Evaluation of altered environmental conditions as a decontamination approach for nonspore-forming biological agents.

AIMS: The purpose of this study was to evaluate the effects of altered environmental conditions on the persistence of Francisella tularensis bacteria and Venezuelan equine encephalitis virus (VEEV), on two material types. METHODS AND RESULTS: Francisella tularensis (F.t.) and VEEV were inoculated (c. 1 × 108 colony-forming units or PFU), dried onto porous and nonporous fomites (glass and paper), and exposed to combinations of altered environmental conditions ranging from 22 to 60°C and 30 to 75% relative humidity (RH). Viability of test organism was assessed after contact times ranging from 30 min to 10 days. Inactivation rates of F.t. and VEEV increased as both temperature and/or RH were increased. Greater efficacy was observed for paper as compared to glass for both test organisms. CONCLUSIONS: The use of elevated temperature and RH increased rate of inactivation for both organisms and greater than six log reduction was accomplished in as little as 6 h by elevating temperature to approximately 60°C. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide information for inactivation of nonspore-forming select agents using elevated temperature and humidity which may aid incident commanders following a biological contamination incident by providing alternative methods for remediation.

Towards development of plasmacytoma cells-based expression systems utilizing alphavirus vectors: An NS0-VEE model.

Plasmacytoma (myeloma) cells have a large protein expression capacity, although their industrial use is confined to stable expression systems. Vectors derived from genomes of viruses from the genus Alphavirus allow obtaining of high yields of target proteins but their use is limited to transient expression. Little information has been published to date on attempts to combine the myeloma cells as hosts with alphaviruses as expression vectors. A plasmid construct which allows rescue of a model alphavirus Venezuelan equine encephalitis virus (VEE) upon transfection of a cell culture was created. Mutations in the capsid and nsP2 genes allow for less cytopathogenic propagation of the virus. A cDNA-copy of the genome was placed in a plasmid under the control of the CMV promoter for virus rescue following DNA transfection. Parameters for the virus rescue by electroporating of the infectious clone in murine myeloma cells (NS0) were optimized. The highest FFU counts (1.2 × 105 FFU per 10 ug DNA) were produced with 2 pulses (voltage 250 V, capacitance 960 u F) and the best electroporation buffer was selected from eight buffers. Self-sustained VEE infection was established in NS0 cultures with high titers (8 × 108 FFU/ml) of the virus, despite a fraction of infected cells dying during 5-days observation. Further development of the NS0-VEE expression system may require addressing of apoptosis induced by VEE.

Protocolo del Sistema Nacional de Vigilancia Epidemiológica: enfermedades zoonóticas. No. 08 / Protocol of the National Epidemiological Surveillance System: zoonotic diseases. No. 08

Estos protocolos están dirigido a personal médico, paramédico y otros profesionales que realizan acciones gerenciales y operativas de vigilancia epidemiológica en los servicios de salud del país, y están divididos en varios tomos para dar a conocer y actualizar la identificación y medidas de control para diversos padecimientos a fin de continuar con el mejoramiento de las capacidades técnicas de los trabajadores de salud, que permita planificar la prestación de servicios con decisiones partiendo de un enfoque epidemiológico comprobado, para responder a los cambios de tendencias epidemiológicas y con ello contribuir al fortalecimiento de prácticas asertivas de la salud pública de nuestro país.

Entry Sites of Venezuelan and Western Equine Encephalitis Viruses in the Mouse Central Nervous System following Peripheral Infection.

UNLABELLED: Venezuelan and western equine encephalitis viruses (VEEV and WEEV; Alphavirus; Togaviridae) are mosquito-borne pathogens causing central nervous system (CNS) disease in humans and equids. Adult CD-1 mice also develop CNS disease after infection with VEEV and WEEV. Adult CD-1 mice infected by the intranasal (i.n.) route, showed that VEEV and WEEV enter the brain through olfactory sensory neurons (OSNs). In this study, we injected the mouse footpad with recombinant WEEV (McMillan) or VEEV (subtype IC strain 3908) expressing firefly luciferase (fLUC) to simulate mosquito infection and examined alphavirus entry in the CNS. Luciferase expression served as a marker of infection detected as bioluminescence (BLM) by in vivo and ex vivo imaging. BLM imaging detected WEEV and VEEV at 12 h postinoculation (hpi) at the injection site (footpad) and as early as 72 hpi in the brain. BLM from WEEV.McM-fLUC and VEEV.3908-fLUC injections was initially detected in the brain's circumventricular organs (CVOs). No BLM activity was detected in the olfactory neuroepithelium or OSNs. Mice were also injected in the footpad with WEEV.McM expressing DsRed (Discosoma sp.) and imaged by confocal fluorescence microscopy. DsRed imaging supported our BLM findings by detecting WEEV in the CVOs prior to spreading along the neuronal axis to other brain regions. Taken together, these findings support our hypothesis that peripherally injected alphaviruses enter the CNS by hematogenous seeding of the CVOs followed by centripetal spread along the neuronal axis. IMPORTANCE: VEEV and WEEV are mosquito-borne viruses causing sporadic epidemics in the Americas. Both viruses are associated with CNS disease in horses, humans, and mouse infection models. In this study, we injected VEEV or WEEV, engineered to express bioluminescent or fluorescent reporters (fLUC and DsRed, respectively), into the footpads of outbred CD-1 mice to simulate transmission by a mosquito. Reporter expression serves as detectable bioluminescent and fluorescent markers of VEEV and WEEV replication and infection. Bioluminescence imaging, histological examination, and confocal fluorescence microscopy were used to identify early entry sites of these alphaviruses in the CNS. We observed that specific areas of the brain (circumventricular organs [CVOs]) consistently showed the earliest signs of infection with VEEV and WEEV. Histological examination supported VEEV and WEEV entering the brain of mice at specific sites where the blood-brain barrier is naturally absent.

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.

Replicon particles of Venezuelan equine encephalitis virus as a reductionist murine model for encephalitis.

Venezuelan equine encephalitis virus (VEE) replicon particles (VRP) were used to model the initial phase of VEE-induced encephalitis in the mouse brain. VRP can target and infect cells as VEE, but VRP do not propagate beyond the first infected cell due to the absence of the structural genes. Direct intracranial inoculation of VRP into mice induced acute encephalitis with signs similar to the neuronal phase of wild-type VEE infection and other models of virus-induced encephalitis. Using the previously established VRP-mRNP tagging system, a new method to distinguish the host responses in infected cells from those in uninfected bystander cell populations, we detected a robust and rapid innate immune response in the central nervous system (CNS) by infected neurons and uninfected bystander cells. Moreover, this innate immune response in the CNS compromised blood-brain barrier integrity, created an inflammatory response, and directed an adaptive immune response characterized by proliferation and activation of microglia cells and infiltration of inflammatory monocytes, in addition to CD4(+) and CD8(+) T lymphocytes. Taken together, these data suggest that a naïve CNS has an intrinsic potential to induce an innate immune response that could be crucial to the outcome of the infection by determining the composition and dynamics of the adaptive immune response. Furthermore, these results establish a model for neurotropic virus infection to identify host and viral factors that contribute to invasion of the brain, the mechanism(s) whereby the adaptive immune response can clear the infection, and the role of the host innate response in these processes.

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.

Venezuelan equine encephalitis virus in the mosquito vector Aedes taeniorhynchus: infection initiated by a small number of susceptible epithelial cells and a population bottleneck.

We evaluated infection of Aedes taeniorhynchus mosquitoes, vectors of Venezuelan equine encephalitis virus (VEEV), using radiolabeled virus and replicon particles expressing green (GFP) or cherry fluorescent protein (CFP). More epidemic VEEV bound to and infected mosquito midguts compared to an enzootic strain, and a small number of midgut cells was preferentially infected. Chimeric replicons infected midgut cells at rates comparable to those of the structural gene donor. The numbers of midgut cells infected averaged 28, and many infections were initiated in only 1-5 cells. Infection by a mixture of GFP- and CFP-expressing replicons indicated that only about 100 midgut cells were susceptible. Intrathoracic injections yielded similar patterns of replication with both VEEV strains, suggesting that midgut infection is the primary limitation to transmission. These results indicate that the structural proteins determine initial infection of a small number of midgut cells, and that VEEV undergoes population bottlenecks during vector infection.

Venezuelan equine encephalitis virus infection of cotton rats.

Venezuelan equine encephalitis virus (VEEV) is an emerging pathogen of equids and humans, but infection of its rodent reservoir hosts has received little study. To determine whether responses to infection vary among geographic populations, we inoculated 3 populations of cotton rats with 2 enzootic VEEV strains (Co97-0054 [enzootic ID subtype] and 68U201 [enzootic IE subtype]). The 3 populations were offspring from wild-caught cotton rats collected in a VEE-enzootic area of south Florida, USA; wild-caught cotton rats from a non-VEE-enzootic area of Texas, USA; and commercially available (Harlan) colony-reared cotton rats from a non-VEE-enzootic region. Although each population had similar early viremia titers, no detectable disease developed in the VEE-sympatric Florida animals, but severe disease and death affected the Texas and Harlan animals. Our findings suggest that the geographic origins of cotton rats are important determinants of the outcome of VEE infection and reservoir potential of these rodents.

Venezuelan equine encephalitis virus infection of mosquito cells requires acidification as well as mosquito homologs of the endocytic proteins Rab5 and Rab7.

Venezuelan equine encephalitis virus (VEEV) is a New World alphavirus that can cause fatal encephalitis in humans. It remains a naturally emerging disease as well as a highly developed biological weapon. VEEV is transmitted to humans in nature by mosquito vectors. Little is known about VEEV entry, especially in mosquito cells. Here, a novel luciferase-based virus entry assay is used to show that the entry of VEEV into mosquito cells requires acidification. Furthermore, mosquito homologs of key human proteins (Rab5 and Rab7) involved in endocytosis were isolated and characterized. Rab5 is found on early endosomes and Rab7 on late endosomes and both are important for VEEV entry in mammalian cells. Each was shown to have analogous function in mosquito cells to that seen in mammalian cells. The wild-type, dominant negative and constitutively active mutants were then used to demonstrate that VEEV requires passage through early and late endosomes before infection can take place. This work indicates that the infection mechanism in mosquitoes and mammals is through a common and ancient evolutionarily conserved pathway.

Adaptation of Venezuelan equine encephalitis virus lacking 51-nt conserved sequence element to replication in mammalian and mosquito cells.

Replication of alphaviruses strongly depends on the promoters located in the plus- and minus-strands of virus-specific RNAs. The most sophisticated promoter is encoded by the 5' end of the viral genome. This RNA sequence is involved in the initiation of translation of viral nsPs, and synthesis of both minus- and plus-strands of the viral genome. Part of the promoter, the 51-nt conserved sequence element (CSE), is located in the nsP1-coding sequence, and this limits the spectrum of possible mutations that can be performed. We designed a recombinant Venezuelan equine encephalitis virus genome, in which the promoter and nsP1-coding sequences are separated. This modification has allowed us to perform a wide variety of genetic manipulations, without affecting the amino acid sequence of the nsPs, and to further investigate 51-nt CSE functioning. The results of this study suggest a direct interaction of the amino terminal domain of nsP2 with the 5' end of the viral genome.

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.

[Study of the effect of culturing system on the phenotypic resistance of Venezuelan equine encephalomyelitis virus]. / Izuchenie vliianiia sistemy kul'tivirovaniia na fenotipicheskuiu ustoichivost' virusa venesuél'skogo éntsefalomielita loshadei.

Comparative assessment of the stability of Venezuelan equine encephalomyelitis virus cultured in BHK-21, Vero, 66-19(1)D, and developing chick embryo cells showed that an increase of the share of phosphatidylcholine and cholesterol in viral envelopes, caused by the conditions of culturing, improved the survival of the virus exposed to heating and some chemicals.

Virulent and attenuated mutant Venezuelan equine encephalitis virus show marked differences in replication in infection in murine macrophages.

One of the first target cells at the site of inoculation with an alphavirus may be monocytes or macrophages. The replication kinetics of virulent and attenuated molecularly cloned Venezuelan equine encephalitis virus (VEE) in murine macrophages were therefore compared. Infection of both quiescent and activated mouse primary peritoneal macrophages with a molecularly cloned, virulent VEE termed V3000 resulted in peak virus titres of 10(4) plaque forming units (PFU)/ml supernatant by 24 h post-infection (pi), followed by rapidly decreasing virus titres. In contrast, a molecularly cloned attenuated VEE mutant, V3032, that differs from V3000 by a single amino acid at E2 glycoprotein position 209 (glu-->lys) replicated more slowly and to higher titres (10(8) PFU/ml supernatant) that peaked at 72 h pi. Replication of V3032, but not V3000, was sharply restricted by prior activation of macrophages with lipopolysaccharide or interferon-gamma. These results indicate that virulent V3000 and attenuated V3032 differ in their growth kinetics in both quiescent and activated macrophages. Thus, macrophages, and their specific activation state, may play a major role in virulent and attenuated VEE replication and pathogenesis.

Spread of Venezuelan equine encephalitis virus in mice olfactory tract.

Spread of Venezuelan equine encephalitis (VEE) virus and damage of the central nervous system (CNS) in mice infected by respiratory route was studied. Virus concentration in organs and blood, "dose-effect" relationships, and ultrastructural lesions in various tissues were examined in immune and normal mice. We showed, via three independent methods--characteristic curve investigations, tissue virus concentration dynamics, and ultrastructural methods--the spread of VEE virus through the olfactory tract into the brain of immune mice. From these experiments it was concluded that in case of respiratory challenge VEE virus can enter the CNS of normal mice by both vascular and olfactory pathways, while in immune mice the main route is olfactory.

Venezuelan equine encephalitis virus propagation in the olfactory tract of normal and immunized mice.

The role of virus spread in the induction of damage to the central nervous system (CNS) of mice infected with Venezuelan equine encephalitis virus (VEEV) via the respiratory route was studied. The virus concentration in various organs and in the blood, the sensitivity to different doses of virus, and ultrastructural lesions in various tissues were examined. It is concluded that VEEV can enter the CNS of nonimmunized mice both by vascular and by olfactory pathways, whereas in immunized mice the olfactory pathway is the main route.

Estimating downwind concentrations of viable airborne microorganisms in dynamic atmospheric conditions.

A Gaussian plume model has been modified to include an airborne microbial survival term that is a best-fit function of laboratory experimental data of weather variables. The model has been included in an algorithm using microbial source strength and local hourly mean weather data to drive the model through a summer- and winter-day cycle. For illustrative purposes, a composite airborne "virus" (developed using actual characteristics from two viruses) was used to show how wind speed could have a major modulating effect on near-source viable concentrations. For example, at high wind speeds such as those occurring during the day, or with short travel times, near-source locations experience high viable concentrations because the microorganisms have not had time to become inactivated. As the travel time increases, because of slow wind speed or longer distances, die-off modulation by sunshine, relative humidity, temperature, etc., potentially becomes increasingly predominant.

Laboratory vector competence of Culex (Melanoconion) cedecei for sympatric and allopatric Venezuelan equine encephalomyelitis viruses.

Laboratory vector competence of Culex (Melanoconion) cedecei was examined for Venezuelan equine encephalomyelitis (VEE) viruses. Colonized adult female mosquitoes originating from a southern Florida population were given bloodmeals from viremic hamsters circulating various titers of 3 hemagglutination inhibition (HI) subtypes of VEE viruses. Following extrinsic incubation of about 3 weeks, mosquitoes were allowed to refeed on uninfected hamsters for transmission trials. Cx. cedecei was highly efficient in becoming infected with and transmitting its sympatric, HI subtype II "Everglades" virus. With bloodmeal titers of 10(0.9) chick embryo cell culture (CEC) plaque forming units (PFU), the infection rate was 9% and transmission occurred following extrinsic incubation. Infection rates were greater than or equal to 80% with oral doses of greater than or equal to 10(1.8), and all infected mosquitoes were capable of transmission following incubation. Cx. cedecei was also highly sensitive to infection with allopatric HI subtype IE Middle American VEE virus isolates. Infection rates were greater than or equal to 50% with bloodmeal titers undetectable by CEC assay. Rates were 100% with oral doses of greater than or equal to 10(0.8) CECPFU. Transmission rates were 100% in all experiments. Similar results were obtained with HI subtype IAB "epizootic" VEE virus isolates from the 1969 Middle American outbreak. Infection rates were 100% with oral doses of greater than or equal to 10(1.2), and transmission rates were 100% after extrinsic incubation. Comparisons with laboratory vector competence of the Middle American enzootic VEE virus vector, Culex (Melanoconion) taeniopus, are discussed.

Replication of alphaviruses in cultures of donkey monocytes.

Representative strains of Venezuelan equine encephalitis virus (VEEV) and Eastern equine encephalitis virus (EEEV) were compared for their ability to grow in cultures of unstimulated leucocytes and monocytes derived from donkey peripheral blood. Replication of epizootic and vaccine strains of VEEV, but not of enzootic strains was observed in this system. Only a minority of monocytes supported virus replication as detected by immunofluorescence, electron microscopy and infectious center assays. EEEV did not appear to replicate in this cell system although virus attached to and was internalized by monocytes.