Reliable detection of St. Louis encephalitis virus by RT-nested PCR

Introducción. El virus de la encefalitis de St. Louis (VESL), arbovirus reemergente en Sudamérica, provocó casos humanos en Argentina y Brasil. Esto pone de manifiesto la necesidad de incrementar el conocimiento sobre arbovirus para poder controlar y prevenir la aparición de futuros casos. Por este motivo, surge la necesidad de realizar exhaustivas investigaciones epidemiológicas y de laboratorio para asegurar la rápida identificación del agente y una apropiada acción de los agentes de salud. En este estudio se describe el desarrollo de una técnica de RT-nested PCR específica para la detección del VESL. Material y métodos. Se procedió a la selección de la región genómica del VESL que aportara mayor información sobre la variabilidad genética natural del virus. Así, se diseñaron cebadores degenerados que amplificaron un fragmento de 234 pb del gen de la envoltura de 9 cepas de VESL (Parton, BeH356964, SPAN11916, AN9275, AN9124 y 78V6507 y tres obtenidas de agrupamientos de mosquitos naturalmente infectados). Resultados. El método amplificó el genoma de todas las cepas del VESL analizadas y no se obtuvo amplificación con otros Flavivirus, tales como el virus de la fiebre amarilla, el virus Ilheus, el virus dengue-2, el virus Bussuquara, el virus del Oeste del Nilo, el virus de la encefalitis japonesa y el virus del valle Murray. Este método fue específico y sensible, con un bajo límite de detección: menos de 10 unidades formadoras de placa. Conclusión. La técnica desarrollada resultó ser confiable y de amplio espectro para la detección del VESL, y puede ser útil para la ejecución de estudios ecológicos, clínicos y de vigilancia virológica (AU)

Introduction. St. Louis encephalitis virus (SLEV) is a re-emerging arbovirus in South America, with reported cases in humans in Argentina and Brazil. This fact indicates that there is an urgent need to increase the current knowledge about this virus in order to control and prevent future cases. Exhaustive epidemiological and laboratory investigation is required to ensure fast, accurate identification of the viral agent and allow prompt surveillance action by health authorities. Herein, we report the development of a species-specific RT-nested PCR to detect SLEV. Material and methods. After selecting the SLEV genomic region providing the greatest information on the natural genetic variability of this virus, degenerated oligonucleotide primers were designed to amplify a 234-bp fragment of the envelope gene from nine SLEV strains (Parton, BeH356964, SPAN11916, AN9275, AN9124, 78V6507 and 3 SLEV strains obtained from naturally infected mosquito pools). Results. The method was able to identify the genome of all the SLEV strains tested and did not amplify unrelated RNA viruses, such as yellow fever virus, Ilheus virus, dengue-2 virus, Bussuquara virus, West Nile virus, Japanese encephalitis virus and Murray Valley encephalitis virus. The method was specific and sensitive, with a lower detection limit of < 10 plaque-forming units. Conclusion. This molecular assay is a reliable procedure with a wide spectrum for detecting the natural diversity of SLEV and may be useful for ecological studies, clinical and laboratory settings and virological surveillance (AU)

Genotype III Saint Louis encephalitis virus outbreak, Argentina, 2005.

Twenty-six years after it was last detected, Saint Louis encephalitis virus (SLEV) genotype III reemerged in 2005 in C6rdoba, Argentina, where it caused an outbreak. Two genotype III SLEV strains were isolated from Culex quinquefasciatus. A 71.43% prevalence for neutralizing antibodies was found in domestic fowl in the homestead of a patient with encephalitis.

[The relationship between mosquito vectors and aquatic birds in the potential transmission of 2 arboviruses]. / Relación entre mosquitos vectores y aves acuáticas en la transmisión potencial de dos arbovirus.

The authors studied for two years the role of the chicks of aquatic birds in the arboviral cycles in coastal lagoons in central Panama in order to determine the relation between Culex (Melanoconion) ocossa and Mansonia (Mansonia) dyari mosquitoes in the transmission and dissemination of the viruses of Saint Louis Encephalitis (SLE) and Venezuelan Equine Encephalitis (VEE). Mosquitoes were captured every fifteen days on two consecutive nights to isolate the virus, using light traps (CDC) and baited traps. The attempts to isolate the virus were made using Vero cell cultures and the determination of antibodies was performed. The results of the serologic tests seem to indicate that four bird species: the ex (?) heron (Bubulcus ibis), the American heron (Casmerodius albus), the spoon-billed duck (Cochlearius cochlearius) and the needle crow (Anhinga anhinga) could function as intermediate hosts in the transmission cycle of SLE. Two species, the ibis (Endocimus albus) and the spoon-billed duck (Cochlearius cochlearius) could also be intermediate hosts of VEE in the coastal lagoons of Panama. The presence of antibodies in chicks could indicate an infection acquired recently, after their birth, in this area. The VEE virus was recovered from blood filled mosquitoes which had fed on a spoon-billed duck probably infected and exposed in a Trinidad #10 trap. No SLE virus was isolated. Other unknown viruses were isolated from mosquitoes selected for these studies, such as C. ocossa and M. dyari. The results obtained with these studies indicate the need for more studies utilizing new field techniques in order to establish a link between SLE and VEE, the vector mosquitoes and the aquatic birds in the coastal lagoons of the area under investigation.

Relación entre mosquitos vectores y aves acuáticas en la transmisión potencial de dos arbovirus / The relationship between mosquito vectors and aquatic birds in the potential transmission of 2 arboviruses

The authors studied for two years the role of the chicks of aquatic birds in the arboviral cycles in coastal lagoons in central Panama in order to determine the relation between Culex (Melanoconion) ocossa and Mansonia (Mansonia) dyari mosquitoes in the transmission and dissemination of the viruses of Saint Louis Encephalitis (SLE) and Venezuelan Equine Encephalitis (VEE). Mosquitoes were captured every fifteen days on two consecutive nights to isolate the virus, using light traps (CDC) and baited traps. The attempts to isolate the virus were made using Vero cell cultures and the determination of antibodies was performed. The results of the serologic tests seem to indicate that four bird species: the ex (?) heron (Bubulcus ibis), the American heron (Casmerodius albus), the spoon-billed duck (Cochlearius cochlearius) and the needle crow (Anhinga anhinga) could function as intermediate hosts in the transmission cycle of SLE. Two species, the ibis (Endocimus albus) and the spoon-billed duck (Cochlearius cochlearius) could also be intermediate hosts of VEE in the coastal lagoons of Panama. The presence of antibodies in chicks could indicate an infection acquired recently, after their birth, in this area. The VEE virus was recovered from blood filled mosquitoes which had fed on a spoon-billed duck probably infected and exposed in a Trinidad #10 trap. No SLE virus was isolated. Other unknown viruses were isolated from mosquitoes selected for these studies, such as C. ocossa and M. dyari. The results obtained with these studies indicate the need for more studies utilizing new field techniques in order to establish a link between SLE and VEE, the vector mosquitoes and the aquatic birds in the coastal lagoons of the area under investigation

Arbovirus investigations in Argentina, 1977-1980. I. Historical aspects and description of study sites.

This is the introductory paper to a series on the ecology of arboviruses in Argentina. Epizootics of equine encephalitis have occurred since at least 1908, principally in the Pampa and Espinal biogeographic zones, with significant economic losses; human cases of encephalitis have been rare or absent. Both western equine and eastern equine encephalitis viruses have been isolated from horses during these epizootics, but the mosquitoes responsible for transmission have not been identified. A number of isolations of Venezuelan equine encephalitis (VEE) virus were reported between 1936 and 1958 in Argentina, but the validity of these findings has been seriously questioned. Nevertheless, serological evidence exists for human infections with a member of the VEE virus complex. Serological surveys conducted in the 1960s indicate a high prevalence of infection of humans and domestic animals with St. Louis encephalitis (SLE), and 2 SLE virus strains have been isolated from rodents. Human disease, however, has rarely been associated with SLE infection. Only 7 isolations of other arboviruses have been described (3 of Maguari, 1 of Aura, 2 of Una, and 1 of an untyped Bunyamwera group virus). In 1977, we began longitudinal field studies in Santa Fe Province, the epicenter of previous equine epizootics, and in 1980 we extended these studies to Chaco and Corrientes provinces. The study sites are described in this paper.

Experimental studies of St. Louis encephalitis virus in vertebrates.

Serologically negative birds and mammals of species, known from other studies to be exposed naturally to St. Louis encephalitis (SLE) virus in Memphis, Tennessee, and other selected species were inoculated experimentally with strains of SLE virus to determine their potential as natural hosts. Mosquitoes (Culex sp.) were allowed to feed on some of the inoculated vertebrate species, held for 14 days, and tested for SLE infection. The cardinals (Richmondena cardinalis), robins (Turdus migratorius), and baby chicks (Gallus gallus) all became viremic; 97% of the bobwhites (Colinus virginianus) and 20% of the Japanese quail (Coturnix coturnix) became viremic. No viremia was detected in raccoons (Procyon lotor), opossums (Didelphis virginiana), or adult cotton rats (Sigmodon hispidus). Only 20% of cottontail rabbits (Sylvilagus audubonii), 50% of wood rats (Neotoma mexicana), and 75% of hamsters (Mesocricetus auratus) but all the young cotton rats and least chipmunks (Eutamias minimus) were susceptible. Robins had the highest titered viremia but were viremic for the shortest period of time. Bobwhites had lower peak viremia titers but for a longer duration. Biologic differences in the response of some vertebrates to different SLE strains were noted. Culex pipiens quinquefasciatus mosquitoes readily became infected after feeding on viremic cardinals. Comparisons of the experimental data with information obtained from field investigations provided a better understanding of the contributions of the various vertebrate species to the transmission and maintenance of SLE virus in nature.

Experimental St. Louis encephalitis virus infection of sloths and cormorants.

Experimental infection of 11 Bradypus variegatus and Choloepus hoffmanni sloths with St. Louis encephalitis (SLE) virus produced detectable viremias of seven to 27 (median 13) days duration and maximum titers of 2.7 to 6.5 (median 5.1) log10 median suckling mouse intracranial lethal doses (SMicLD50) per ml. Experimental SLE viremia onset was delayed and maximum titer depressed in two sloths concurrently infected with naturally acquired viruses. SLE viremias in four experimentally inoculated cormorants Phalacrocorax olivaceus were shorter, and of equal or lower titer, than in sloths. Colonized Culex pipiens quinquefasciatus mosquitoes were infected by feeding on sloths circulating at least 4.8 log10 SMicLD50 of SLE virus per ml, and subsequently transmitted the infection to mice and chicks. An uninoculated baby Bradypus became infected by contact transmission from its mother. The antibody response of sloths to SLE virus was slow, being undetectable until several weeks post-inoculation. However, both sloth species developed high and long-lasting neutralizing and hemagglutination-inhibition antibody titers. The complement-fixation antibody response in Bradypus was lower and slower to develop than in Choloepus. Sloths with naturally acquired SLE virus antibody did not become detectably viremic after experimental inoculation. Neither sloths nor cormorants become overly ill from SLE virus infection.

Mode of entry of a neurotropic arbovirus into the central nervous system. Reinvestigation of an old controversy.

The mechanism by which neurotropic arboviruses gain access to the central nervous system remains uncertain, although it is generally assumed that viremic infection results in growth across or passive diffusion through brain capillaries. In contrast to the natural reservoir hosts of these arboviruses, clinical hosts (e.g., horses, humans) have viremias of very brief duration and low magnitude. We investigated the question of neuroinvasion in 5- to 6-week-old Syrian hamsters infected with St. Louis encephalitis virus (strain TBH-28). This model shares with the human disease low or undetectable viremia and many clinical and pathoanatomical features. The mortality rate after intraperitoneal inoculation of a moderate viral dose was 88%. No viremia was detectable by a sensitive assay in 31% of the animals. In the remaining hamsters, the mean peak viremia was 1.0 log10 plaque-forming units/0.05 ml and the mean duration 1 to 2 days. There was no correlation between viremia and outcome of infection, length of incubation period, or brain virus titer. Tissue infectivity studies showed a rise in titer in the olfactory neuroepithelium on day 4 postinoculation, then in the olfactory bulbs (day 5 postinoculation), and finally in the remainder of the brain (day 6 postinoculation). Specific immunofluorescence was demonstrated in the bipolar neurons of the olfactory epithelium, their dendrites, and in axon bundles of the olfactory nerves in the submucosa. By electron microscopy, virus particles and associated tubular structures were demonstrated within dendrites, perikarya, and axons of olfactory neurons, and to a lesser extent in macrophages and Bowman's gland cells in the lamina propria. In cells of Bowman's glands large numbers of virions were sequestered within secretory granules. Virus was recovered from nasal washings on day 4 postinoculation. Similar findings were obtained in weanling mice inoculated intraperitoneally with another (mouse-virulent) St. Louis encephalitis viral strain (77V-12908). These data taken together indicate that the olfactory pathway is the principal route of viral entry into the central nervous system. After peripheral inoculation a low-level viremia results in infection of highly susceptible cells in the olfactory neuroepithelium, allowing centripetal axonal transport of virus to the olfactory bulb, whence spread is unimpeded throughout the neuropil of the central nervous system. Infection of Bowman's gland cells in the olfactory mucosa and shedding of virus in nasal mucus may be an adaptation for nonarthropod-borne transmission, a feature of many flaviviruses.

Isolation of St. Louis encephalitis virus from a patient in Belém, Brazil.

St. Louis encephalitis (SLE) virus was isolated from a 21-year-old female hospitalized on 4 October 1978 in Belém, Brazil. Symptomatology on admission included fever, chills, severe headache, abdominal pain, myalgia, arthralgia and jaundice. SLE virus was isolated from her blood drawn on the 8th day of illness and subsequent seroconversion was documented. Serological tests showed the isolate to be closely related to the Belém prototype of SLE virus but distinct from other flaviviruses tested. The patient was discharged without sequelae after 16 days of hospitalization. Epidemiological investigations where the patient worked and lived revealed no evidence of extensive transmission of SLE virus.

Geographic variation among St. Louis encephalitis virus strains in the viremic responses of avian hosts.

We studied the capacity of 44 strains of St. Louis encephalitis (SLE) virus to induce viremia in an epidemiologically important wild avian host, the House Sparrow (Passer domesticus). Selected virus strains were also inoculated at varying doses into 3-week-old chicks. Viremic responses were analyzed in terms of the proportion of inoculated nestling and adult birds which became viremic, the mean duration and the mean peak titer of viremia. Infectivity of avian sera was determined by plaque assay in primary duck embryo cell cultures. The susceptibility of the House Sparrow to viremic infection with different SLE virus strains varied markedly. Nestling sparrows 6-10 days of age were generally more susceptible than adult birds. All virus strains isolated during Culex pipiens-borne epidemics in the eastern United States were highly viremogenic [viremia in 80% of birds with a mean duration of greater than or equal to 1.6 days in adults or greater than or equal to 2.7 days in nestlings and a mean peak titer of greater than or equal to 10(3.0) plaque-forming units (PFU)/ml in adults and 10(4.0) PFU/ml in nestlings]. All virus strains isolated from Culex tarsalis in the western United States, strains isolated from rodents in South America. and six of 16 strains isolated from various sources elsewhere in tropical America were partially or fully attenuated. A high degree of concordance was demonstrated between experimental viremia in sparrows, viremia in 3-week-old chicks, and neurovirulence for weanling mice. The epidemilogic significance of these findings is discussed.

Arboviruses in New York State: an attempt to determine the role of arboviruses in patients with viral encephalitis and meningitis.

In a reveiw of 2,963 patients with signs of infections of the central nervous system in New York State in 1966--1977, arboviruses were found to be the confirmed or presumptive etiologic agents in 60 patients. California encephalitis (CE) virus was the most common (44 patients), followed by Powassan (POW) virus (8), St. Louis encephalitis virus (7), and eastern equine encephalitis (EEE) virus (1). Most patients (47) were children. The incidence of encephalitis was higher in patients with arbovirus findings than in infections with any other of the common neurotropic viruses. The disease was fatal in two patients, one infected with POW virus, the other with EEE virus. Most patients with CE virus infections resided in suburban areas. All POW infections were contracted in six rural counties known for their recreational facilities.

Isolation of St. Louis encephalitis virus in South Brazil.

Eight strains of a flavivirus identified as St. Louis encephalitis (SLE) virus were isolated from wild rodents, birds, and sentinel mice in three locations in the State of São Paulo, Brazil from 1967--1969. No illness attributable to SLE virus infection was detected among the local inhabitants, although about 5% of the local population had neutralizing antibodies to this virus.

Survival of arboviruses in trypanosome-infected triatomines.

The potential of triatomines to maintain arboviruses was demonstrated by the ability of Rhodinius prolixus with experimentally punctured abdomen to harbor Venezuelan equine encephalitis (VEE) virus for at least 4 months and St. Louis encephalitis virus for 1 month. At 30 days after infection VEE virus was found at low titers in Trypanosoma cruzi-infected R. prolixus and at moderate titers in T. rangeli-infected R. neglectus. Transmission of VEE virus by bite of punctured bugs was successful 2 weeks after virus ingestion; attempts at 30 days failed.

St. Louis encephalitis in southern Ontario: laboratory studies for arboviruses.

The first reported outbreak of St. Louis encephalitis in Canada occurred in the summer of 1975 in southern Ontario -- in the Windsor-Sarnia-Chatham area, the Niagara region and the city of Toronto. Hemmagglutination inhibition and complement fixation testing of serum samples collected during the outbreak confirmed that St. Louis encephalitis virus was the etiologic agent. Furthermore, this virus was isolated from brain tissue of a patient who died. This outbreak was probably an extension of the outbreak that occurred in the United States that summer. It was the first outbreak of arbovirus encephalitis in the province of Ontario.