Venezuelan Encephalitis Virus Research Articles

Dose-response models for eastern US, western US and Venezuelan equine encephalitis viruses in mice–Part II: Quantification of the effects of host age on the dose response

Many infectious disease hazards demonstrate higher susceptibility with regards to younger host ages. This trend of increased susceptibility with decreasing host age can also lead to an increased likelihood of mortality, and prolonged/chronic health effects. For quantitative microbial risk assessment (QMRA) modeling, the ability to quantify the effect of host age in the dose response model can allow modelers to account for these effects mechanistically. Additionally, QMRA modelers using age-dependent dose response models can model entire populations within the dose-response itself rather than modeling age ranges using susceptibility factors. This research developed host-age dependent exponential and beta Poisson dose response models for Eastern, Western and Venezuelan encephalitis viruses (EEV, WEV and VEV respectively) for two routes – intracranial and intraperitoneal. Improvement in fit was statistically tested as a means of assessing the benefit of including age dependency into the dose response models. EEV demonstrated improvement in fit using host-age dependency only for the exponential model except for intracranial exposure. EEV demonstrated an improvement in fit when using age dependency in the beta Poisson dose response model for both exposure routes. VEV demonstrated an improvement in fit using age dependency for both exposure routes. WEV demonstrated an improvement in fit for intracranial exposure, but neither of the age dependent dose-response models provided a good fit for WEV intraperitoneal exposure.

Rift Valley fever: still an emerging infection after 3500 years.

Rift Valley fever: still an emerging infection after 3500 years.

Identification, molecular cloning, and analysis of full-length hepatitis C virus transmitted/founder genotypes 1, 3, and 4.

ABSTRACTHepatitis C virus (HCV) infection is characterized by persistent replication of a complex mixture of viruses termed a “quasispecies.” Transmission is generally associated with a stringent population bottleneck characterized by infection by limited numbers of “transmitted/founder” (T/F) viruses. Characterization of T/F genomes of human immunodeficiency virus type 1 (HIV-1) has been integral to studies of transmission, immunopathogenesis, and vaccine development. Here, we describe the identification of complete T/F genomes of HCV by single-genome sequencing of plasma viral RNA from acutely infected subjects. A total of 2,739 single-genome-derived amplicons comprising 10,966,507 bp from 18 acute-phase and 11 chronically infected subjects were analyzed. Acute-phase sequences diversified essentially randomly, except for the poly(U/UC) tract, which was subject to polymerase slippage. Fourteen acute-phase subjects were productively infected by more than one genetically distinct virus, permitting assessment of recombination between replicating genomes. No evidence of recombination was found among 1,589 sequences analyzed. Envelope sequences of T/F genomes lacked transmission signatures that could distinguish them from chronic infection viruses. Among chronically infected subjects, higher nucleotide substitution rates were observed in the poly(U/UC) tract than in envelope hypervariable region 1. Fourteen full-length molecular clones with variable poly(U/UC) sequences corresponding to seven genotype 1a, 1b, 3a, and 4a T/F viruses were generated. Like most unadapted HCV clones, T/F genomes did not replicate efficiently in Huh 7.5 cells, indicating that additional cellular factors or viral adaptations are necessary for in vitro replication. Full-length T/F HCV genomes and their progeny provide unique insights into virus transmission, virus evolution, and virus-host interactions associated with immunopathogenesis.

Vascular mineralization in the brain of horses

Vascular mineralization (siderocalcinosis) in the brain of horses has been usually assumed to be an incidental age-related finding with no clinic significance. In the present study, eight 15-32-year-old horses of different breeds with cerebral siderocalcinosis were studied. Four of these horses had acute and severe central nervous system clinical signs of unknown etiology, 2 horses had neurological signs of known cause, and 2 horses did not have neurological signs. Gross examination of the brains in 4 animals revealed symmetrical foci of malacia in the cerebellar white matter. Histologically, moderate to severe mineralization of blood vessels and parenchyma were observed in all 8 horses, occasionally associated with necrosis of the adjacent tissue. Some horses were tested by virus isolation, polymerase chain reaction, immunohistochemistry, and serology to investigate Rabies virus; West Nile virus; Equid herpesvirus 1 and 4; Eastern, Western, Venezuelan, and Saint Louis encephalitis virus; and Sarcocystis neurona infection. These tests were negative in all samples analyzed. Brain cholinesterase activity and heavy metal screening were also unremarkable. The significance of the vascular and parenchymal mineralization in the brains of some of these horses remains undetermined. However, the severity of the lesions observed in the brains of some of the animals in the present study, coupled with the negative results for other common causes of neurological disease in horses, suggests a possible relationship between siderocalcinosis and the clinical signs observed.

The development of HIV-1 subtype C vaccines for Southern Africa.

One in nine people in South Africa are estimated to be HIV-1 infected, with the majority of these infections being due to HIV-1 subtype C. Until recently, most HIV-1 candidate vaccines were not based on subtype C genes. In response to this epidemic, therefore, the South African AIDS Vaccine Initiative (SAAVI) was established to facilitate the development and testing of candidate HIV-1 subtype C vaccines. The first HIV-1 subtype C candidate vaccine is due to be, tested at the end of 2002, and is based on Venezuelan encephalitis virus replicons expressing Gag protein. The next candidate vaccines to be tested will be DNA and modified vaccinia Ankara vaccines expressing subtype C genes.

Vector competence of Culex (Melanoconion) taeniopus for allopatric and epizootic Venezuelan equine encephalomyelitis viruses.

The vector competence of Culex (Melanoconion) taeniopus was examined in the laboratory for "enzootic" allopatric and "epizootic" strains of Venezuelan equine encephalomyelitis viruses of hemagglutination inhibition subtypes I, II, III, and IV. Following bloodmeals from viremic hamsters, and extrinsic incubation of 20-22 days, mosquitoes were allowed to refeed for transmission attempts. Infection rates never exceeded 50% with oral doses of less than 10(4) chick embryo cell culture plaque forming units (CECPFU), and approached 100% only after ingestion of greater than or equal to 10(5.5) PFU. Transmission was achieved for some "epizootic" subtype IABC and "enzootic" subtype ID strains after bloodmeals containing greater than or equal to 10(3.4) CECPFU; subtypes II, III, and IV were never transmitted despite oral doses up to 10(5.0) CECPFU. These data contrast sharply with those reported previously for sympatric "enzootic" subtype IE Middle American Venezuelan encephalitis viruses.

Virus-induced alterations in cyclic adenosine monophosphate generation in hamster islets of Langerhans.

Inoculation of golden Syrian hamsters with Venezuelan encephalitis (VE) virus results in a sustained diminution in glucose-stimulated insulin release that is correctable by cyclic (c) AMP analogs and phosphodiesterase inhibitors. This suggested the importance of directly measuring cAMP content in VE-infected and control islets in response to insulin secretagogues. The basal cAMP content of VE-infected islets (0.14 +/- 0.02 pmol/micrograms islet DNA) was approximately half that of control islets (0.27 +/- 0.02 pmol/micrograms islet DNA) (P less than 0.05). In the presence of 10 microM glucagon (and 3 mM glucose), the rate of cAMP generation in VE-infected islets was only half that of control islets. With 10 mM alpha-ketoisocaproic acid, the rates of cAMP generation were indistinguishable between control and experimental groups. In response to 20 mM glucose and 3-isobutyl-1-methylxanthine (IBMX) (a phosphodiesterase inhibitor), cAMP generation in VE-infected islets was 81% (NS) of the control rate. When a more specific phosphodiesterase inhibitor, RO 20-1724, was used with 20 mM glucose, cAMP generation in the infected islets was only 44% (P less than 0.001) of the control value. Insulin secretion over the perifusion period paralleled the cAMP levels. In the presence of 10 mM alpha-ketoisocaproic acid, there was no difference in insulin secretion between VE-infected and control islets, while there was a statistically significant (P less than 0.05) difference with 10 microM glucagon or 20 mM glucose (in 1 mM RO 20-1724). These data point to a defect in the cAMP generation system of VE-infected islets, although additional factors involved in insulin secretion may also be impaired by the virus.

Markers of Venezuelan encephalitis virus which distinguish enzootic strains of subtype I-D from those of I-E.

Strains of Venezuelan encephalitis virus isolated from enzootic habitats during interepizootic periods in Middle America and northern South America can be distinguished from each other antigenically by hemagglutination inhibition. This test has provided the basis for the classification of these virus strains into subtypes I-E and I-D, respectively. Virus strains of these two subtypes have been found to differ profoundly with respect to virulence for English short hair guinea pigs. Studies are described which confirm that virus strains of the I-D subtype are guinea pig virulent, and that virulence is not the result of cocycling subpopulations of epizootic subtype I-AB or I-C virions. Two additional markers were found which distinguish subtype I-D and I-E Venezuelan encephalitis virus strains. Firstly, hydroxylapatite chromatography of intact virions at pH 6.5 showed differential elution of I-D and I-E prototype strains. Virions of subtype I-D strains eluted at 0.08 to 0.11 M phosphate, while those of subtype I-E strains eluted at 0.15 to 0.20 M phosphate. Secondly, the isoelectric points of the E1 envelope glycoproteins of the I-D and I-E prototype strains were significantly different; pH 6.85 to 7.00 and pH 7.25 to 7.30, respectively. There was no significant difference in the isoelectric points of the E2 envelope glycoproteins. These distinguishing characteristics most likely reflect a fundamental difference in virion surface structure.

Ecologic observations of Venezuelan encephalitis virus in vertebrates and isolations of Nepuyo and Patois viruses from sentinel hamsters at Pacific and Atlantic habitats in Guatemala, 1968-1980.

La Avellana and Puerto Barrios, two enzootic foci of Venezuelan encephalitis (VE) virus on the Pacific and Caribbean lowlands (respectively) of Guatemala have been studied over a 13-year period. Data from sentinel hamsters and guinea pigs and wild and domestic vertebrates are reported. VE virus strains were isolated from hamsters each period they were exposed during the rainy seasons 1968-1980 and at the end of the dry season 1974. Rates of isolation of VE virus ranged from 0.2%-5.7% hamster/days/exposure. All strains tested were free of epizootic virions. Although virus was isolated from sentinel guinea pigs, their deaths were not attributable to infection with VE virus. Antibody titers in 26 of 28 terrestrial mammals bled at La Avellana in 1971 were higher to enzootic than to epizootic VE strains. Thirty-seven percent of 109 residents of Puerto Barrios had antibody to VE virus. In 13 of 20 tested, antibodies were engendered by the enzootic strain. Nepuyo and Patois viruses were isolated from sentinel hamsters at both La Avellana and Puerto Barrios.

Barriers to dissemination of Venezuelan encephalitis viruses in the Middle American enzootic vector mosquito, Culex (Melanoconion) taeniopus.

Barriers to dissemination of Middle American epizootic hemagglutination inhibition subtype I-AB, and enzootic, subtype IE, Venezuelan encephalitis (VE) viruses were examined in a colony of the enzootic vector mosquito, Culex (Melanoconion) taeniopus. This species is highly susceptible to oral infection with enzootic, but not epizootic, virus strains. Adult female mosquitoes were intrathoracically inoculated with epizootic virus suspensions to ascertain whether a mesenteron infection barrier exists to these subtype I-AB strains. All inoculated mosquitoes became infected, including those receiving only 10 chick embryo cell culture plaque-forming units (CEC pfu). This confirmed that a mesenteron infection barrier exists to epizootic, but not enzootic Middle American VE strains. Mosquitoes were also given high titer hamster bloodmeals of epizootic viruses and dissected at 2-day intervals to determine the location of virus in the few infected individuals. With mean bloodmeal titers of up to 10(5.3) CEC pfu, only 20% or less of the mosquitoes became infected, and virus replication was confined to the mesenteron. This indicated that a mesenteron escape barrier to epizootic VE viruses exists in this mosquito. Mosquitoes were also given large and small oral doses of enzootic virus strains to compare viral replication patterns. With high titer bloodmeals, virus disseminated from the mesenteron within 4 days of infection, and titers in mosquitoes peaked 7-9 days after infection. All mosquitoes that ingested large doses became infected. Mosquitoes receiving small oral doses of enzootic viruses showed a different pattern of virus replication.(ABSTRACT TRUNCATED AT 250 WORDS)

An unusual strain of Venezuelan encephalitis virus existing sympatrically with subtype I-D strains in a Peruvian rain forest.

In 1971, an unusual strain of Venezuelan encephalitis (VE) virus (71D1252) was recovered from the same small area of a rain forest in the western Amazon basin of South America near Iquitos, Loreto, Peru, from which strains of subtype I-D were recovered. The marker characteristics of this strain resembled most closely those of VE subtype III (Mucambo) and were distinctly different from coexisting I-D strains. Thus the concurrent presence of two different VE virus subtypes in one place was a striking exception to the usual geographic allopatry of VE virus subtypes. Strain 71D1252 also contained temperature sensitive (ts) (37 degrees C versus 39 degrees C) virions in the original mosquito suspension and first suckling mouse passage brain tissue suspensions. It thus represents one of the few so-far-reported ts strains of viruses found in nature, and the only natural ts strain of VE virus.

Japanese encephalitis virus replication: Studies on host cell nuclear involvement

Replication, as measured by virus production, of both the flavivirus Japanese encephalitis virus (JEV) and the alphavirus Venezuelan encephalitis virus (VEV) was unaffected by short pulses of actinomycin D (act D) at early times postinfection (PI). Replication of JEV was found to be partially inhibited by continuous exposure to act D under conditions where VEV replication was equally sensitive to the drug. JEV replication proceeded normally in the presence of mitomycin C, a DNA synthesis inhibitor. Autoradiographic analysis revealed that virus-specific RNA was present only in the cytoplasm at both early and late times PI. When infected cell membranes were separated on a discontinuous sucrose gradient, most of the virus-specific RNA was associated with the endoplasmic reticulum fraction.

Viruses may be etiologic agents for non-insulin-dependent (type II) diabetes.

Although diabetes mellitus is generally subclassified into either of two categories, insulin-dependent (type I) and non-insulin-dependent (type II), the heterogeneity of its clinical expressions, genetics, and etiologies may make a clear distinction in classification difficult. Evidence to date for the involvement of viruses in the etiology of the disease has involved only type I diabetes. However, clinical data derived from the subtle chronicity of type II diabetes, the lack of pathological alterations in the pancreas in some patients with type II diabetes, and animal and human studies with Venezuelan encephalitis virus and rubella virus suggest a possible role for viruses in the pathogenesis of non-insulin-dependent diabetes.

Marker characteristics of Venezuelan encephalitis virus strains isolated before and after epidemics and equine epizootics in Middle America.

Ninety-four strains of Venezuelan encephalitis (VE) virus isolated from sentinel hamsters exposed in the Middle American countries of Mexico, Guatemala, Belize, and Honduras were examined for the presence of virions with marker characteristics of strains that cause large epidemics and equine epizootics. Thirty-four strains came from before and 60 strains came from after the Middle American epidemics and equine epizootics of 1966 and 1969-1972. Twenty-three virion clones that resembled epizootic strains by hydroxylapatite chromatography and Vero monkey kidney cell plaque size determinations were characterized further. However, the predominant virions in these clones were like enzootic strains from Middle America north of the Panama Canal region, and not like Middle American epizootic VE strains, since they were in hemagglutination-inhibition antigenic subtype IE, usually had optimal pH of hemagglutination at 6.2, and were avirulent for English shorthair guinea pigs inoculated subcutaneously. These results provide evidence against the theory of origin of epidemic-equine epizootic VE virus strains that posits that epizootic virions emerge in Middle America from strains containing mixtures of enzootic and epizootic virions in enzootic habitats.

Influence of enviromental factors on onset of diabetes Mellitus (II): Studies with nicotine in prediabetic mice.

Some enviromental substances to which we are exposed in everyday life are involved in the onset of chronic disease. This study was performed to clarify whether nicotine plays a role in the development of diabetes mellitus(DM). We used mice in two phases of DM. Prediabetic mice (SZ mice) induced by streptozoto-cin(SZ) at a subdiabetogenic dose(50 mg/kg, iv) had normal blood glucose levels(BGL) and impared glucose tolerance (IGT). Supre-diabetic mice (CP-44) induced by the infection with a pancreatropic variant of Venezuelan encephalitis (VE) virus, CP-44, had normal BGL and diminished insulin release(DIR) in GTT. Nicotine administration was performed by 2 routes: ip (0.03-0.4 mg/kg/day) and po, ad libitum (ab. 10 and 42 mg/kg/day). In ip-groups, prediabetic SZ mice (ICR) showed significantly high BGL at 0.1 mg of nicotine for 1 month;but 6 months later, SZ mice administered nicotine at the dose of 0.4 mg/kg/day recovered. Supredi a bet ic CP-44 mice (ICR) showed significantly high BGL at 0.1 mg of nicotine for 1 month and recovered at higher doses of nicotine by 6 months. In po-groups in which C D F -j mice were used, all SZ mice administered nicotine showed significantly higher BGL. CP-44 mice showed significantly higher BGL, regardless of nicotine administration. The pathology and other biochemical alterations are under the study.

Mesenteronal infection threshold of an epizootic strain of Venezuelan encephalitis virus in Culex (Melanoconion) taeniopus mosquitoes and its implication to the apparent disappearance of this virus strain from an enzootic habitat in Guatemala.

Culex (Melanoconion) taeniopus is a vector of Venezuelan encephalitis (VE) virus at a marsh focus in Guatemala and has low mesenteronal thresholds for infection by and transmission of two enzootic strains of VE virus. In contrast, samples of natural populations and subsequent F2 and F4 generations of these mosquitoes have a high mesenteronal threshold for infection by an epizootic VE strain isolated at the same marsh during the end of the 1969 VE epidemic-equine epizootic. The resistance of Cu. (Mel). taeniopus to mesenteronal infection by this VE strain probably represents a key factor in the apparent disappearance of epizootic VE virus from the marsh focus following the 1969 outbreak.

A comparison of the 26 S mRNAs and structural proteins of an equine virulent Venezuelan encephalitis virus and its vaccine derivative

Fingerprints of the oligonucleotides generated by RNase T 1 digestion of the equine virulent Venezuelan equine encephalitis virus, Trinidad donkey (TRD), intracellular 26 S RNA were similar to those of its vaccine derivative, TC-83 virus. Three oligonucleotides present in the fingerprint of TRD virus 26 S RNA were missing from that of the TC-83 26 S RNA which had three new oligonucleotides. To determine if these genetic changes are expressed in the three structural proteins of the two VEE viruses, we compared the individual proteins from each virus by peptide mapping the trypsin fragments by high-performance liquid chromatography. The nucleocapsid proteins and E, envelope glycoprotein from both viruses produced identical peptide maps; however, analysis of the peptide fragments of the E 2 envelope glycoproteins revealed four different peptide fragments between the two viruses.

Virus-induced alterations in insulin release in hamster islets of Langerhans.

After the inoculation of Golden Syrian hamsters with the TC-83 vaccine strain of Venezuelan encephalitis (VE) virus, a sustained diminution in glucose-stimulated insulin release and glucose intolerance of shorter duration develops. To understand better the mechanism of this defect in insulin release, we examined insulin secretion in response to several test agents in isolated perifused islets from control and 24-d post-VE virus-infected hamsters. 50 islets were used in all perifusion experiments, and data were expressed as total insulin released as well as peak response for each test agent during a 30-min perifusion period from control and VE-infected islets. After perifusion with 20 mM glucose, a 45% diminution of insulin release was noted in VE-infected islets in comparison with control islets, which in turn was similar to in vivo findings. However, following 1-mM tolbutamide stimulation, insulin release was similar in control and VE-infected islets. In separate studies, 1 mM tolbutamide, 10 mM theophilline, 1 mM dibutyryl cyclic (c)AMP, and 1 mM 8-bromo-cAMP resulted in statistically similar insulin-release curves in control and VE-infected islets. Additional experiments assessing [5-3H]glucose use in control and infected islets after 20 min of perifusion with 20 mM glucose revealed virtually identical values (239 +/- 30-control; and 222 +/- 27-VE-infected islets). Morphological and morphometric evaluation of VE-infected islets (21 d following virus inoculation) showed no changes in islet volume density, beta cell density, and beta cell granulation. Thus, VE virus induces a defect in glucose-stimulated insulin release from hamster beta cells that can be corrected by cAMP analogues and does not alter islet glucose use.

Intestinal threshold of an enzootic strain of Venezuelan encephalitis virus in Culex (Melanoconion) taeniopus mosquitoes and its implications to vector competency and vertebrate amplifying hosts.

The minimal intestinal dose of an enzootic strain of Venezuelan encephalitis (VE) virus for Culex (Melanoconion) taeniopus mosquitoes caught at a marsh habitat of VE virus in Guatemala was less than five plaque forming units (pfu) of virus. Ingestion of this dose of virus in blood of viremic hamsters resulted in transmission of virus to other hamsters. This low intestinal threshold of an enzootic strain of VE virus indicates that the natural Guatemalan population of Cu. (Mel.) taeniopus can acquire VE virus from vertebrates that have viremia levels as low as 1,000-5,000 pfu/ml of blood, provided other factors do not limit virus interchange between mosquitoes and vertebrates.

An attenuated mutant of Venezuelan encephalitis virus: Biochemical alterations and their genetic association with attenuation

A temperature-sensitive mutant of Venezuelan encephalitis virus derived by chemical mutagenesis from the hamster-virulent 68U201 wild-type ( wt) strain was previously found to be attenuated. This mutant, is 126, replicated in infected hamsters and elicited production of protective antibodies. Further phenotypic differences between is 126 and 68U201 wt have been characterized in an attempt to localize the genetic basis of the mutant's attenuated virulence. The mutant was shown to differ from the parent virus with respect to virion surface structure-dependent characteristics: temperature lability, plaque sizes in Vero cells, and binding properties to hydroxylapatite. The surface difference was identified by isoelectric focusing of the virion envelope glycoproteins as an alteration in the E 1 glycoprotein. The common genetic basis of all these phenotypic differences was demonstrated by the isolation of independently arising, stable genetic revertants of ts 126, which exhibited characteristics identical in every respect to 68U201 wt. It appears from these studies that the mutation which gave rise to the is 126 mutant virus occurred in the structural gene coding for the E 1 envelope glycoprotein and that the resultant phenotypic alteration in this glycoprotein is genetically associated with the mutant's lack of virulence.