootic and enzootic VEE virus isolates. Mosquito collections were made to identify possible vectors, and clinical findings were determined by direct observation of patients visiting hospitals and clinics in affected regions, and by inspecting patient records. Equine vaccination and vector control were used in an attempt to halt the spread of the outbreak. FINDINGS: Most affected people had an acute, self-limited febrile illness of 3 to 4 days duration. However, convulsions were often seen in children, and abortions and fetal deaths occurred in pregnant women infected with VEE virus. Antigenic characterisation of 12 virus isolates spanning the temporal and spatial range of the outbreak indicated that all are VEE serotype IC. Phylogenetic analysis revealed that all of the 1995 viruses were closely related to serotype IC viruses isolated during a large VEE outbreak that occurred in the same regions of Colombia and Venezuela from 1962-1964. A 1983 mosquito isolate from north central Venezuela was also closely related to the 1995 isolates. INTERPRETATION: This outbreak was remarkably similar to one that occurred in same regions of Venezuela and Colombia during 1962-1964. Symptoms of infected patients, estimated mortality rates, meteorological conditions preceding the epidemic, and seasonal patterns of transmission were all very similar to those reported in the previous outbreak. In addition, viruses isolated during 1995 were antigenically and genetically nearly identifical to those obtained during 1962-1964. These findings suggest that the epidemic resulted from the re-emergence of an epizootic serotype IC VEE virus. Identification of a similar virus isolate in mosquitoes in Venezuela in 1983, 10 years after epidemic/epizootic VEE activity ceased, raises the possibility of a serotype IC enzootic transmission cycle in northern Venezuela.     

Glycoproteins E2 of the Venezuelan and eastern equine encephalomyelitis viruses contain multiple cross-reactive epitopes

Enzyme immunoassay (EIA) with sixty types of monoclonal antibodies (MAbs) was used to study cross-reactive epitopes on the attenuated and virulent strains of the Eastern equine encephalomyelitis (EEE) and Venezuelan equine encephalomyelitis (VEE) viruses. All three structural proteins of the EEE and VEE viruses were demonstrated to have both cross-reactive and specific antigenic determinants. The glycoprotein E1 of EEE and VEE viruses possesses three cross-reactive epitopes for binding to MAbs. The glycoprotein E2 has a cluster of epitopes for 20 cross-reacting MAbs produced to EEE and VEE viruses. Cross-reactive epitopes were localised within five different sites of glycoprotein E2 of VEE virus and within four sites of that of the EEE virus. There are no cross-neutralising MAbs to the VEE and EEE viruses. Only one type of the protective Mabs was able to cross-protect mice against lethal infection by the virulent strains of the VEE and EEE viruses. Eight MAbs blocked the hemagglutination activity (HA) of both viruses. Antigenic alterations of neutralising and protective sites were revealed for all attenuated strains of the VEE and EEE viruses. Comparative studies of the E2 proteins amino acid sequences show that the antigenic modifications observed with the attenuated strains of the VEE virus may be caused by multiple amino acid changes in positions 7, 62, 120, 192 and 209-213. The escape-variants of the VEE virus obtained with cross-reactive MAbs 7D1, 2D4 and 7A6 have mutations of the E2 protein at positions 59, 212-213 and 232, respectively. Amino acid sequences in these regions of the VEE and EEE viruses are not homologous. These observations indicate that cross-reactive MAbs are capable of recognising discontinuous epitopes on the E2 glycoprotein.     

Improved protection against Venezuelan equine encephalitis by genetic engineering of a recombinant vaccinia virus

An improved vaccine is needed against Venezuelan equine encephalitis (VEE) virus because the existing live attenuated vaccine, TC-83, causes a high incidence of adverse effects, and the Formalin-inactivated vaccine, C-84, does not protect against airborne infection. A recombinant vaccine had previously been constructed in which the VEE structural proteins were expressed by vaccinia virus. Although protection against subcutaneous challenge with VEE was achieved, the vaccine had limited efficacy against aerosolized virus. We made a similar construct (WR100) and compared its performance with that of a recombinant vaccinia virus which had been altered in two ways (WR103) in order to improve its performance as a vaccine: a synthetic promoter was inserted upstream of the VEE coding sequence to increase the amount of VEE proteins produced, and a single nucleotide in the E2 glycoprotein gene was altered to enhance immunogenicity. The WR103 virus expressed greater amounts of VEE proteins on the surface of infected cells than did WR100, and this difference was found to correspond to a 3.5-fold increase in VEE protein production. Sera from mice immunized with WR103 contained elevated levels of antibody to VEE, and enhanced protection against subcutaneous challenge with the pathogenic Trinidad donkey strain was achieved. This altered construct could form the basis for a better vaccine against VEE.     

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.     

Sampling methods for potential epidemic vectors of eastern equine encephalomyelitis virus in Massachusetts

The neurotoxic effect of monosodium L-glutamate (MSG) on the morphologies in the darkly stained sexually dimorphic nucleus of the preoptic area (SDN-POA) and the lighter-staining surrounding area (non-SDN-POA) within the medial preoptic nucleus (MPN) was evaluated. Male and female Long-Evans rats were used. MSG (4 mg/g of body weight) was administered subcutaneously to pups on days 1 and 3 postnatally. Normal saline was used as the vehicle. At the age of 6 months, the rats were sacrificed and the brain tissues were fixed for histological examination. The morphological changes, i.e., total volume, density, total neuron number, neuronal nuclear volume (NNV) and ratio of pyknosis, of the SDN-POA and non-SDN-POA within the MPN, were estimated using the AMS VIDS III semiautomatic image-analytic system. The results indicate that neonatal MSG treatment caused significant neuronal loss and decreases in total volume of the SDN-POA and non-SDN-POA of male and female rats. However, only the SDN-POA of MSG-treated male rats showed a significant increase of pyknosis and decrease of neuronal density. A significant enlargement of NNV in the SDN-POA and non-SDN-POA was observed in the MSG-treated male rats. These results indicate that the MPN shows sex-specific and area-specific changes after neonatal neurotoxicity due to MSG.     

Clinical aspects of human Venezuelan equine encephalitis in Texas

The Venezuelan equine encephalitis epidemic which occurred in Texas in 1971 produced a wide range of predominantly mild clinical symptoms. This epidemic, which peaked on 13-14 July, was most intensely felt in the far-south counties of Cameron and Hidalgo. In all, 88 laboratory-confirmed human cases were reported to the U.S. Center for Disease Control by the Texas State Department of Health. The ratio of male to female cases was about two to one. An attack of 20.8 cases per 100,000, observed in both the 20-29 and 30-39 age groups, was higher than attack rates experienced by other age groups and by the population at large. Together, Cameron and Hidalgo counties experienced a much higher overall attack rate (21.7 cases por 100,000) than did affected counties in the Corpus Christi area (4.9 cases per 100,000). Knowledge about when various patients were first exposed points to an incubation period ranging from 27.5 hours to four days. In those 79 cases for which clinical data were available, the most common clinical manifestations were found to be fever, severe headache, myalgia, and chills. Evidence of mild to moderate central nervous system involvement was found in 10 out of 25 children and young people under 17 years of age, and in six out of 54 adults. Two children still had residual paralysis six weeks after onset of illness, but by 10 months these sequelae had disappeared. Seven of the 54 adults, however, still complained of tiring easily a year after onset of illness. Leukopenia, as demonstrated by a count of less rhan 4,500 white blood cells per cubic millimeter, was observed in 75 per cent of the patients examined.     

Venezuelan equine encephalitis and Oropouche virus infections among Peruvian army troops in the Amazon region of Peru

An outbreak of a febrile illness characterized by headache, ocular pain, myalgia, and arthralgia occurred during June 1994 among Peruvian army troops in Northern Peru. On June 14-16, 1994, clinical data and blood samples were obtained from eight soldiers with a febrile illness, and from 26 others who had a history of febrile illness during the past three months. A follow-up blood sample was obtained 107 days later from four of the febrile and seven of the afebrile soldiers. Serum samples were tested for dengue (DEN), Oropouche (ORO), and Venezuelan equine encephalitis (VEE) IgM and IgG antibodies by an enzyme-linked immunosorbent assay (ELISA). Virus isolation was performed by inoculation of newborn mice and Vero cell cultures. Viral isolates were identified by immunofluorescence, ELISA, and nucleotide sequencing. A VEE virus infection was confirmed in three of the eight febrile soldiers, two by virus isolation, and one by serology. Antigenic analysis indicated that one of the virus isolates was similar to VEE subtype I, variety ID, viruses previously isolated in Colombia and Venezuela. Nucleotide sequence data showed that both viral isolates were identical to one another and closely related to VEE ID viruses previously isolated in Peru, Colombia, and Venezuela. Serologic results showed that two of 26 afebrile soldiers had IgM antibody to VEE and four had IgG antibody to VEE; two febrile soldiers had IgG antibody in their first serum samples. Oropouche-specific IgM antibody was detected in one of the eight febrile and five of the afebrile soldiers, and 18 of the 34 soldiers had low titers of ORO IgG antibody titers, which did not meet the diagnostic criteria for confirmed cases. All soldiers were negative for DEN IgM antibody, and 10 had flavivirus IgG antibody that reacted with DEN antigens. These data indicated that VEE ID virus was one of the causes of illness among Peruvians soldiers and that this was the first association of this VEE subtype with human disease in Peru.     

Lymphoreticular and myeloid pathogenesis of Venezuelan equine encephalitis in hamsters

Ultrastructural, histopathologic, and virologic studies of adult hamsters infected with virulent Venezuelan equine encelphalomyelitis (VEE) virus (Subtype I-B) demonstrated precise chronologic and topographic progression of lesions and viral replication in extraneural sites. Thymus contained the earliest lesions and the highest initial and subsequent viral titers. No particular cytotropism was observed as highly efficient viral replication and severe cytonecrosis proceded. Early cortical necrosis of splenic periarteriolar lymphocytic sheath was followed by lymphoblastoid repopulation of the peripheral zone. Massive bone marrow necrosis was accompained by ultrastructural evidence of VEE viral particle production in reticulum cells, rubricytes, myeloid cells, lymphoblastoid cells, and megakaryocytes. Speed, efficiency, destructiveness, and relative sensitivity of virtually all lymphoreticular and hematopoetic cells were hallmarks of virulent VEE infection in the hamster.     

Use of telemetry to assess vaccine-induced protection against parenteral and aerosol infections of Venezuelan equine encephalitis virus in non-human primates

Two investigational vaccines, TC-83 (live-attenuated) and C-84 (formalin-inactivated), are currently available to immunize at-risk individuals against Venezuelan equine encephalitis virus (VEE). Ideally, such vaccines should protect against both the natural mosquito-borne route of infection and from aerosol, the most common route of laboratory infection. Whereas considerable data on vaccine efficacy following parenteral challenge are available, the efficacy of these vaccines against disease caused by aerosol exposure is not well established in primates. We compared the immunogenicity and protective capacity of TC-83 and C-84 against either subcutaneous or aerosol routes of infection in cynomolgus monkeys implanted with temperature-monitoring radiotelemetry devices. A single s.c. dose of TC-83, or three s.c. doses (days 0, 7, 28) of C-84, elicited similar serum virus-neutralizing antibody responses. Animals immunized with either TC-83 or C-84 were protected against s.c. infection. In contrast, after aerosol infection, 40% of the animals vaccinated with either TC-83 or C-84 developed signs nearly as severe as those seen in unvaccinated animals. Protection was not entirely consistent with the measured preinfection immune responses: unprotected animals had serum virus-neutralizing antibody titers and lymphoproliferative responses similar to those seen in protected animals. In this study, C-84 (three doses) protected monkeys as well as TC-83 (one dose) against either a s.c. or aerosol VEE challenge.     

Replicon-helper systems from attenuated Venezuelan equine encephalitis virus: expression of heterologous genes in vitro and immunization against heterologous pathogens in vivo

A replicon vaccine vector system was developed from an attenuated strain of Venezuelan equine encephalitis virus (VEE). The replicon RNA consists of the cis-acting 5' and 3' ends of the VEE genome, the complete nonstructural protein gene region, and the subgenomic 26S promoter. The genes encoding the VEE structural proteins were replaced with the influenza virus hemagglutinin (HA) or the Lassa virus nucleocapsid (N) gene, and upon transfection into eukaryotic cells by electroporation, these replicon RNAs directed the efficient, high-level synthesis of the HA or N proteins. For packaging of replicon RNAs into VEE replicon particles (VRP), the VEE capsid and glycoproteins were supplied in trans by expression from helper RNA(s) coelectroporated with the replicon. A number of different helper constructs, expressing the VEE structural proteins from a single or two separate helper RNAs, were derived from attenuated VEE strains Regeneration of infectious virus was not detected when replicons were packaged using a bipartite helper system encoding the VEE capsid protein and glycoproteins on two separate RNAs. Subcutaneous immunization of BALB/c mice with VRP expressing the influenza HA or Lassa virus N gene (HA-VRP or N-VRP, respectively) induced antibody responses to the expressed protein. After two inoculations of HA-VRP, complete protection against intranasal challenge with influenza was observed. Furthermore, sequential immunization of mice with two inoculations of N-VRP prior to two inoculations of HA-VRP induced an immune response to both HA and N equivalent to immunization with either VRP construct alone. Protection against influenza challenge was unaffected by previous N-VRP immunization. Therefore, the VEE replicon system was characterized by high-level expression of heterologous genes in cultured cells, little or no regeneration of plaque-forming virus particles, the capability for sequential immunization to multiple pathogens in the same host, and induction of protective immunity against a mucosal pathogen.     

Vector competence of mosquitoes (Diptera:Culicidae) from Massachusetts for a sympatric isolate of eastern equine encephalomyelitis virus

This study examined the characteristics of students with specific learning disabilities in either reading and spelling or arithmetic. Based on scores obtained using the revised Woodcock-Johnson Psycho-Educational Battery, students with a marked weakness in arithmetic relative to reading and spelling were designated as Group A. Group R-S showed the opposite pattern. Each group included 30 participants ranging in age from 7 to 16 years, with a mean age of 10 years. The boy-to-girl ratios were 16:14 and 19:11 in Group A and Group R-S, respectively. Comparisons using measures from the Wechsler Intelligence Scale for Children-Third Edition (WISC-III) indicated that Group A was weaker in nonverbal skills than Group R-S, despite equivalent overall IQ scores between the two groups. Group R-S showed a within-group strength in nonverbal versus verbal skills. Group A students were more likely than Group R-S students to have counseling provided as part of their Individualized Education Program, suggesting greater socioemotional difficulty among Group A students. The present study supports the connection between nonverbal skills and socioemotional functioning noted by previous researchers, and generalizes findings from earlier studies to more current test editions.     

Kinetics of heat aggregation of proteins

In this case report, the anaesthetic management for a removal of phaeochromocytoma undertaken immediately following Caesarean section is described. A 32-year-old female patients was given epidural anaesthesia for Caesarean section, and thereafter, general anaesthesia for a resection of phaeochromocytoma. During surgery, phentolamine, nitroglycerine and prostaglandin E1 were electively administered to decrease blood pressure and heart rate. A live infant was delivered and the supra-adrenal tumour was excised successfully. The patient's post-operative recovery was uneventful.     

Effect of salt concentration in larval rearing water on susceptibility of Aedes Mosquitoes (Diptera: Culicidae) to eastern equine and Venezuelan equine encephalitis viruses

The effect of salt concentration in larval rearing water on the susceptibility of adult Aedes taeniorhynchus (Wiedemann) and Aedes sollicitans (Skuse) to infection with eastern equine encephalomyelitis (EEE) virus was tested in the laboratory. Ae. sollicitans was more susceptible to infection (79%, n = 82) and viral dissemination (16%) with EEE virus than was Ae. taeniorhynchus (42%, n = 184) and (5%), respectively, when fed on a chick with a viremia of 10(7) +/- 0.1 plaque-forming units/ml; however, infection rates in adults were not affected by rearing in salt concentrations ranging from fresh water to brackish water containing 2.4% sea salts (1 part fresh water and 2 parts seawater). When fed on the same viremic 6-d-old chicken, all 48 Aedes albopictus (Skuse), reared in fresh water, became infected. Similarly, Venezuelan equine encephalitis viral infection or dissemination rates did not vary among Ae. taeniorhynchus adults that were reared in water containing 0, 1, or 2% sea salts.     

Viremia in young herons and ibis infected with Venezuelan encephalitis virus

Fifty-seven of 61 nestling, 8- to 30-day-old herons of three species (Black-crowned Night Heron, Great Egret, and Snowy Egret), developed viremia lasting one to three days following subcutaneous inoculation with small doses of endemic or epidemic strains of Venezuelan encephalitis virus from Mexico, Guatemala or Venezuela. Two epidemic strains from Guatemala or Venezuela stimulated levels of viremia similar to those following infection with enzootic strains. Great Egrets, Striated and Boat-billed Herons and Scarlet Ibis older than 30 days of age developed viremias of lower levels and shorter durtions than did young birds. Marked differences in levles of viremia were not observed among Black-crowned Night Herons, Great Egrets, or Snowy Egrets. Over 50% of viremic blood samples from herons 8-30 days of age contained 1000 or more chick embryo cell culture plaque forming units of Venezuelan encephalitis per ml, levels sufficient to infect some vector species mosquitoes.     

Kinetics of inactivation of Penaeus penicillatus acid phosphatase during inhibition by N-bromosuccinimide

1. Effects of the synthetic vitamin D analogue EB1089 on indices of apoptosis in cultured human breast cancer cells and in nitrosomethylurea-induced rat mammary tumours in vivo were investigated. 2. At a dose of 0.5 microg kg(-1) body weight, EB1089 caused significant inhibition of tumour progression over the 28 day treatment period in the absence of a significant increase in serum calcium concentration. Higher doses of EB1089 (1 and 2.5 microg kg(-1)) produced substantial regression of the experimental tumours which was accompanied by a striking change in the histological appearance of tumours consistent with induction of tumour cell death. 3. Fragmentation of genomic DNA is a characteristic feature of apoptosis. With the terminal transferase (TdT) assay, 3' DNA breaks indicative of DNA fragmentation were detected histochemically in mammary tumour cells from animals treated with EB1089 (2.5 microg kg(-1)) for 14 days. 4. Effects of the vitamin D analogue on induction of apoptosis were examined in vitro using the MCF-7 human breast cancer cell line. Using the TUNEL method, positive nuclear staining indicative of DNA fragmentation was detected in cells treated for 4 days with 10 nM EB1089. Apoptosis was also quantitated using a cell death ELISA which revealed a time and dose dependent induction of apoptosis by EB1089. 5. The effects of EB1089 on the expression of two oncoproteins which may regulate apoptosis, bcl-2 and bax were examined by Western analysis. In MCF-7 cell cultures treated with 1,25(OH)2D3 or EB1089 (1 x 10(-8) M), bcl-2 protein levels were decreased in a time-dependent manner relative to control levels. In contrast bax protein was not markedly regulated by these compounds. Densitometric analyses indicate that the vitamin D compounds lower the bcl-2/bax ratio favouring increased susceptibility of MCF-7 cells to undergo apoptosis. 6. These results suggest that the synthetic vitamin D analogue EB1089 may promote tumour regression by inducing active cell death.     

Dose-dependent inactivation of extracellular human immunodeficiency virus by miramistin

BACKGROUND: There is a need for an atraumatic, fast, reliable, inexpensive, reversible-on-demand method for female sterilization which is also free from side-effects. The use of an Nd:YAG laser for occlusion of human fallopian tubes in vitro was assessed for achieving these aims. METHODS: An in vitro study was performed on coagulation of fallopian tube tissue using continuous wave Nd:YAG laser. Posthysterectomy human uteri were exposed to laser radiation either directly through an optical fibre or through a sapphire contact probe at the ostia at different laser powers and inter-action times. RESULTS: Laser-induced tissue coagulation plugged the ostia in a clean, controlled and predictable manner. Microscopic examination of the coagulated tissue showed about 50 microns wide blind holes without any continuous channel; thus eliminating the possibility of passage of sperms through such a plug. The depth of coagulation along the lumen of the fallopian tubes increased linearly with the interaction time of the laser beam at a constant power, either by direct irradiation or through a contact probe. The maximum depth of coagulation was found to be about 3 mm in case of direct irradiation at a laser power of about 6.5 W and interaction time of 50 seconds. Beyond these values, charring occurred at the surface of the tissue. CONCLUSION: Nd:YAG laser might be a suitable means for female sterilization. Further studies in experimental and clinical settings would be required to confirm its utility.