Characterization of arenaviruses using a family-specific primer set for RT-PCR amplification and RFLP analysis. Its potential use for detection of uncharacterized arenaviruses.

Arenaviruses are enveloped viruses with a genome composed of two ssRNA species, designated L and S. The arenaviruses were divided in two major groups (Old World and New World), based on serological properties and genetic data, as well as geographic distribution. A sequence alignment analysis of all reported arenavirus S RNAs yielded 17 conserved regions in addition to a reported conserved region at the end of both RNAs. The consensus sequences of these regions were used to design generalized primers suitable for RT-PCR amplification of a set of overlapping nucleotide sequence fragments comprising the complete S RNA of any arenavirus. A restriction analysis (RFLP) was designed to rapidly typify the amplified fragments. This RT-PCR-RFLP approach was tested with Old World (LCM) and New World (Junin and Tacaribe) arenaviruses. Furthermore, using this procedure the whole S RNA of a novel arenavirus isolate obtained from a rodent trapped in central Argentina, was amplified and characterized. Partial nucleotide sequence data were used for phylogenetic analyses that showed the relationships between this arenavirus and the rest of the members of the family. This relatively simple methodology will be useful both in basic studies and epidemiological survey programs.

The role of mononuclear blood cells in experimental Junin virus spread to the central nervous system.

The neuroinvasiveness of Junin virus depends on the viral strain, animal species, and age. The role of infected blood cells in hematogenous Junin virus spread to the central nervous system (CNS) was studied by determining the growth in pheripheral mononuclear cells and brain tissue of Candid 1 and XJCL3 laboratory strains, in Calomys musculinus and guinea pigs. The present study demonstrated that Junin virus replicates in circulating peripheral lymphocytes and macrophages of 11-day-old guinea pigs and 6 +/- 1-day-old Calomys musculinus. Moreover, the observation that mononuclear phagocyte depletion did block Junin virus neuroinvasion firmly indicates that the cellular viremia (circulating monocytes) is one of the mechanisms of Junin virus attenuated strains spread to the CNS in animal hosts.

Western equine encephalomyelitis: virulence markers and their epidemiologic significance.

Comparative studies are described on the virulence of the western equine encephalomyelitis (WEE) complex viruses for mice. Three epizootic WEE virus strains (McMillan, Cba 87, and Cba CIV 180) and five enzootic WEE complex viruses (Highlands J [HJ], Y62-33, Aura, Fort Morgan [FM], and WEE AG80-646) were examined. The neurovirulence and the neuroinvasiveness of these viruses for adult mice were established and correlated with viremia and virus replication in brain tissue. Adult mice inoculated intraperitoneally showed differential responses that corresponded to the epidemiologic attributes of WEE viruses. Viruses associated with equine epizootics were neurovirulent and neuroinvasive, whereas enzootic viruses were neither neuroinvasive nor neurovirulent. In North America, HJ virus appears to be an antigenic link with an intermediate virulence between epizootic WEE virus and the enzootic FM virus. The HJ virus has been associated with rare cases of sporadic equine and human diseases. In South America, no virus with intermediate virulence characteristics has been described. We speculate that epizootics may arise from nonpathogenic strains such as AG80-646 maintained in enzootic transmission cycles.

Cross-neutralization tests among Cache Valley virus isolates revealing the existence of multiple subtypes.

Maguari virus has been classified as a subtype of Cache Valley virus. Seven Bunyamwera serogroup viruses (including prototypes Cache Valley and Maguari viruses), 4 viruses shown in previous tests as close antigenic relatives of Maguari or Cache Valley viruses, and Xingu virus were cross-tested by serum dilution-plaque reduction neutralization. All viruses were distinguishable from prototypes Cache Valley and Maguari viruses. The close antigenic relationships of the Cache Valley-like viruses demonstrate that multiple subtypes of Cache Valley virus exist and suggest that such antigenic variation is a phenotypic expression of considerable genetic diversity.

Kairi virus identified from a febrile horse in Argentina.

A virus isolated from the blood of a febrile horse in Argentina was identified as a strain of Kairi virus. This is the fifth Bunyamwera serogroup virus isolated from livestock and wild animals in the Americas. Bunyamwera serogroup viruses have been isolated from febrile humans in the Americas and Africa.

Arbovirus investigations in Argentina, 1977-1980. II. Arthropod collections and virus isolations from Argentine mosquitoes.

Prospective surveys for arboviruses were carried out in Santa Fe, Corrientes, and Chaco provinces, Argentina, aperiodically during 1977-1980. A total of 313,233 mosquitoes and 598 biting flies other than mosquitoes were collected and tested for virus in 5,197 and 45 pools, respectively. Forty virus strains were isolated, all from mosquitoes, as follows: Santa Fe Province: 4 Gamboa group viruses from Aedeomyia squamipennis, 1 strain each of St. Louis encephalitis virus from Culex pipiens quinquefasciatus and Culex (Culex) spp.; Corrientes Province: a single strain of a newly discovered Anopheles A serogroup virus, Las Maloyas, from Anopheles albitarsis; and Chaco Province: 4 Gamboa group viruses from Ad. squamipennis, 6 strains of new Bunyaviridae (1 Antequera, 1 Barranqueras, and 4 Resistencia) from Culex (Melanoconion) delpontei, 3 strains of a new subtype of western equine encephalitis virus and 1 strain of Para virus from the Cx. (Mel.) ocossa group, 12 strains of a newly discovered subtype (VI) of the Venezuelan equine encephalitis complex from Cx. (Mel.) delpontei, and 1 strain each from Ad. squamipennis, Aedes scapularis, Ae. spp., Cx. (Cux.) spp., Cx. (Mel.) ocossa group, Mansonia spp., and Psorophora spp. Bloodmeals from 265 engorged mosquitoes were identified by precipitin test. These data, coupled with data on engorgement rates for 25,995 mosquitoes from bait collections, provide information on the host feeding patterns of several mosquito species. This information is discussed, along with data on relative abundance of mosquito species, within the context of the vector relationships of the species from which viruses were isolated. The association of Cx. (Mel.) delpontei with 18 strains of 4 different viruses in Chaco Province, plus its catholic feeding habits, clearly indicate for the first time the importance of this species as an arbovirus vector.

Arbovirus investigations in Argentina, 1977-1980. III. Identification and characterization of viruses isolated, including new subtypes of western and Venezuelan equine encephalitis viruses and four new bunyaviruses (Las Maloyas, Resistencia, Barranqueras, and Antequera).

Forty viruses isolated from mosquitoes between 1977 and 1980 in Argentina have been identified and characterized. Nineteen strains of VEE virus, identical by neutralization (N) tests, were shown by hemagglutination-inhibition tests with anti-E2 glycoprotein sera to represent a new subtype VI of the VEE complex. RNA oligonucleotide fingerprints of this virus were distinct from subtype I viruses. The virus was not lethal for English short-haired guinea pigs, indicating that it is probably not equine-virulent. Three strains of a member of the WEE virus complex were shown to differ by N tests in 1 direction from prototype WEE virus. The new WEE subtype was also found to be distinct by RNA oligonucleotide mapping. Its vector relationships indicate that it is an enzootic virus, and it has not been associated with equine disease. A new member of the Anopheles A serogroup was identified, shown to be most closely related to Lukuni and Col An 57389 viruses, and given the name Las Maloyas virus. A strain of Para virus (Bunyaviridae, Bunyavirus) was identified. Six isolates, representing 3 new viruses morphologically resembling bunyaviruses are described; the names Antequera, Barranqueras, and Resistencia are proposed for these agents, which were all isolated from Culex (Melanoconion) delpontei in Chaco Province. No serologic relationships between these viruses and other bunyaviruses were found. Since they are antigenically interrelated, they form a new (Antequera) serogroup. Eight Gamboa serogroup viruses and 2 strains of St. Louis encephalitis virus were also identified.

Arbovirus investigations in Argentina, 1977-1980. IV. Serologic surveys and sentinel equine program.

Serologic surveys of wild and domestic birds, wild mammals, and horses were conducted during arbovirus field studies in Argentina from 1977 through 1980, a non-epizootic interval. The prevalence of neutralizing antibodies to eastern equine encephalitis (EEE) was consistently higher than to western equine encephalitis (WEE) virus in all species and all areas. The presence of antibodies in short-lived avian species and in young unvaccinated horses and the demonstration of seroconversions in horses during the period, indicated that these viruses are either enzootic in, or annually reintroduced into, Argentina. Antibodies to AG80-646, a new subtype of WEE virus isolated in the subtropical north (Chaco Province) from Culex (Melanoconion) mosquitoes, were found in horses and rodents in that region. Antibodies to the TC-83 strain of Venezuelan equine encephalitis (VEE) virus were found in all areas studied. The presence of antibodies in some horses was probably related to vaccination, but the demonstration of seroconversions in sentinel horses and of antibodies in birds and wild mammals indicates active transmission of VEE virus. In 1980 a new enzootic subtype of VEE virus (AG80-663) was isolated from mosquitoes in Chaco; neutralizing antibodies to this virus were prevalent in horses and rodents in this area. Infections with Aura and Una viruses were most common in the subtropical northern provinces. Infection with St. Louis encephalitis was prevalent and widespread, and birds, principally passerine and columbiform species, appear to be the principal hosts. An interesting and unexplained finding was the absence of arbovirus antibodies, in particular SLE antibodies in house sparrows (Passer domesticus). Antibody prevalences in horses exceeded 50% in all areas, and 12% of horses surveyed in Santa Fe Province developed antibody in a 17-month period. Antibodies to other flaviviruses were rare. A high prevalence of immunity to Maguari virus was found in horses; this agent is considered to be a potential equine pathogen. Antibodies to 2 new viruses, Barranqueras and Resistencia, which had been isolated from Cx. (Melanoconion) in Chaco Province, were found in rodents there. Immunity to Gamboa group viruses was prevalent, and birds were implicated as principal hosts.

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.

Viruses isolated from Aedeomyia squamipennis mosquitoes collected in Panama, Ecuador, and Argentina: establishment of the Gamboa serogroup.

Twenty-four virus strains were isolated from Aedeomyia squamipennis mosquitoes collected in Ecuador. One additional strain each was isolated from this species from Panama and ARgentina. All 26 isolates were shown to be related serologically to prototype Gamboa virus, originally isolated from Ad. squamipennis mosquitoes collected in Panama. Antigenic comparisons of eight strains, including prototype Gamboa virus, indicated the existence of four distinct viruses. Neutralization tests with sera from a variety of mammalian and avian species from Argentina provided further evidence that Gamboa serogroup viruses are transmitted between Ad. squamipennis and birds.

Arbovirus isolations from mosquitoes collected during and after the 1982-1983 epizootic of western equine encephalitis in Argentina.

Mosquitoes were collected in Santa Fe and Rio Negro provinces, Argentina, in 1982-1983 during a western equine encephalitis (WEE) epizootic. Totals of 153,084 mosquitoes from Santa Fe Province and 484 from Rio Negro Province were tested for virus in 2,351 pools. Seventeen virus strains were isolated, all from Santa Fe collections, as follows: 4 WEE, 6 Venezuelan equine encephalitis, 1 St. Louis encephalitis, 2 Antequera, 1 Maguari, 1 Melao, 1 new vesiculovirus (Calchaqui), and 1 Gamboa. The WEE virus isolates were from Aedes albifasciatus, Anopheles albitarsis, Mansonia species, and Psorophora pallescens. Collections during the spring and summer (1983-1984) following the epizootic yielded 49,707 mosquitoes from Santa Fe, 15,961 from Rio Negro, and 2,019 from Chubut provinces. Twenty-two virus strains were isolated, all from Santa Fe mosquitoes, as follows: 3 strains of SLE virus and 19 strains of Turlock (TUR) virus. All but one of the TUR virus isolates appear to have come from mosquitoes that engorged on a viremic chicken following entry into a bait trap. The vector relationships of each virus isolated during and after the WEE epizootic are discussed.

A newly recognized vesiculovirus, Calchaqui virus, and subtypes of Melao and Maguari viruses from Argentina, with serologic evidence for infections of humans and horses.

In 1983, 17 virus strains were isolated from mosquitoes collected during an outbreak of western equine encephalitis in Santa Fe Province, Argentina. Strains of western equine encephalitis, Venezuelan equine encephalitis, St. Louis encephalitis, and Antequera viruses were isolated, as were several bunyaviruses of the California and Bunyamwera serogroups and a new vesiculovirus. Complement fixation and neutralization tests were used to identify the California serogroup virus as a subtype of Melao virus, the Bunyamwera serogroup virus as a subtype of both Maguari and Playas viruses, and the vesiculovirus as a newly recognized agent for which the name Calchaqui virus is proposed. A limited serosurvey of horses and humans in Santa Fe Province and horses from the adjacent Santiago del Estero Province was performed to determine the prevalence of neutralizing antibody to the subtypes of Melao and Maguari viruses and to Calchaqui virus. The high prevalence of antibodies to these three agents indicates the need for further studies of their disease potential in horses, because they are closely related to several other viruses that are known equine pathogens.

Transmission of western equine encephalomyelitis virus by Argentine Aedes albifasciatus (Diptera: Culicidae).

Aedes albifasciatus (Macquart) has been considered a potential vector of western equine encephalitis (WEE) virus in Argentina because it has been found naturally infected, it is susceptible to infection per os, and its distribution and feeding habits are compatible with those of an epizootic and epidemic vector. Ae. albifasciatus females collected in the vicinity of Cordoba, Argentina, were highly susceptible to WEE virus perorally (ID50 less than 0.5 Vero cell plaque-forming units) when fed on viremic chicks. Virus transmission trials were done 9 to 16 d after infection by feeding potentially infected mosquitoes individually on chicks. Among 31 mosquitoes that survived the incubation period, six refed on susceptible chicks. Virus transmission by bite was demonstrated by five of six (83%) mosquitoes. Therefore, the final piece of evidence incriminating Ae. albifasciatus as a natural vector of WEE virus has been obtained. This is the first mosquito species to be incriminated as a vector of WEE virus in South America.

Culex species (Diptera: Culicidae) from central Argentina: identification by electrophoretic zymograms and genetic relationships.

Starch gel electrophoretic zymograms were used for the taxonomic separation of mosquitoes of the genus Culex, possible vectors of encephalitis in Argentina. Catalase, hexokinase, malic enzyme, isocitrate and malate dehydrogenases, and leucine aminopeptidase were species markers that allowed the clear identification of individual Culex apicinus Philippi, Cx. bidens Dyar, Cx. brethesi Dyar, Cx. declarator Dyar and Knab, Cx. dolosus Arribalzaga, Cx. maxi Dyar, Cx. quinquefasciatus Say, Cx. saltanensis Dyar, and Cx. tatoi Casal & García. Intraspecific polymorphism was revealed but did not preclude species separation. On the basis of allele frequencies, an index of genetic distance between species was calculated. Genetic affinities determined by electrophoretic data agreed with taxonomic affinities ascertained by conventional systematics. Thus, electrophoretic zymograms proved to be a powerful tool for the study of the genus Culex in Argentina.

Prevalence of Legionella spp. in swimming pool environment.

A study was carried out to evaluate the prevalence of bacteria of the Legionella genus in the water from the pools and showers of 12 swimming pools in the city of Bologna (Italy). Sampling took place four times for every establishment, once for each season of the year. Legionella spp. were found in 2 of the 48 pool water samples (L. micdadei and L. bozenanii) and in 27 of the 48 samples taken from the hot water of the showers: 19 were positive for L. pneumophila (10-19,250 cfu/l) and 18 for other species (20-6000 cfu/l). The contamination was seen to be consistent throughout the year, since the same species of legionellae were isolated at different samplings and in concentrations that did not reveal any seasonal trend. The legionellae and the Gram negative bacteria were not found in shower water with a temperature above 43 degrees C. However, in the samples of shower water with a lower temperature a statistically significant inverse correlation was seen between legionellae and Pseudomonas aeruginosa (r = -0.51; p<0.01) as well as between legionellae and Gram negative bacteria (r = -0.70; p<0.01). The potential risk of contracting infections from Legionella spp. in the swimming pool environment does not seem to be linked to the pool water, but to that of the showers. The water temperature of showers should therefore be maintained at a level high enough to prevent the reproduction of these bacteria.

The use of an animal-baited net trap for collecting mosquitoes during western equine encephalitis investigations in Argentina.

A large net trap was used to sample mosquito populations attracted to horses at three sites each in Santa Fe and Rio Negro Provinces, Argentina, during the austral summer of 1984. These provinces, as well as others in Argentina, were affected by a severe epizootic of western equine encephalitis (WEE) during 1982-83. Totals of 2,752 and 6,929 mosquitoes were collected in Santa Fe and Rio Negro Provinces during five and three trap nights, respectively. Culex mosquitoes of the subgenus Culex were predominant (45.8% of total) in the Santa Fe collections, although Aedes albifasciatus also was prevalent (21.7%). The latter species was predominant (95.7% of total) in the Rio Negro collections. The mosquito fauna was less complex (minimum of 6 species) in Rio Negro Province as compared to Santa Fe Province (minimum of 18 species). The advantages of the net trap indicate that this trap can become a useful tool in arbovirus ecology studies in other areas.

Biological characteristics of an enzootic subtype of western equine encephalomyelitis virus from Argentina.

In order to expand our knowledge on the biological characteristics of an enzootic South American subtype of western equine encephalomyelitis (WEE) virus, strain AG80-646, we inoculated guinea pigs, rabbits, newborn chickens and Vero and chick embryo cell cultures with this and other WEE and Wee-related viruses. AG80-646 was found apathogenic for guinea pigs even when inoculated intracranially (i.e.) or intraperitoneally (i.p.), and the animals did not develop viraemia. AG80-646 killed rabbits and the animals developed high viraemia (peak titer was 7.0 log PFU/0.1 ml). These data and previous serological evidence led us to look for a mammal as a natural host. AG80-646 was found lethal for newborn chickens inoculated subcutaneously (s.c.) (peak viraemia titer was 6.6 log PFU/0.1 ml). AG80-646 produced plaques (diameter 0.8-1.0 mm) in Vero and chick embryo cells 3-4 days post infection (p.i.) A comparison of AG80-646 with other WEE complex virus strains led to the following observations: (1) The lethality for guinea pigs was high for the two epizootic Argentinian strains, Cba 87 and Cba CIV 180, zero for the two enzootic strains, AG80-646 and BeAr 10315 (virus Aura), and intermediate for the Russian strain Y62-33 (low by i.c. route and zero by i.p. route); (2) AG80-646 was more virulent for rabbits inoculated i.p. than the three epizootic strains Cba 87, Cba CIV 180 and McMillan; (3) AG80-646 was less virulent for new-born chickens than the Argentinian epizootic strain Cba CIV 180; (4) The viraemia level correlated always with the strain virulence in each animal host. This study provides tools for the differentiation of WEE complex viruses and strains in the future ecological work on WEE in South America.

Rapid vascular clearance of two strains of Junin virus in Calomys musculinus: selective macrophage clearance.

Clearance of Junin (JUN) virus strains with different virulence for Calomys musculinus (Cm) was followed using the Candid #1 virulent and CbaFHA 5069 attenuated strains. In addition, virulent virus albino mice (AM) were included as control host and Venezuelan equine encephalitis (VEE-VI) virus as control virus. The virus inoculum (Vo) and the blood samples (Vt) obtained at different times post-inoculation (p.i.) were titrated on Vero cells and the cleared plaque forming-units (PFU) were calculated as the log Vt/Vo. In Cm both JUN virus strains were cleared rapidly (within 5 min the Candid #1 strain and within 10 min the CbaFHA 5069 strain); meanwhile, VEE-VI virus could be recovered from blood until 30 min p. i. Furthermore, JUN and VEE-VI viruses showed the same behaviour in Am as in Cm. We conclude that the JUN virus strains of different virulence for Cm did not show differences in their clearance from the blood of these animals. Moreover, the rapid clearance observed was independent of the animal host and viral dose.

Peroral susceptibility of Aedes albifasciatus and Culex pipiens complex mosquitoes (Diptera: Culicidae) from Argentina to western equine encephalitis virus.

The transmission cycle of western equine encephalitis (WEE) virus in South America is unknown. A WEE virus strain was isolated from Aedes albifasciatus in Argentina during the WEE epizootic of 1982-83. Also, Culex pipiens from Argentina was reported to be able to transmit WEE virus experimentally, but other results indicate that Cx. pipiens from the USA is refractory to this virus. We determined the susceptibility of Argentina strains of Ae. albifasciatus and Culex pipiens complex mosquitos to infection by WEE virus by the oral route. Adult females were fed on chicks infected with a WEE virus strain isolated in Cordoba Province, Argentina, or were fed on a blood/virus suspension. Each mosquito ingested between 10(1.6) to 10(6.4) vero cell plaque-forming units of virus. Each of 28 Ae. albifasciatus was positive for virus from the fourth day postfeeding, and there was evidence for virus replication. In contrast, 0/44 Cx. p. quinquefasciatus and only 1/15 Cx. p. pipiens was positive. Aedes albifasciatus is susceptible to infection by WEE virus and should be considered a potential vector of this virus in Argentina. Both subspecies of Cx. pipiens are refractory to peroral infection by WEE virus and probably do not play a role in the WEE virus cycle in Argentina.

[Isolation of lymphocytic choriomeningitis virus from human individuals]. / Aislamiento del virus de la coriomeningitis linfocitaria de seres humanos.

The activity of lymphocytic choriomeningitis virus (LCMv) in Argentina has been previously reported on the basis of serological evidence in rodents and humans and the isolation of only one strain of LCMv from a Mus domesticus captured in the province of Córdoba. The aim of this paper was to register patients with serological diagnosis of LCM, to isolate and to identify human strains of LCMv in Argentina. During the last 19 years, 15 cases were diagnosed as LCM by immunoflourescent indirect assay (IFI) and enzyme-linked immunosorbent assay (ELISA) but when neutralizing assay (NT) was incorporated, eight cases were classified as confirmed, three as probable and four as negative. The geographic distribution of the cases included three provinces: Córdoba, Buenos Aires and Santa Fe. Viral isolation was attempted in five patients classified as confirmed and only two resulted positive (P5226 and P8573). They were identified as LCMv by IFI and NT. The coexistence of LCMv with other arenaviruses, such as Junin and Oliveros viruses, in the same area, raises the probability of interactions between them, which could modify the virulence and/or pathogenicity for humans associated to genomic changes. Future studies of antigenic, genomic and virulence variability of different Argentine strains of LCMv, as well as the systematic search for human infection, will contribute to define the importance of this viral agent in our country and to implement control measures.