Characterization of Three New Insect-Specific Flaviviruses: Their Relationship to the Mosquito-Borne Flavivirus Pathogens.

Three novel insect-specific flaviviruses, isolated from mosquitoes collected in Peru, Malaysia (Sarawak), and the United States, are characterized. The new viruses, designated La Tina, Kampung Karu, and Long Pine Key, respectively, are antigenically and phylogenetically more similar to the mosquito-borne flavivirus pathogens, than to the classical insect-specific viruses like cell fusing agent and Culex flavivirus. The potential implications of this relationship and the possible uses of these and other arbovirus-related insect-specific flaviviruses are reviewed.

Characterization of the Punta Toro species complex (genus Phlebovirus, family Bunyaviridae).

Punta Toro virus (PTV), a member of the PTV complex, is a relatively common causative agent of febrile illness in Panama that is often misdiagnosed as 'dengue' or 'influenza'. Currently, only two named members make up this species complex, PTV and Buenaventura virus (BUEV). Genomic and antigenic characterization of 17 members of the PTV complex, nine of which were isolated from human acute febrile illness cases, reveals that this species complex is composed of six distant viruses. We propose to add four additional new viruses, designated Leticia virus, Cocle virus, Campana virus and Capira virus.

New genome sequences of gamboa viruses (family bunyaviridae, genus orthobunyavirus) isolated in panama and Argentina.

We describe here the nearly complete open reading frame (ORF) of five Gamboa virus strains isolated in Panama and Argentina. The viruses with complete ORF showed the regular genome organization observed in other orthobunyaviruses with exception to the presence of NSs protein. All predicted proteins showed homology with viruses belonging to members of the family Bunyaviridae.

Genetic and biological characterization of selected Changuinola viruses (Reoviridae, Orbivirus) from Brazil.

The genus Orbivirus of the family Reoviridae comprises 22 virus species including the Changuinola virus (CGLV) serogroup. The complete genome sequences of 13 CGLV serotypes isolated between 1961 and 1988 from distinct geographical areas of the Brazilian Amazon region were obtained. All viral sequences were obtained from single-passaged CGLV strains grown in Vero cells. CGLVs are the only orbiviruses known to be transmitted by phlebotomine sandflies. Ultrastructure and molecular analysis by electron microscopy and gel electrophoresis, respectively, revealed viral particles with typical orbivirus size and morphology, as well as the presence of a segmented genome with 10 segments. Full-length nucleotide sequencing of each of the ten RNA segments of the 13 CGLV serotypes provided basic information regarding the genome organization, encoded proteins and genetic traits. Segment 2 (encoding VP2) of the CGLV is uncommonly larger in comparison to those found in other orbiviruses and shows varying sizes even among different CGLV serotypes. Phylogenetic analysis support previous serological findings, which indicate that CGLV constitutes a separate serogroup within the genus Orbivirus. In addition, six out of 13 analysed CGLV serotypes showed reassortment of their genome segments.

Venezuelan equine encephalitis virus activity in the Gulf Coast region of Mexico, 2003-2010.

Venezuelan equine encephalitis virus (VEEV) has been the causative agent for sporadic epidemics and equine epizootics throughout the Americas since the 1930s. In 1969, an outbreak of Venezuelan equine encephalitis (VEE) spread rapidly from Guatemala and through the Gulf Coast region of Mexico, reaching Texas in 1971. Since this outbreak, there have been very few studies to determine the northward extent of endemic VEEV in this region. This study reports the findings of serologic surveillance in the Gulf Coast region of Mexico from 2003-2010. Phylogenetic analysis was also performed on viral isolates from this region to determine whether there have been substantial genetic changes in VEEV since the 1960s. Based on the findings of this study, the Gulf Coast lineage of subtype IE VEEV continues to actively circulate in this region of Mexico and appears to be responsible for infection of humans and animals throughout this region, including the northern State of Tamaulipas, which borders Texas.

Isolation and characterization of sylvatic mosquito-borne viruses in Trinidad: enzootic transmission and a new potential vector of Mucambo virus.

Mosquito surveillance was carried out in three forested regions of Trinidad during July 2007-March 2009. A total of 185,397 mosquitoes representing at least 46 species was collected, divided into pools of 1-50 mosquitoes according to species and sex, and screened for arboviruses using cytopathic effect assays on Vero cell monolayers. Eighty-five viruses were isolated, including members of the genera Alphavirus (Mucambo virus; MUCV) and Orthobunyavirus (Caraparu, Oriboca, Bimiti, and Wyeomyia viruses). Species of the Culex subgenus Melanoconion accounted for 56% of the total number of mosquitoes collected and 97% of the viruses isolated; Cx. (Mel.) portesi accounted for 92% of virus isolations. Our results also implicate for the first time Aedes (Ochlerotatus) hortator as a potential vector of MUCV. Phylogenetic analyses of 43 MUCV strains suggest population subdivision within Trinidad, consistent with the hypothesis of enzootic maintenance in localized rodent populations.

Isolation and phylogenetic analysis of Mucambo virus (Venezuelan equine encephalitis complex subtype IIIA) in Trinidad.

In the 1950s and 1960s, alphaviruses in the Venezuelan equine encephalitis (VEE) antigenic complex were the most frequently isolated arboviruses in Trinidad. Since then, there has been very little research performed with these viruses. Herein, we report on the isolation, sequencing, and phylogenetic analyses of Mucambo virus (MUCV; VEE complex subtype IIIA), including 6 recently isolated from Culex (Melanoconion) portesi mosquitoes and 11 previously isolated in Trinidad and Brazil. Results show that nucleotide and amino acid identities across the complete structural polyprotein for the MUCV isolates were 96.6-100% and 98.7-100%, respectively, and the phylogenetic tree inferred for MUCV was highly geographically- and temporally-structured. Bayesian analyses suggest that the sampled MUCV lineages have a recent common ancestry of approximately 198 years (with a 95% highest posterior density (HPD) interval of 63-448 years) prior to 2007, and an overall rate of evolution of 1.28 x 10(-4) substitutions/site/yr.

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.

West Nile virus infection of primary mouse neuronal and neuroglial cells: the role of astrocytes in chronic infection.

Primary cultures of embryonic murine neurons and newborn mouse astrocytes were inoculated with West Nile virus (WNV) strain NY385-99 to compare the pathogenesis of WNV infection in these types of CNS cells. Two different outcomes were observed. WNV infection in the neurons was rapidly progressive and destructive; within 5 days, all of the neurons were destroyed through apoptosis. WNV infection in the astrocytes evolved more slowly and did not seem to be highly lethal to the cells. The infected astrocytes continued to produce infectious virus (10(4.6)-10(6.5) PFU/mL) for 114 days, in a permissive, persistent infection. During this period, WNV antigen could be shown in the cytoplasm of the infected astrocytes by immunocytochemical assay, transmission electron microscopy of ultrathin sections, and in the cell culture medium by complement fixation test. Our results with this in vitro experimental murine cell model indicate that astrocytes can develop chronic or persistent infection with WNV, suggesting that these cells may play a role in the maintenance of WNV in the CNS.

Oropouche virus isolation, southeast Brazil.

An Oropouche virus strain was isolated from a novel host (Callithrix sp.) in Arinos, Minas Gerais State, southeastern Brazil. The virus was identified by complement fixation test and confirmed by reverse transcription-polymerase chain reaction. Phylogenetic analysis identified this strain as a genotype III isolate previously recognized only in Panama.

Araguari virus, a new member of the family Orthomyxoviridae: serologic, ultrastructural, and molecular characterization.

This paper reports the results of serologic, structural, biochemical, and genetic studies indicating that Araguari virus, a previously unassigned viral agent, is a member of the family Orthomyxoviridae and genus Thogotovirus. Araguari virus has six RNA fragments; biologically, it shares several properties with other viruses in the family Orthomyxoviridae. Nucleotide sequencing of the RNA segments 4 (glycoprotein) and 5 (nucleoprotein) of Araguari virus aligned with the orthomyxoviruses, showing the closest relationship with Thogoto virus (sequence similarity = 61.9% and 69.1%, respectively, for glycoprotein and nucleoprotein), but also sharing a more distant similarity with Dhori and Influenza C viruses, especially for the glycoprotein gene. Based on these results, we propose that Araguari virus should be assigned as a new member of the family Orthomyxoviridae and genus Thogotovirus.

Molecular epidemiology of group C viruses (Bunyaviridae, Orthobunyavirus) isolated in the Americas.

To date, no molecular studies on group C viruses (Bunyaviridae, Orthobunyavirus) have been published. We determined the complete small RNA (SRNA) segment and partial medium RNA segment nucleotide sequences for 13 group C members. The full-length SRNA sequences ranged from 915 to 926 nucleotides in length, and revealed similar organization in comparison with other orthobunyaviruses. Based on the 705 nucleotides of the N gene, group C members were distributed into three major phylogenetic groups, with the exception of Madrid virus, which was placed outside of these three groups. Analysis of the Caraparu virus strain BeH 5546 revealed that it has an SRNA sequence nearly identical to that of Oriboca virus and is a natural reassortant virus. In addition, analysis of 345 nucleotides of the Gn gene for eight group C viruses and for strain BeH 5546 revealed a different phylogenetic topology, suggesting a reassortment pattern among them. These findings represent the first evidence for natural reassortment among the group C viruses, which include several human pathogens. Furthermore, our genetic data corroborate previous relationships determined using serologic assays (complement fixation, hemagglutination inhibition, and neutralization tests) and suggest that a combination of informative molecular, serological, and ecological data is a helpful tool to understand the molecular epidemiology of arboviruses.

Short report: serological evidence of West Nile virus activity in El Salvador.

Epizootics of encephalitis in El Salvador killed 203 equines between November 2001 and April 2003. During an investigation of the outbreaks, 18 (25%) of 73 serum samples collected from stablemates of deceased animals in 2003 had antibodies to West Nile virus. Ten of these infections were confirmed by plaque reduction neutralization tests, suggesting West Nile virus has extended its range and spread to Central America.

Endemic Venezuelan equine encephalitis in northern Peru.

Since Venezuelan equine encephalitis virus (VEEV) was isolated in Peru in 1942, >70 isolates have been obtained from mosquitoes, humans, and sylvatic mammals primarily in the Amazon region. To investigate genetic relationships among the Peru VEEV isolates and between the Peru isolates and other VEEV strains, a fragment of the PE2 gene was amplified and analyzed by single-stranded conformation polymorphism. Representatives of seven genotypes underwent sequencing and phylogenetic analysis. The results identified four VEE complex lineages that cocirculate in the Amazon region: subtypes ID (Panama and Colombia/Venezuela genotypes), IIIC, and a new, proposed subtype IIID, which was isolated from a febrile human, mosquitoes, and spiny rats. Both ID lineages and the IIID subtype are associated with febrile human illness. Most of the subtype ID isolates belonged to the Panama genotype, but the Colombia/Venezuela genotype, which is phylogenetically related to epizootic strains, also continues to circulate in the Amazon basin.

Diagnóstico sorológico de infecções por dengue e febre amarela em casos suspeitos no Estado do Pará, Brasil, 1999 / Serological diagnosis of dengue and yellow fever infections in suspected cases from Pará State, Brazil, 1999

De junho a dezembro de 1999, foram coletadas 785 amostras de soro de pacientes com suspeita clínica de dengue e/ou febre amarela. Os pacientes foram atendidos nas unidades de saúde distribuídas pelas seis mesorregiöes do Estado do Pará, Brasil. As amostras de soro foram testadas pelo método de inibiçäo da hemaglutinaçäo para detecçäo de anticorpos para Flavivirus e pelo ensaio imunoenzimático para detecçäo de imunoglobulina M para dengue e febre amarela. Das amostras coletadas, 563 (71,7 por cento) foram positivas pelo IH, e dentre estas 150 (26,6 por cento) foram positivas pelo ELISA-IgM. O vírus dengue foi responsável pela maioria das infecçöes recentes em todas as mesorregiöes e os casos de febre amarela detectados neste estudo foram restritos às mesorregiöes Marajó e Sudeste

Ultrastructural, antigenic and physicochemical characterization of the Mojuí dos Campos (Bunyavirus) isolated from bat in the Brazilian Amazon region.

The Mojuí dos Campos virus (MDCV) was isolated from the blood of an unidentified bat (Chiroptera) captured in Mojuí dos Campos, Santarém, State of Pará, Brazil, in 1975 and considerated to be antigenically different from other 102 arboviruses belonging to several antigenic groups isolated in the Amazon region or another region by complement fixation tests. The objective of this work was to develop a morphologic, an antigenic and physicochemical characterization of this virus. MDCV produces cytopathic effect in Vero cells, 24 h post-infection (p.i), and the degree of cellular destruction increases after a few hours. Negative staining electron microscopy of the supernatant of Vero cell cultures showed the presence of coated viral particles with a diameter of around 98 nm. Ultrathin sections of Vero cells, and brain and liver of newborn mice infected with MDCV showed an assembly of the viral particles into the Golgi vesicles. The synthesis kinetics of the proteins for MDCV were similar to that observed for other bunyaviruses, and viral proteins could be detected as early as 6 h p.i. Our results reinforce the original studies which had classified MDCV in the family Bunyaviridae, genus Bunyavirus as an ungrouped virus, and it may represent the prototype of a new serogroup.

[Serological diagnosis of dengue and yellow fever infections in suspected cases from Pará State, Brazil, 1999]. / Diagnóstico sorológico de infecções por dengue e febre amarela em casos suspeitos no Estado do Pará, Brasil, 1999.

From June to December 1999, 785 serum samples were obtained from patients clinically suspected of having dengue or yellow fever. The patients were referred by public health centers distributed within the six mesoregions of Par State, Brazil. Serum samples were tested for Flavivirus antibodies by hemagglutination inhibition test and for dengue and yellow fever viruses by enzyme-linked immunosorbent assay for IgM detection. Of the sera collected, 563 (71.7%) were positive by HI test and out of these 150 (26.6%) were positive by ELISA-IgM. Dengue virus was responsible for most of the recent infections in all regions; yellow fever cases detected in the current study were restricted to the Maraj and Southeast regions.

Inadequate management of natural ecosystem in the Brazilian Amazon region results in the emergence and reemergence of arboviruses

Um total de 187 diferentes espécies de arbovírus, além de outros vírus de vertebrados, foram identificados pelo Instituto Evandro Chagas (IEC) no período de 1954 a 1998, entre as mais de 10.000 cepas de vírus isoladas de seres humanos, insetos hematófagos e vertebrados-sentinela e silvestres. Apesar dos estudos intensivos realizados na Amazônia brasileira, sobretudo no Estado do Pará, pouco se sabe a respeito da maioria desses vírus, com exceçäo de dados a respeito de data, hora, fonte e método de isolamento, assim como a capacidade de infectar animais laboratoriais. Fazem uma revisäo dos dados ecológicose epidemiológicos e procuram associar o impacto, sobre os diversos vírus, das mudanças populacionais dos vetores e hospedeiros induzidas por profundas alteraçöes no meio ambiente. O desmatamento, o uso do subsolo, a construçäo de represas e de rodovias, a colonizaçäo humana e a urbanizaçäo foram as principais modificaçöes ambientais introduzidas pelo homem e associadas à emergência ou reermegência de importantes arbovírus, inclusive alguns com atividade patogênica em seres humanos.