Glycosaminoglycan binding properties of natural venezuelan equine encephalitis virus isolates

Equine-virulent, epidemic/epizootic strains of Venezuelan equine encephalitis (VEE) virus (VEEV) arise via mutation of progenitor enzootic strains that replicate poorly in equines. Sequencing studies have implicated positively charged amino acids on the surface of the E2 envelope glycoprotein in the acquisition of equine virulence and viremia potential, suggesting that changes in binding to cell surface glycosaminoglycans (GAGs) may mediate VEE emergence. Therefore, we evaluated the binding of natural enzootic and epizootic VEEV isolates to Chinese hamster ovary (CHO) cells expressing normal, high levels of GAGs as well as to mutant CHO cells lacking GAG expression. Binding to GAGs was not consistently associated with the epizootic phenotype, and cell culture passages resulted in increased GAG binding. The low levels of GAG binding exhibited by some low-passage, equine-virulent subtype IC VEEV strains indicate that the positive-charge E2 mutations implicated in VEE subtype IC emergence are not artifacts of laboratory passage and suggest that GAG binding does not play a major role in mediating VEE emergence. The increased GAG binding exhibited by VEEV strain CPA201 from the 1993 Mexican epizootic, when compared to that of closely related enzootic subtype IE strains, was shown to result from a Glu-to-Lys mutation at position 117 of the E2 envelope glycoprotein.     

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.     

Positively Charged Amino Acid Substitutions in the E2 Envelope Glycoprotein Are Associated with the Emergence of Venezuelan Equine Encephalitis Virus

Epidemic-epizootic Venezuelan equine encephalitis (VEE) viruses (VEEV) have emerged repeatedly via convergent evolution from enzootic predecessors. However, previous sequence analyses have failed to identify common sets of nucleotide or amino acid substitutions associated with all emergence events. During 1993 and 1996, VEEV subtype IE epizootics occurred on the Pacific Coast of the states of Chiapas and Oaxaca in southern Mexico. Like other epizootic VEEV strains, when inoculated into guinea pigs and mice, the Mexican isolates were no more virulent than closely related enzootic strains, complicating genetic studies of VEE emergence. Complete genomic sequences of 4 of the Mexican strains were determined and compared to those of closely related enzootic subtype IE isolates from Guatemala. The epizootic viruses were less than 2% different at the nucleotide sequence level, and phylogenetic relationships confirmed that the equine-virulent Mexican strains probably evolved from enzootic progenitors on the Pacific Coast of Mexico or Guatemala. Of 35 amino acids that varied among the Guatemalan and Mexican isolates, only 8 were predicted phylogenetically to have accompanied the phenotypic change. One mutation at position 117 of the E2 envelope glycoprotein, involving replacement of Glu by Lys, resulted in a small-plaque phenotype characteristic of epizootic VEEV strains. Analysis of additional E2 sequences from representative enzootic and epizootic VEEV isolates implicated similar surface charge changes in the emergence of previous South American epizootic phenotypes, indicating that E2 mutations are probably important determinants of the equine-virulent phenotype and of VEE emergence. Maximum-likelihood analysis indicated that one change at E2 position 213 has been influenced by positive selection and convergent evolution of the epizootic phenotype.     

Genetic and anatomic determinants of enzootic Venezuelan equine encephalitis virus infection of Culex (Melanoconion) taeniopus

Venezuelan equine encephalitis (VEE) is a re-emerging, mosquito-borne viral disease with the potential to cause fatal encephalitis in both humans and equids. Recently, detection of endemic VEE caused by enzootic strains has escalated in Mexico, Peru, Bolivia, Colombia and Ecuador, emphasizing the importance of understanding the enzootic transmission cycle of the etiologic agent, VEE virus (VEEV). The majority of work examining the viral determinants of vector infection has been performed in the epizootic mosquito vector, Aedes (Ochlerotatus) taeniorhynchus. Based on the fundamental differences between the epizootic and enzootic cycles, we hypothesized that the virus-vector interaction of the enzootic cycle is fundamentally different from that of the epizootic model. We therefore examined the determinants for VEEV IE infection in the enzootic vector, Culex (Melanoconion) taeniopus, and determined the number and susceptibility of midgut epithelial cells initially infected and their distribution compared to the epizootic virus-vector interaction. Using chimeric viruses, we demonstrated that the determinants of infection for the enzootic vector are different than those observed for the epizootic vector. Similarly, we showed that, unlike A. taeniorhynchus infection with subtype IC VEEV, C. taeniopus does not have a limited subpopulation of midgut cells susceptible to subtype IE VEEV. These findings support the hypothesis that the enzootic VEEV relationship with C. taeniopus differs from the epizootic virus-vector interaction in that the determinants appear to be found in both the nonstructural and structural regions, and initial midgut infection is not limited to a small population of susceptible cells.     

Vector infection determinants of Venezuelan equine encephalitis virus reside within the E2 envelope glycoprotein

Epizootic subtype IAB and IC Venezuelan equine encephalitis viruses (VEEV) readily infect the epizootic mosquito vector Aedes taeniorhynchus. The inability of enzootic subtype IE viruses to infect this mosquito species provides a model system for the identification of natural viral determinants of vector infectivity. To map mosquito infection determinants, reciprocal chimeric viruses generated from epizootic subtype IAB and enzootic IE VEEV were tested for mosquito infectivity. Chimeras containing the IAB epizootic structural gene region and, more specifically, the IAB PE2 envelope glycoprotein E2 precursor gene demonstrated an efficient infection phenotype. Introduction of the PE2 gene from an enzootic subtype ID virus into an epizootic IAB or IC genetic backbone resulted in lower infection rates than those of the epizootic parent. The finding that the E2 envelope glycoprotein, the site of epitopes that define the enzootic and epizootic subtypes, also encodes mosquito infection determinants suggests that selection for efficient infection of epizootic mosquito vectors may mediate VEE emergence.     

Potential sources of the 1995 Venezuelan equine encephalitis subtype IC epidemic

Venezuelan equine encephalitis viruses (VEEV) belonging to subtype IC have caused three (1962-1964, 1992-1993 and 1995) major equine epizootics and epidemics. Previous sequence analyses of a portion of the envelope glycoprotein gene demonstrated a high degree of conservation among isolates from the 1962-1964 and the 1995 outbreaks, as well as a 1983 interepizootic mosquito isolate from Panaquire, Venezuela. However, unlike subtype IAB VEEV that were used to prepare inactivated vaccines that probably initiated several outbreaks, subtype IC viruses have not been used for vaccine production and their conservation cannot be explained in this way. To characterize further subtype IC VEEV conservation and to evaluate potential sources of the 1995 outbreak, we sequenced the complete genomes of three isolates from the 1962-1964 outbreak, the 1983 Panaquire interepizootic isolate, and two isolates from 1995. The sequence of the Panaquire isolate, and that of virus isolated from a mouse brain antigen prepared from subtype IC strain P676 and used in the same laboratory, suggested that the Panaquire isolate represents a laboratory contaminant. Some authentic epizootic IC strains isolated 32 years apart showed a greater degree of sequence identity than did isolates from the same (1962-1964 or 1995) outbreak. If these viruses were circulating and replicating between 1964 and 1995, their rate of sequence evolution was at least 10-fold lower than that estimated during outbreaks or that of closely related enzootic VEEV strains that circulate continuously. Current understanding of alphavirus evolution is inconsistent with this conservation. This subtype IC VEEV conservation, combined with phylogenetic relationships, suggests the possibility that the 1995 outbreak was initiated by a laboratory strain.     

Resistance to Alpha/Beta Interferons Correlates with the Epizootic and Virulence Potential of Venezuelan Equine Encephalitis Viruses and Is Determined by the 5′ Noncoding Region and Glycoproteins

We compared the alpha/beta interferon (IFN-α/β) sensitivities of the TC-83 vaccine strain and 24 enzootic and epizootic Venezuelan equine encephalitis (VEE) isolates. The IFN-resistant or -sensitive phenotype correlated well with epizootic or enzootic potential. IFN-α/β resistance of Trinidad donkey (TRD) virus correlated with virulence determinants in the 5′ noncoding region and glycoproteins. Infection of mice lacking a functional IFN system with the IFN-sensitive TC-83 virus resulted in disease equivalent to that produced by the virulent, IFN-resistant TRD virus, further demonstrating that IFN resistance contributes to VEE virus virulence and is a biological marker of epizootic potential.     

The use of chimeric Venezuelan equine encephalitis viruses as an approach for the molecular identification of natural virulence determinants

Venezuelan equine encephalitis (VEE) virus antigenic subtypes and varieties are considered either epidemic/epizootic or enzootic. In addition to epidemiological differences between the epidemic and enzootic viruses, several in vitro and in vivo laboratory markers distinguishing the viruses have been identified, including differential plaque size, sensitivity to interferon (IFN), and virulence for guinea pigs. These observations have been shown to be useful predictors of natural, equine virulence and epizootic potential. Chimeric viruses containing variety IAB (epizootic) nonstructural genes with variety IE (enzootic) structural genes (VE/IAB-IE) or IE nonstructural genes and IAB structural genes (IE/IAB) were constructed to systematically analyze and map viral phenotype and virulence determinants. Plaque size analysis showed that both chimeric viruses produced a mean plaque diameter that was intermediate between those of the parental strains. Additionally, both chimeric viruses showed intermediate levels of virus replication and virulence for guinea pigs compared to the parental strains. However, IE/IAB produced a slightly higher viremia and an average survival time 2 days shorter than the VE/IAB-IE virus. Finally, IFN sensitivity assays revealed that only one chimera, VE/IAB-IE, was intermediate between the two parental types. The second chimera, containing the IE nonstructural genes, was at least five times more sensitive to IFN than the IE parental virus and greater than 50 times more sensitive than the IAB parent. These results implicate viral components in both the structural and nonstructural portions of the genome in contributing to the epizootic phenotype and indicate the potential for epidemic emergence from the IE enzootic VEE viruses.     

Genetic and phenotypic changes accompanying the emergence of epizootic subtype IC Venezuelan equine encephalitis viruses from an enzootic subtype ID progenitor

Recent studies have indicated that epizootic Venezuelan equine encephalitis (VEE) viruses can evolve from enzootic, subtype ID strains that circulate continuously in lowland tropical forests (A. M. Powers, M. S. Oberste, A. C. Brault, R. Rico-Hesse, S. M. Schmura, J. F. Smith, W. Kang, W. P. Sweeney, and S. C. Weaver, J. Virol. 71:6697-6705, 1997). To identify mutations associated with the phenotypic changes leading to epizootics, we sequenced the entire genomes of two subtype IC epizootic VEE virus strains isolated during a 1992-1993 Venezuelan outbreak and four sympatric, subtype ID enzootic strains closely related to the predicted epizootic progenitor. Analysis by maximum-parsimony phylogenetic methods revealed 25 nucleotide differences which were predicted to have accompanied the 1992 epizootic emergence; 7 of these encoded amino acid changes in the nsP1, nsP3, capsid, and E2 envelope glycoprotein, and 2 were mutations in the 3' untranslated genome region. Comparisons with the genomic sequences of IAB and other IC epizootic VEE virus strains revealed that only one of the seven amino acid changes associated with the 1992 emergence, a threonine-to-methionine change at position 360 of the nsP3 protein, accompanied another VEE virus emergence event. Two changes in the E2 envelope glycoprotein region believed to include the major antigenic determinants, both involving replacement of uncharged residues with arginine, are also candidates for epizootic determinants.     

Eastern and Venezuelan equine encephalitis viruses differ in their ability to infect dendritic cells and macrophages: impact of altered cell tropism on pathogenesis

Eastern and Venezuelan equine encephalitis viruses (EEEV and VEEV, respectively) cause severe morbidity and mortality in equines and humans. Like other mosquito-borne viruses, VEEV infects dendritic cells (DCs) and macrophages in lymphoid tissues, fueling a serum viremia and facilitating neuroinvasion. In contrast, EEEV replicates poorly in lymphoid tissues, preferentially infecting osteoblasts. Here, we demonstrate that infectivity of EEEV for myeloid lineage cells including DCs and macrophages was dramatically reduced compared to that of VEEV, whereas both viruses replicated efficiently in mesenchymal lineage cells such as osteoblasts and fibroblasts. We determined that EEEV infection of myeloid lineage cells was restricted after attachment, entry, and uncoating of the genome. Using replicon particles and translation reporter RNAs, we found that translation of incoming EEEV genomes was almost completely inhibited in myeloid, but not mesenchymal, lineage cells. Alpha/beta interferon (IFN-alpha/beta) responses did not mediate the restriction, as infectivity was not restored in the absence of double-stranded RNA-dependent protein kinase, RNase L, or IFN-alpha/beta receptor-mediated signaling. We confirmed these observations in vivo, demonstrating that EEEV is compromised in its ability to replicate within lymphoid tissues, whereas VEEV does so efficiently. The altered tropism of EEEV correlated with an almost complete avoidance of serum IFN-alpha/beta induction in vivo, which may allow EEEV to evade the host's innate immune responses and thereby enhance neurovirulence. Taken together, our data indicate that inhibition of genome translation restricts EEEV infectivity for myeloid but not mesenchymal lineage cells in vitro and in vivo. In this regard, the tropisms of EEEV and VEEV differ dramatically, likely contributing to observed differences in disease etiology.     

Genetic Characterization of Venezuelan Equine Encephalitis Virus from Bolivia, Ecuador and Peru: Identification of a New Subtype ID Lineage

Venezuelan equine encephalitis virus (VEEV) has been responsible for hundreds of thousands of human and equine cases of severe disease in the Americas. A passive surveillance study was conducted in Peru, Bolivia and Ecuador to determine the arboviral etiology of febrile illness. Patients with suspected viral-associated, acute, undifferentiated febrile illness of <7 days duration were enrolled in the study and blood samples were obtained from each patient and assayed by virus isolation. Demographic and clinical information from each patient was also obtained at the time of voluntary enrollment. In 2005–2007, cases of Venezuelan equine encephalitis (VEE) were diagnosed for the first time in residents of Bolivia; the patients did not report traveling, suggesting endemic circulation of VEEV in Bolivia. In 2001 and 2003, VEE cases were also identified in Ecuador. Since 1993, VEEV has been continuously isolated from patients in Loreto, Peru, and more recently (2005), in Madre de Dios, Peru. We performed phylogenetic analyses with VEEV from Bolivia, Ecuador and Peru and compared their relationships to strains from other parts of South America. We found that VEEV subtype ID Panama/Peru genotype is the predominant one circulating in Peru. We also demonstrated that VEEV subtype ID strains circulating in Ecuador belong to the Colombia/Venezuela genotype and VEEV from Madre de Dios, Peru and Cochabamba, Bolivia belong to a new ID genotype. In summary, we identified a new major lineage of enzootic VEEV subtype ID, information that could aid in the understanding of the emergence and evolution of VEEV in South America.     

Replication and clearance of Venezuelan equine encephalitis virus from the brains of animals vaccinated with chimeric SIN/VEE viruses

Venezuelan equine encephalitis virus (VEEV) is an important, naturally emerging zoonotic pathogen. Recent outbreaks in Venezuela and Colombia in 1995, involving an estimated 100,000 human cases, indicate that VEEV still poses a serious public health threat. To develop a safe, efficient vaccine that protects against disease resulting from VEEV infection, we generated chimeric Sindbis (SIN) viruses expressing structural proteins of different strains of VEEV and analyzed their replication in vitro and in vivo, as well as the characteristics of the induced immune responses. None of the chimeric SIN/VEE viruses caused any detectable disease in adult mice after either intracerebral (i.c.) or subcutaneous (s.c.) inoculation, and all chimeras were more attenuated than the vaccine strain, VEEV TC83, in 6-day-old mice after i.c. infection. All vaccinated mice were protected against lethal encephalitis following i.c., s.c., or intranasal (i.n.) challenge with the virulent VEEV ZPC738 strain (ZPC738). In spite of the absence of clinical encephalitis in vaccinated mice challenged with ZPC738 via i.n. or i.c. route, we regularly detected high levels of infectious challenge virus in the central nervous system (CNS). However, infectious virus was undetectable in the brains of all immunized animals at 28 days after challenge. Hamsters vaccinated with chimeric SIN/VEE viruses were also protected against s.c. challenge with ZPC738. Taken together, our findings suggest that these chimeric SIN/VEE viruses are safe and efficacious in adult mice and hamsters and are potentially useful as VEEV vaccines. In addition, immunized animals provide a useful model for studying the mechanisms of the anti-VEEV neuroinflammatory response, leading to the reduction of viral titers in the CNS and survival of animals.     

Antibody to the E3 glycoprotein protects mice against lethal venezuelan equine encephalitis virus infection

Six monoclonal antibodies were isolated that exhibited specificity for a furin cleavage site deletion mutant (V3526) of Venezuelan equine encephalitis virus (VEEV). These antibodies comprise a single competition group and bound the E3 glycoprotein of VEEV subtype I viruses but failed to bind the E3 glycoprotein of other alphaviruses. These antibodies neutralized V3526 virus infectivity but did not neutralize the parental strain of Trinidad donkey (TrD) VEEV. However, the E3-specific antibodies did inhibit the production of virus from VEEV TrD-infected cells. In addition, passive immunization of mice demonstrated that antibody to the E3 glycoprotein provided protection against lethal VEEV TrD challenge. This is the first recognition of a protective epitope in the E3 glycoprotein. Furthermore, these results indicate that E3 plays a critical role late in the morphogenesis of progeny virus after E3 appears on the surfaces of infected cells.     

Vector-Borne Transmission Imposes a Severe Bottleneck on an RNA Virus Population

RNA viruses typically occur in genetically diverse populations due to their error-prone genome replication. Genetic diversity is thought to be important in allowing RNA viruses to explore sequence space, facilitating adaptation to changing environments and hosts. Some arboviruses that infect both a mosquito vector and a mammalian host are known to experience population bottlenecks in their vectors, which may constrain their genetic diversity and could potentially lead to extinction events via Muller''s ratchet. To examine this potential challenge of bottlenecks for arbovirus perpetuation, we studied Venezuelan equine encephalitis virus (VEEV) enzootic subtype IE and its natural vector Culex (Melanoconion) taeniopus, as an example of a virus-vector interaction with a long evolutionary history. Using a mixture of marked VEEV clones to infect C. taeniopus and real-time RT-PCR to track these clones during mosquito infection and dissemination, we observed severe bottleneck events that resulted in a significant drop in the number of clones present. At higher initial doses, the midgut was readily infected and there was a severe bottleneck at the midgut escape. Following a lower initial dose, the major bottleneck occurred at initial midgut infection. A second, less severe bottleneck was identified at the salivary gland infection stage following intrathoracic inoculation. Our results suggest that VEEV consistently encounters bottlenecks during infection, dissemination and transmission by its natural enzootic vector. The potential impacts of these bottlenecks on viral fitness and transmission, and the viral mechanisms that prevent genetic drift leading to extinction, deserve further study.     

Recombinant sindbis/Venezuelan equine encephalitis virus is highly attenuated and immunogenic

Venezuelan equine encephalitis virus (VEEV) is an important, naturally emerging zoonotic virus. VEEV was a significant human and equine pathogen for much of the past century, and recent outbreaks in Venezuela and Colombia (1995), with about 100,000 human cases, indicate that this virus still poses a serious public health threat. The live attenuated TC-83 vaccine strain of VEEV was developed in the 1960s using a traditional approach of serial passaging in tissue culture of the virulent Trinidad donkey (TrD) strain. This vaccine presents several problems, including adverse, sometimes severe reactions in many human vaccinees. The TC-83 strain also retains residual murine virulence and is lethal for suckling mice after intracerebral (i.c.) or subcutaneous (s.c.) inoculation. To overcome these negative effects, we developed a recombinant, chimeric Sindbis/VEE virus (SIN-83) that is more highly attenuated. The genome of this virus encoded the replicative enzymes and the cis-acting RNA elements derived from Sindbis virus (SINV), one of the least human-pathogenic alphaviruses. The structural proteins were derived from VEEV TC-83. The SIN-83 virus, which contained an additional adaptive mutation in the nsP2 gene, replicated efficiently in common cell lines and did not cause detectable disease in adult or suckling mice after either i.c. or s.c. inoculation. However, SIN-83-vaccinated mice were efficiently protected against challenge with pathogenic strains of VEEV. Our findings suggest that the use of the SINV genome as a vector for expression of structural proteins derived from more pathogenic, encephalitic alphaviruses is a promising strategy for alphavirus vaccine development.     

Pegylated alpha interferon is an effective treatment for virulent venezuelan equine encephalitis virus and has profound effects on the host immune response to infection

Venezuelan equine encephalitis virus (VEEV) is a highly infectious alphavirus endemic in parts of Central and South America. The disease is transmitted by mosquitoes, and the natural reservoir is the small rodent population, with epidemics occurring in horses and occasionally humans. Following infection, VEEV replicates in lymphoid tissues prior to invasion of the central nervous system. Treatment of VEEV-infected BALB/c mice with polyethylene glycol-conjugated alpha interferon (PEG IFN-alpha) results in a greatly enhanced survival from either a subcutaneous or an aerosol infection. Virus is undetectable within PEG IFN-alpha-treated individuals by day 30 postinfection (p.i.). Treatment results in a number of changes to the immune response characteristics normally associated with VEEV infection. Increased macrophage activation occurs in PEG IFN-alpha-treated BALB/c mice infected with VEEV. The rapid activation of splenic CD4, CD8, and B cells by day 2 p.i. normally associated with VEEV infection is absent in PEG IFN-alpha-treated mice. The high tumor necrosis factor alpha production by macrophages from untreated mice is greatly diminished in PEG IFN-alpha-treated mice. These results suggest key immunological mechanisms targeted by this lethal alphavirus that can be modulated by prolonged exposure to IFN-alpha.     

Isolation and characterisation of a human-like antibody fragment (scFv) that inactivates VEEV in vitro and in vivo

Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus genus and several species of this family are pathogenic to humans. The viruses are classified as potential agents of biological warfare and terrorism and sensitive detection as well as effective prophylaxis and antiviral therapies are required.In this work, we describe the isolation of the anti-VEEV single chain Fragment variable (scFv), ToR67-3B4, from a non-human primate (NHP) antibody gene library. We report its recloning into the bivalent scFv-Fc format and further immunological and biochemical characterisation.The scFv-Fc ToR67-3B4 recognised viable as well as formalin and ?-propionolactone (?-Pl) inactivated virus particles and could be applied for immunoblot analysis of VEEV proteins and immuno-histochemistry of VEEV infected cells. It detected specifically the viral E1 envelope protein of VEEV but did not react with reduced viral glycoprotein preparations suggesting that recognition depends upon conformational epitopes. The recombinant antibody was able to detect multiple VEEV subtypes and displayed only marginal cross-reactivity to other Alphavirus species except for EEEV. In addition, the scFv-Fc fusion described here might be of therapeutic use since it successfully inactivated VEEV in a murine disease model. When the recombinant antibody was administered 6 hours post challenge, 80% to 100% of mice survived lethal VEEV IA/B or IE infection. Forty to sixty percent of mice survived when scFv-Fc ToR67-3B4 was applied 6 hours post challenge with VEEV subtypes II and former IIIA. In combination with E2-neutralising antibodies the NHP antibody isolated here could significantly improve passive protection as well as generic therapy of VEE.     

Analysis of intrahost variation in Venezuelan equine encephalitis virus reveals repeated deletions in the 6-kilodalton protein gene

RNA viruses exist as a spectrum of mutants that is generated and maintained during replication within the host. Consensus sequencing overlooks minority genotypes present in the viral sample that may impact the population's phenotype. In-depth sequencing of an original field isolate of subtype IE Venezuelan equine encephalitis virus (VEEV) demonstrated the presence of multiple deletions within the 6,000-molecular-weight (6K) protein gene. Using in vitro and in vivo experiments, similar deletions were generated in an additional VEEV strain originating from an infectious cDNA clone. Time course experiments demonstrated that the deletions are produced during acute infection although not until 24 h postinfection. Molecular clones containing some of these deletions were generated, and although the larger deletions appear to be noninfectious, viruses with the smaller deletions were viable and formed small plaques. Serial passages provided no evidence that these deletion mutants function as defective interfering particles. Furthermore, since wild-type infections generally occur at a low multiplicity of infection, it is unlikely that these deletions are propagated in natural transmission cycles. However, they could affect pathogenesis at later stages of infection. Because they are ubiquitously generated both in vivo and in vitro, further investigation is warranted to understand the generation of these deletions and their significance for disease.     

Venezuelan Equine Encephalitis Virus in Iquitos,Peru: Urban Transmission of a Sylvatic Strain

Enzootic strains of Venezuelan equine encephalitis virus (VEEV) have been isolated from febrile patients in the Peruvian Amazon Basin at low but consistent levels since the early 1990s. Through a clinic-based febrile surveillance program, we detected an outbreak of VEEV infections in Iquitos, Peru, in the first half of 2006. The majority of these patients resided within urban areas of Iquitos, with no report of recent travel outside the city. To characterize the risk factors for VEEV infection within the city, an antibody prevalence study was carried out in a geographically stratified sample of urban areas of Iquitos. Additionally, entomological surveys were conducted to determine if previously incriminated vectors of enzootic VEEV were present within the city. We found that greater than 23% of Iquitos residents carried neutralizing antibodies against VEEV, with significant associations between increased antibody prevalence and age, occupation, mosquito net use, and overnight travel. Furthermore, potential vector mosquitoes were widely distributed across the city. Our results suggest that while VEEV infection is more common in rural areas, transmission also occurs within urban areas of Iquitos, and that further studies are warranted to identify the precise vectors and reservoirs involved in urban VEEV transmission.