Caracterización biológica de cepas del virus Encefalitis San Luis en células de la inmunidad innata / Biological characterization of St. Louis Encephalitis virus strains in cells of innate immunity

Los flavivirus transmitidos por mosquitos son una amenaza actual y emergente en todo el mundo. Dentro de este género, el virus Encefalitis San Luis (VESL) causa una forma severa de enfermedad neuroinvasiva donde la respuesta inmune es un componente crucial de la defensa del huésped. En este trabajo se investigó la interacción entre VESL y células de la inmunidad innata, en un modelo de infección in vitro de monocitos humanos (células U937) con cepas de distinta virulencia y condiciones epidemiológicas de aislamiento (CbaAr-4005 y 78V-6507). Se evaluó la capacidad de infectar y replicar del virus, como también el efecto citopático y la cinética de viabilidad de monocitos durante la infección. Los resultados demostraron la susceptibilidad de los monocitos a la infección, replicación y muerte por ambas cepas virales. Sin embargo, se hallaron diferencias significativas entre ellas. La cepa epidémica y de mayor virulencia CbaAr-4005 registró una tasa de infección y replicación superior a la de la cepa endémica y de menor virulencia 78V-6507. Se comprobó también que el VESL indujo la muerte de monocitos humanos, dependiendo del tiempo post-infección (pi) y de la cepa. Así, CbaAr-4005 provocó a partir del día 3 pi el doble de mortalidad celular que 78V-6507. Además, en los monocitos infectados se observaron alteraciones de parámetros morfológicos que podrían relacionarse con el tipo de mecanismo de muerte celular asociado a la infección por VESL.

Mosquitoes borne Flavivirus infections are an actual and emergent worldwide threat to human health. Within this genus, Saint Louis Encephalitis Virus (SLEV) causes a severe neuroinvasive disease where immune response is crucial for host survival. In this study the interaction between SLEV and innate immune cells was evaluated. An in vitro infection model with human monocytes (U937 cells) and strains with variations in virulence and isolation conditions (CbaAr-4005 and 78V-6507) were used. Infection capacity, replication capacity, cytopathic effect and monocyte viability kinetics were measured. The results showed susceptibility to infection and replication to both strains. However, significant differences were found among them. CbaAr-4005, the epidemic and more virulent strain, showed higher infection and replication ratios compared to 78V-6507. SLEV infection that induces cell death of human monocytes was also found in a post-infection time and in a strain dependent manner. Since day 3 post-infection, twice the mortality in CbaAr-4005 infected cells was observed. Furthermore, infected monocytes showed alterations in morphologic parameters that could be related with apoptosis mechanisms associated to SLEV infections.

Os Flavivírus transmitidos por mosquitos são uma ameaça atual e emergente no mundo todo. Nesse gênero, o vírus Encefalite Saint Louis (VESL) causa uma forma grave de doença neuroinvasiva onde a resposta imune é um componente crucial da defesa do hospedeiro. Neste trabalho nos investigamos a interação entre VESL e células de imunidade inata em um modelo de infecção in vitro de monócitos humanos (células U937) com estirpe de diferentes virulências e condições epidemiológicas de isolamento (CbaAr-4005 e 78V-6507). Foi avaliada a capacidade do vírus de infectar e replicar , assim como o efeito citopático e a viabilidade cinética dos monócitos durante a infecção. Os resultados demonstraram a suscetibilidade dos monócitos à infecção, replicação e morte por ambas as estirpes virais. No entanto, foram detectadas diferenças significativas entre eles. A estirpe epidémica e de maior virulenta CbaAr-4005 teve uma maior taxa de infecção e replicação do que a estirpe endémica e menos virulenta 78V-6507. Foi comprovado também que o VESL induziu a morte de monócitos humanos, dependendo do tempo pós-infecção (pi) e da estirpe. Assim, a CbaAr-4005 causou a partir do dia 3 pi o dobro da mortalidade celular o que a 78V- 6507. Além disso, alterações nos parâmetros morfológicos foram observadas nos monócitos infectados que poderiam estar relacionadas ao tipo de mecanismo de morte celular associado à infecção pelo VESL.

The ecology and epidemiology of Ross River and Murray Valley encephalitis viruses in Western Australia: examples of One Health in Action.

Arboviruses are maintained and transmitted through an alternating biological cycle in arthropods and vertebrates, with largely incidental disease in humans and animals. As such, they provide excellent examples of One Health, as their health impact is inextricably linked to their vertebrate hosts, their arthropod vectors and the environment. Prevention and control requires a comprehensive understanding of these interactions, and how they may be effectively and safely modified. This review concentrates on human disease due to Ross River and Murray Valley encephalitis viruses, the two major arboviral pathogens in Australia. It describes how their pattern of infection and disease is influenced by natural climatic and weather patterns, and by anthropogenic activities. The latter includes human-mediated environmental manipulations, such as water impoundment infrastructures, human movements and migration, and community and social changes, such as urban spread into mosquito larval habitats. Effective interventions need to be directed at the environmental precursors of risk. This can best be achieved using One Health approaches to improve collaboration and coordination between different disciplines and cross-sectoral jurisdictions in order to develop more holistic mitigation and control procedures, and to address poorly understood ecological issues through multidisciplinary research.

[Viral encephalitis virus, a new bioterrorist menace]. / Les virus des encéphalites virales, une nouvelle menace bioterroriste.

Often responsible for little known infections, today viral encephalitis viruses appear as a new bioterrorist menace, because of their easy production and their great pathogenic potential. Spraying is the best way to permit the rapid diffusion of certain encephalitis viruses. Diagnosis of viral encephalitis, predominating in tropical surroundings, is difficult. In the majority of cases, symptoms differ little from those of common flu. With supplementary examinations, the biological abnormalities are usually non-specific. There are no characteristic images on scans or MRI. Identification of the virus in the nasopharynx, blood or cerebrospinal fluid, in serology, PCR or RT-PCR permits confirmation of the virus. Treatment is essentially symptomatic and relies on appropriate reanimation measures. Ribavirin can be indicated in some cases such as the Rift Valley fever, but is formally contraindicated in West Nile encephalitis. The aim of terrorist groups who would use this type of weapon is more to provoke panic and disorganisation than to kill as many people as possible.

Detection of Israel turkey meningo-encephalitis virus from mosquito (Diptera: Culicidae) and Culicoides (Diptera: Ceratopogonidae) species and its survival in Culex pipiens and Phlebotomus papatasi (Diptera: Phlebotomidae).

Israel turkey meningo-encephalitis (ITME) virus was detected in pools of Ochlerotatus caspius Pallas and Culicoides imicola Kieffer trapped at a turkey run at Nir David during an outbreak in August 1995. Experimental membrane feeding on a blood ITME suspension showed that Culex pipiens L. became harbored virus for at least 14 d. When Phlebotomus papatasi Scopoli were fed on an infected turkey, they became infected and harbored the virus for at least 7 d. Because Phlebotomines are trapped frequently at turkey runs in Israel, they should be suspected as potential vectors of ITME.

Replication and plaque formation of swine hemagglutinating encephalomyelitis virus (67N) in swine cell line, SK-K culture.

Swine hemagglutinating encephalomyelitis virus (HEV), 67N strain, adapted to suckling mouse brain, grew readily in a porcine cell line, SK-K cell culture with cytopathic effect (CPE) consisting of syncytium formation and detachment of fused cells and round cells from glass surface. After further passages in SK-K cell monolayers with undiluted culture fluid, CPE developed earlier and became complete within 48 h postinoculation (p.i.). Viral specific antigen was detected in the cytoplasm of the infected SK-K cells by indirect immunofluorescence using rabbit antiserum against the mouse-passaged virus. The SK-K-passaged virus as well as the original mouse-passaged virus formed clear plaques on SK-K cell monolayers under simple overlay medium. The plaque assay system for HEV 67N was established by studying various factors influencing the plaque formation in the SK-K cell cultures. By this system more than 10(6) PFU/0.2 ml of the virus yield was detected in the fluid phase of the infected cultures at 48 h p.i. The SK-K-passaged virus caused fatal infection in 4-week-old mice by intracerebral inoculation, but was inhibited by rabbit antiserum against the mouse-passaged virus. Plaque formation and hemagglutinating activity of the virus were specifically inhibited by antisera against the mouse-passaged and SK-K-passaged 67N virus.

Role of antibodies and host cells in plaque enhancement of Murray Valley encephalitis virus.

Chicken antisera to Murray Valley encephalitis (MVE) virus, when incubated with virus and assayed for plaques on chicken embryo (CE) monolayers, neutralized MVE virus at high concentrations of antibody, but caused increases in plaque counts at low concentrations of antibody. Plaque enhancement did not occur when the same virus-antibody mixtures were assayed on a continuous line of rhesus monkey kidney cells (LLC-MK2), nor when the anti-MVE antibody was of mammalian origin and the assay system was CE monolayers. Correspondingly, chicken anti-MVE did not enhance the plaque formation of MVE virus in a stable line of mouse macrophages, P-388D1, whereas rabbit and mouse anti-MVE did enhance plaque formation. This enhancing activity was associated with noncytophilic immunoglobulin G (IgG). The Fc terminus of the IgG molecule was required, as no plaque enhancement occurred with chicken anti-MVE Fab. These data indicate that there is a requirement for taxonomic complementarity between Fc termini and Fc receptors in the above systems. CE cell monolayers were found to contain approximately 2% of Fc receptor-bearing cells among the fibroblast-like cells. Fc receptor-bearing cells in CE monolayers were isolated and found to be of the mononuclear phagocytic lineage. These mononuclear phagocytes, which originate in lymphoid tissues and blood associated with CE tissue fragments, are integrated into primary CE monolayers and form infectious centers in the presence of virus and low dilutions of antibody.

Replication of Borna disease virus in cell cultures.

Borna disease (BD) virus from infected brain tissue of horses or rabbits could be grown in embryonic brain cells from rabbits or rats with high virus yields. The cells became persistently infected and could be subcultivated without loss of infectivity. Cocultivation of infected rabbit brain (ERB) cells with GMK-, Vero-, or MDCK-cells led to persistently infected cell lines. BD virus grown in MDCK cells after cocultivation became adapted to this cell type and could be used directly for further infection of MDCK cells.

Infection rates of Ascocystis-infected Aedes triseriatus following ingestion of La Crosse virus by the larvae.

The La Crosse (LAC) virus infection rate of Aedes triseriatus larvae that ingest LAC virus does not appear to be increased by concomitant infection of larvae by the gregarine parasite, Ascocystis barretti. Infection rates ranged only from 0--2.6% in adult Ae. triseriatus reared from groups of A. barretti-infected larvae that had ingested LAC virus (California encephalitis group) at dosages of 2.0--7.7 log10 SMICLD50/ml. Females resulting from orally infected larvae transmitted LAC virus to suckling mice. Larvae that were infected with A. barretti and devoured carcasses of adult mosquitoes containing 4.7 log10 SMICLD50/ml failed to become infected. A. barretti spores developing in transovarially infected mosquitoes did not harbor LAC virus; thus, A. barretti does not appear to be a mechanism for virus dispersal.

Higher venereal infection and transmission rates with La Crosse virus in Aedes triseriatus engorged before mating.

Infection of colonized female Aedes triseriatus by La Crosse (LAC) virus occurred more frequently when females were inseminated by infected males after the females engorged blood (49% of 39) than when mating took place before engorgement (4% of 554). Salivary transmission of LAC virus to mice also was more frequent in females venerally infected after engorgement on a normal mouse (35% of 34) than in females mated before engorgement (2% of 49). LAC virus was transovarially transmitted by 40% of 10 females mated by infected males, and in 64% of 279 progeny reared from eggs of second or later ovarian cycles.

Aedes triseriatus and La Crosse virus: lack of infection in eggs of the first ovarian cycle following oral infection of females.

La Crosse (LAC) virus filial infection rates were 0% for 279 first ovarian cycle larvae, 43% for 380 second ovarian cycle larvae, and 58% for 363 third ovarian cycle larvae from orally infected mosquitoes representing 14 Wisconsin populations of Aedes triseriatus. LAC virus was not detected in 72 pools representing 2,250 first ovarian cycle larvae, while 35 pools and 16 pools each containing 30 second and third ovarian cycle larvae, respectively, were all positive for LAC virus. Similar results were obtained when the extrinsic incubation period temperature was 25 degrees C, 27 degrees C, or variable (17, 23, and 29 degrees C for 8 hours each). LAC virus was not detected in 240 second ovarian cycle larvae in which the bloodmeal for the first ovarian cycle was non-infectious. Infection was not detected in 337 first ovarian cycle larvae from female mosquitoes that had been injected intrathoracically with LAC virus concomitantly with receiving a non-infectious bloodmeal. After an extrinsic incubation temperature of 25 degrees C, LAC virus was discovered in dissected mosquito ovarian tissue 7 days postfeeding on an infectious bloodmeal. The epidemiological implications of these findings are discussed.

Bunyavirus development in arctic and Aedes aegypti mosquitoes as revealed by glucose oxidase staining and immunofluorescence.

Northway virus replication has been detected in salivary glands of wild-caught Culiseta inornata and Aedes communis mosquitoes from the western Canadian Arctic after incubation at 4 degrees C for 9 to 11 months, and after incubation at 13 degrees C for 3 to 4 months after they received virus by oral ingestion or intrathoracic injection. Aedes hexodontus supported Northway virus replication after incubation at 13 degrees C for one month after intrathoracic injection. Aedes aegypti supported Northway virus replication after incubation at 13 degrees C or 23 degrees C for 6 to 28 days following intrathoracic injection. A larval isolate of California encephalitis virus (snowshoe hare subtype) multiplied in all 3 species of arctic mosquito after incubation at 13 degrees C for 1 to 3 months after virus was administered by oral ingestion or intrathoracic injection. Virus was detected in salivary glands of Cs. inornata after 329 days incubation at 4 degrees C after intrathoracic injection. Bunyavirus antigens in salivary glands of arctic and domestic mosquitoes were detected by the glucose oxidase immunoenzyme technique somewhat less frequently than by assay for virus infectivity.

Experimental infection of Putorius eversmanni polecats and Martes foina martens with Tahyna virus.

Three Putorius eversmanni pole-cats and two Martes foina martens aged about 9 months were subcutaneously infected with about 260 suckling mouse LD50 of the extraneurally passaged "236" strain of Tahyna virus (California group, genus Bunyavirus). Viraemia with maximal titres of 1.32 (pole-cats) and 1.28 (martens) dex intraperitoneal (i.p.) mouse LD50/0.02 ml was demonstrated from 48 to 96 hr after inoculation (p.i.). By the plaque-reduction neutralization test, seroconversion was demonstrated 15 days p.i. (from less than 4 to titres of 8192 in pole-cats and 4096 in martens).

Infection rates of Aedes triseriatus following ingestion of La Crosse virus by the larvae.

Infection rates ranged from 0-2.1% in adults of Aedes triseriaus reared from groups of larvae that had ingested La Crosse (LAC) virus (Clifornia encephalitis group) at dosages of 7.0-8.3 log 10 SMICLD50/ml. Females form orally infected larvae transmitted the virus to suckling mice. Larvae that devoured carcasses of transovarially infected larvae containing 3.0 log 10 SMICLD 50/ml failed to become infected. Ingestion by larvae of infected carcasses appears, therefore, to be unimportant as a method of horizontal amplification of LAC virus.

Transmission of Northway and St. Louis encephalitis viruses by arctic mosquitoes.

Transmission of a Canadian arctic isolate of Northway virus has been demonstrated after incubation of arctic Aedes communis mosquitoes at 13 degrees C for 27 days after intrathoracic injection of 300 plaque forming units of virus. Replication has also been demonstrated after intrathoracic injection of domestic A. aegypti mosquitoes of this virus. Virions of Northway virus, 84--92 nm diameter were morphologically typical of a bunyavirus after propagation in salivary glands of A. communis or in tissue cultures of baby hamster kidney (BHK-21) cells. An Ontario isolate of St. Louis encephalitis was transmitted by bites of A. communis after 27 days incubation at 13 degrees C after oral ingestion of 3 or 30 mouse LD50 virus. This mosquito species transmitted virus after 13 to 76 days incubation at 13 degrees C following intrathoracic injection of 3 mouse LD50 or higher virus doses.

The role of the nucleus in the replication of measles virus.

The replication of measles and SSPE viruses in enucleate BSC-1 cells was measured by plaque assay, and the synthesis of virus-specific antigen was measured by indirect immunofluorescence. Titres of infectious virus produced in enucleate cells at 24 hours post inoculation (p.i.) were consistently 100-fold less, and the number of enucleate cells containing virus antigen at 30 hours p.i. was 10-fold less, than in nucleate control cultures. Enucleate cells at this time had 30% of the protein synthetic capacity of control nucleate cells, and supported the growth of Semliki Forest virus to titres equivalent to nucleate cell production.

Venereal transmission of La Crosse (California encephalitis) arbovirus in Aedes triseriatus mosquitoes.

Veneral transmission of La Crosse virus by males of Aedes triseriatus was demonstrated. La Crosse virus was detected in the bursa of females after induced copulation, and disseminated infection was shown to occur occasionally. Since males of Aedes triseriatus have transovarial filial infection rates similar to those of females and can repeatedly mate, veneral transmission may be an important supplement to other natural endemic maintenance mechanisms.