Dengue virus serotypes in Jamaica, 2003-2007.

BACKGROUND: Dengue virus (DENV) infection is increasing in prevalence and severity globally. The severity of dengue is influenced by several factors including the immune response, viral and host genetic factors. METHOD: The DENV serotypes were determined in 770 serum samples from dengue immunoglobulin (Ig) M antibody positive (n = 469), dengue IgM negative (n = 185) and dengue antibody negative (n = 116) patients with suspected dengue who presented during (n = 150) or after (n = 620) the acute phase of illness during 2003-2007. Dengue antibodies were detected by enzyme-linked immunosorbent assays and DENV RNA by reverse transcriptase-polymerase chain reaction (RT-PCR) performed on serum and cell culture supernatants of C6/36 mosquito cells inoculated with acute phase serum (n = 150). RESULTS: Based on serological profiles, 41% of acute phase sera and 66% of post acute sera were from patients with current primary or secondary dengue, while 41% and 35% of acute and post-acute phase sera, respectively, were from patients with secondary dengue or past exposure only. Dengue virus RNA was found in 20/770 samples (2.6%). Only 1.5% (9/620) of sera collected after the acute phase of illness tested positive for DENV RNA compared with 2.6% (4/150) of sera collected during the acute phase and 7.3% of cell culture supernatants inoculated with acute phase serum (11/150, p = 0.001). All four serotypes including DENV-1 (3/20, 15%), DENV-2 (7/20, 35%), DENV-3 (3/20, 15%) and DENV-4 (7/20, 35%) were identified over the five-year period. These results also showed that DENV-1, 2 and 4 were present during 2007 and that DENV-2 and DENV- 4 were the likely causative viruses of the 2007-2008 dengue outbreak in Jamaica. The three strains of DENV-3 were isolated from infants less than three years of age with primary infection during 2006. CONCLUSION: This study highlights the increasing threat of dengue and severe dengue disease to the Jamaican population. Preventative measures including laboratory surveillance and vector control should be strictly maintained at the highest level.

Molecular epidemiology of dengue in Jamaica dengue virus genotypes in Jamaica, 2007.

The genotypes of dengue viruses (DENV) isolated from patients with dengue in Jamaica during 2007 were determined using DNA sequencing and phylogenetic analysis of the C-prM gene junction. The 17 DENV analysed included strains of DENVserotypes 1 (DENV-1, n = 3), DENV-2 (n = 7) and DENV-4 (n = 7). All strains ofDENV-1 were classified as genotype III, while 1 of 7 strains of DENV-2 belonged to the Asian American/Asian genotype, genotype I/III (Jamaica genotype), 2 were genotype V, the American genotype and 4 strains clustered with reference strains belonging to genotype IV. The 6 DENV-4 strains from Jamaica and the control strain clustered together in a separate clade from Caribbean/American reference strains, which belong to genotype II and Asian strains, classified as genotypes I and III. There has been little evolution in the DENV-1 strains circulating in Jamaica over the years and this might reduce the risk of outbreaks due to this serotype. In contrast, the high genetic diversity in strains of DENV-2 viruses in circulation, the presence of more recently introduced genotypes and a new clade of DENV-4 might contribute to the epidemic potential of these DENV serotypes. These preliminary data clearly indicate the need to maintain laboratory surveillance, and other control measures against hyperendemicity of dengue in Jamaica.

Dengue HLA associations in Jamaicans.

BACKGROUND: Polymorphisms in the human leukocyte antigen (HLA) genes might predispose certain individuals to dengue fever (DF) and the severe forms of the disease: dengue haemorrhagic fever/ dengue shock syndrome (DHF/DSS). SUBJECTS AND METHOD: A DNA-based HLA typing method was used to determine the HLA class I and II alleles in 50 patients with dengue, including 45 cases of DF 5 cases of DHF and 177 healthy individuals in Jamaica. RESULTS: HLA-A*24 and - DRbeta5*01/02 were significantly associated with dengue infection while possession of HLA-A*23, -CW*04, -DQbeta*02, -DQbeta*03 and DQbeta*06 were protective. No other significant associations were found after correction for the number of alleles tested at each HLA-locus. CONCLUSION: This is the first study to report a significant association with HLA-A*24 and DF although this allele is associated with DHF and DSS in Vietnamese patients. The other HLA associations observed in the Jamaican cohort also are different from those reported in other ethnic groups. Further studies which involve larger numbers of patients with DHF and explore functional aspects of HLA allelic associations with dengue in Jamaicans are necessary.

Seroprevalence of dengue in Trinidad using rapid test kits: a cord blood survey.

A cross-sectional sero-epidemiological study was conducted to determine the prevalence of dengue in Trinidad. Two commercial rapid test kits, PanBio Dengue Duo IgM and IgG Rapid Strip Test and the Bio-Check Plus Dengue G/M Cassette Test (Brittney) were used. The immunosorbent assay (ELISA) (FOCUS Technologies, California) was used as the control. One hundred and twenty five cord blood samples were collected (46 from Mt. Hope Women's Hospital (MH) and 79 from the San Fernando General Hospital (SF)). All blood samples were tested in accordance with the two rapid kits and ELISA assay manufacturer's instructions. From 125 cord blood samples, the IgG FOCUS ELISA results showed 93.5 and 95% infections at MH and SF, respectively. Whereas the Brittney and PanBio kits showed 10.9 and 5.1%, and 26.1 and 50.6% for MH and SF, respectively. Based on the FOCUS ELISA (control) assays, the combined seroprevalence rate from north and south Trinidad was 94.4%. IgG and IgM sensitivity and specificity levels were higher in the PanBio than Brittney test kits. The high seroprevalence rates observed in Trinidad are discussed to stimulate more research to explain this phenomenon and to prevent the Southeast Asian scenario from developing in the Americas.

A retrospective study of the 1996 DEN-1 epidemic in Trinidad: demographic and clinical features

A retrospective analysis of the 1996 DEN-1 epidemic in Trinidad was undertaken to better understand the clinical and demographic expression of dengue infection in the island during one of the larger epidemics in the past 10 years and following the reintroduction of DEN-1 into the island in 1991 after a gap of 14 years. A total of 393 laboratory-confirmed cases were identified. Of these, notes for 157 patients were available for analysis. The epidemic was island-wide, though most cases occurred in the most densely populated county of St. George. There was a slight predominance of females (51.6 per cent) among the cases, and while all age groups were affected, older children and adults comprised the majority. South Asians among the population predominated. Overall, 27 clinical symptoms were reported. The most common were: fever (98.7 per cent), generalized pain (96.2 per cent) and anorexia (63.1 per cent). Rash, arthralgia, retro-orbital pain and haemorrhage (all mentioned in the WHO clinical description for dengue fever) were reported in <50 per cent of cases. Gastrointestinal symptoms were also very common and occurred in over two-thirds of cases at presentation. Bleeding manifestations were reported in 30 per cent of patients and commonly involved the gastrointestinal tract. Features of DHF were noted in only six (4 per cent)

Ecological studies of enzootic Venezuelan equine encephalitis in north-central Venezuela, 1997-1998.

From 1997-1998, we investigated the possible continuous circulation of epizootic Venezuelan equine encephalitis (VEE) virus suggested by a 1983 subtype IC interepizootic mosquito isolate made in Panaquire, Miranda State, Venezuela. The study area was originally covered by lowland tropical rainforest but has been converted into cacao plantations. Sentinel hamsters, small mammal trapping, mosquito collections, and human serosurveys were used to detect active or recent virus circulation. Six strains of subtype ID VEE virus were isolated from hamsters that displayed no apparent disease. Four other arboviruses belonging to group A (Togaviridae: Alphavirus), two Bunyamwera group (Bunyaviridae), and three Gamboa group (Bunyaviridae) arboviruses were also isolated from hamsters, as well as 8 unidentified viruses. Venezuelan equine encephalitis-specific antibodies were detected in 5 small mammal species: Proechimys guairae, Marmosa spp., and Didelphis marsupialis. Mosquito collections comprised of 38 different species, including 8 members of the subgenus Culex (Melanoconion), did not yield any virus isolates. Sera from 195 humans, either workers in the cacao plantation or nearby residents, were all negative for VEE virus antibodies. Sequences of 1,677 nucleotides from the P62 gene of 2 virus isolates indicated that they represent a subtype ID lineage that is distinct from all others characterized previously, and are unrelated to epizootic VEE emergence.

Extreme genetic diversity among Pirital virus (Arenaviridae) isolates from western Venezuela.

Pirital-like virus isolates from rodents collected in a variety of habitats within a six-state area of central Venezuela were analyzed genetically by amplifying a portion of the nucleocapsid protein gene using RT-PCR. Comparisons of the sequences from 30 selected Pirital-like virus isolates demonstrated up to 26% divergence in nucleotide sequences and up to 16% divergence in deduced amino acid sequences. Within the Pirital monophyletic group, 14 distinct lineages or genotypes, differing by at least 6% in nucleotide sequences, were identified. Although sample sizes were small for some lineages, many of the different genotypes were sampled in only one region or locality, suggesting allopatric divergence. Complement fixation tests with representatives of the most divergent Pirital virus lineages failed to delineate multiple species or subtypes within the Pirital clade. These results indicate that the previously proposed 12% nucleocapsid protein amino acid sequence divergence cutoff value for delineating arenavirus species is not appropriate for the entire family. When individual clones were examined from PCR amplicons, a mean of 0.17% sequence diversity vs the consensus sequences was detected, suggesting diverse quasispecies populations within infected rodent hosts. Possible explanations for the extreme genetic diversity within and among Pirital virus populations in infected rodents are discussed.

Potential sources of the 1995 Venezuelan equine encephalitis subtype IC epidemic.

Venezuelan equine encephalitis viruses (VEEV) belonging to subtype IC have caused three (1962-1964, 1992-1993 and 1995) major equine epizootics and epidemics. Previous sequence analyses of a portion of the envelope glycoprotein gene demonstrated a high degree of conservation among isolates from the 1962-1964 and the 1995 outbreaks, as well as a 1983 interepizootic mosquito isolate from Panaquire, Venezuela. However, unlike subtype IAB VEEV that were used to prepare inactivated vaccines that probably initiated several outbreaks, subtype IC viruses have not been used for vaccine production and their conservation cannot be explained in this way. To characterize further subtype IC VEEV conservation and to evaluate potential sources of the 1995 outbreak, we sequenced the complete genomes of three isolates from the 1962-1964 outbreak, the 1983 Panaquire interepizootic isolate, and two isolates from 1995. The sequence of the Panaquire isolate, and that of virus isolated from a mouse brain antigen prepared from subtype IC strain P676 and used in the same laboratory, suggested that the Panaquire isolate represents a laboratory contaminant. Some authentic epizootic IC strains isolated 32 years apart showed a greater degree of sequence identity than did isolates from the same (1962-1964 or 1995) outbreak. If these viruses were circulating and replicating between 1964 and 1995, their rate of sequence evolution was at least 10-fold lower than that estimated during outbreaks or that of closely related enzootic VEEV strains that circulate continuously. Current understanding of alphavirus evolution is inconsistent with this conservation. This subtype IC VEEV conservation, combined with phylogenetic relationships, suggests the possibility that the 1995 outbreak was initiated by a laboratory strain.

Guanarito virus (Arenaviridae) isolates from endemic and outlying localities in Venezuela: sequence comparisons among and within strains isolated from Venezuelan hemorrhagic fever patients and rodents.

Despite intensive surveillance, Venezuelan hemorrhagic fever (VHF), caused by Guanarito (GTO) virus, has been detected in only a small region of western Venezuela. To determine whether VHF is associated with a particular regional GTO virus strain(s), 29 isolates from rodents and humans throughout the surrounding regions were analyzed by partial sequencing of the nucleocapsid protein gene. Phylogenetic trees delineated nine distinct GTO genotypes that differ by 4-17% in nucleotides and up to 9% in amino acid sequences; most appeared to be restricted to discrete geographic regions, although a few genotypes were isolated in several locations. Each genotype included at least one strain recovered from a rodent, but only two genotypes were isolated from VHF cases. The presence outside of the endemic/epidemic region of two genotypes isolated also from VHF cases suggests that human pathogenic viruses occur outside of the endemic zone, but do not frequently infect people and/or cause apparent disease there. VHF does not appear to be associated with a GTO virus genotype that is restricted to a certain rodent species. When quasispecies diversity was examined, rodent isolates had higher sequence variation than human isolates. One rodent isolate included a mixture of two phylogenetically distinct genotypes, suggesting a dual infection.

Natural rodent host associations of Guanarito and pirital viruses (Family Arenaviridae) in central Venezuela.

The objective of this study was to elucidate the natural rodent host relationships of Guanarito and Pirital viruses (family Arenaviridae) in the plains of central Venezuela. Ninety-two arenavirus isolates from 607 animals, representing 10 different rodent species, were characterized to the level of serotype. The 92 isolates comprised 19 Guanarito virus strains and 73 Pirital virus strains. The 19 Guanarito virus isolates were from Zygodontomys brevicauda; 72 (98.6%) of the 73 Pirital virus isolates were from Sigmodon alstoni. These results indicate that the natural rodent associations of these 2 sympatric arenaviruses are highly specific and that Z brevicauda and S. alstoni are the principal rodent hosts of Guanarito and Pirital viruses, respectively.

Venezuelan hemorrhagic fever: clinical and epidemiological studies of 165 cases.

Epidemiological and clinical data are presented on 165 cases of Venezuelan hemorrhagic fever (VHF), a newly emerging viral zoonosis caused by Guanarito virus (of the family Arenaviridae). The disease is endemic in a relatively circumscribed area of central Venezuela. Since its first recognition in 1989, the incidence of VHF has peaked each year between November and January, during the period of major agricultural activity in the region of endemicity. The majority of cases have involved male agricultural workers. Principal symptoms among the patients with VHF included fever, malaise, headache, arthralgia, sore throat, vomiting, abdominal pain, diarrhea, convulsions, and a variety of hemorrhagic manifestations. The majority of patients also had leukopenia and thrombocytopenia. The overall fatality rate among the 165 cases was 33.3%, despite hospitalization and vigorous supportive care.

[Serotypes and genotypes of dengue virus circulating in Venezuela, 1990-1997]. / Serotipos y genotipos de virus dengue circulantes en Venezuela, 1990-1997.

Due to the increasing severity of hemorrhagic dengue epidemics during the last years in Venezuela, a retrospective analysis was conducted to identify the behaviour of the dengue virus serotypes circulating in the country and the molecular evolution of dengue virus serotype 2. The data presented here indicates that dengue virus serotypes 1, 2 and 4 are endemic in Venezuela, they circulate simultaneously around the year in the biggest urban cities, however, one particular serotype is predominant during an epidemic period and replaces the virus serotype dominant during the previous epidemic period. The increased severity of dengue fever since 1989 in Venezuela might be associated to the introduction of the Asiatic genotype of virus which replaced the autochthonous Caribbean genotype. The Asiatic genotype is recognised as a more virulent virus.

[Venezuelan hemorrhagic fever: eight years of observation]. / Fiebre hemorrágica venezolana: ocho años de observación.

Venezuelan hemorrhagic fever (VHF) is a severe disease characterised by fever, malaise, sore throat, followed by abdominal pain, diarrhea, a variety of hemorrhagic manifestations and convulsions. The arenavirus Guanarito is the causal agent and the virus natural reservoir is the rodent Zygodontomys brevauda (cane mouse). The disease affect agricultural male workers, between 14-54 years of age, mainly from Guanarito municipality of Portuguesa state and adjacent regions of Barinas State. Since VHF emergency in 1989 up till 1997, 220 cases have been reported with a fatality rate of 33%. Epidemiological informations suggest that VHF has a cyclic behaviour, with epidemic periods of high incidence, every 4-5 years. During the interepidemic periods few VHF cases are reported.

Isolation and characterization of pirital virus, a newly discovered South American arenavirus.

Specific rodent species are principal hosts for each of the well-characterized members of the virus family Arenaviridae. Guanarito virus (Arenaviridae) is the etiologic agent of Venezuelan hemorrhagic fever. A previous study on the epidemiology of Venezuelan hemorrhagic fever revealed extensive arenavirus infection (presumed to be caused by Guanarito virus) in two rodent species. Sigmodon alstoni and Zygodontomys brevicauda, collected from the region of Venezuela in which the disease is endemic. In the present study, four arenavirus isolates recovered from the Municipality of Guanarito (two isolates each from S. alstoni and Z. brevicauda) were characterized to learn more about the natural rodent host relationships of Guanarito virus. Serologic tests and analyses of nucleocapsid protein gene sequence data indicated that the two isolates from Z. brevicauda are strains of Guanarito virus and that the two isolates from S. alstoni are representatives of a novel New World arenavirus (proposed name Pirital) that is antigenically and phylogenetically distinct from all known New World arenaviruses. The results of the present study provide further evidence that the cane mouse Z. brevicauda is a natural host of Guanarito virus and suggest that the cotton rat S. alstoni is the natural reservoir host of Pirital but not Guanarito virus.

Origins of dengue type 2 viruses associated with increased pathogenicity in the Americas.

The recent emergence and spread of dengue hemorrhagic fever in the Americas have been a major source of concern. Efforts to control this disease are dependent on understanding the pathogenicity of dengue viruses and their transmission dynamics. Pathogenicity studies have been hampered by the lack of in vitro or in vivo models of severe dengue disease. Alternatively, molecular epidemiologic studies which associate certain dengue virus genetic types with severe dengue outbreaks may point to strains with increased pathogenicity. The comparison of nucleotide sequences (240 bp) from the E/NS1 gene region of the dengue virus genome has been shown to reflect evolutionary relationships and geographic origins of dengue virus strains. This approach was used to demonstrate an association between the introduction of two distinct genotypes of dengue type 2 virus and the appearance of dengue hemorrhagic fever in the Americas. Phylogenetic analyses suggest that these genotypes originated in Southeast Asia and that they displaced the native, American genotype in at least four countries. Vaccination and other control efforts should therefore be directed at decreasing the transmission of these "virulent" genotypes.

Isolation, characterization and geographic distribution of Caño Delgadito virus, a newly discovered South American hantavirus (family Bunyaviridae).

Rodents collected from the Venezuelan llanos (plains) during field studies of viral hemorrhagic fever were tested for evidence of hantavirus infection. Hantavirus antibody was found in one (7.7%) of 13 Oryzomys bicolor, one (3.4%) of 29 Rattus rattus, 10 (6.0%) of 166 Sigmodon alstoni and one (2.2%) of 45 Zygodontomys brevicauda. Hantavirus-specific RNA was detected in lung tissues from four antibody-positive rodents: two S. alstoni from Portuguesa State and one S. alstoni each from Cojedes and Barinas States. A hantavirus isolate (herein identified as VHV-574) was recovered from lung tissue from a hantavirus RNA-positive S. alstoni collected from Portuguesa State. The results of serological tests and analyses of small and medium RNA segment nucleotide sequence data indicated that VHV-574 represents a novel hantavirus (proposed name 'Caño Delgadito') that is distinct from all previously characterized hantaviruses. The results of analyses of nucleotide sequence data from the four hantavirus RNA-positive S. alstoni suggested that Caño Delgadito virus is widely distributed in the Venezuelan llanos.

Emergence of a new epidemic/epizootic Venezuelan equine encephalitis virus in South America.

One of the most important questions in arbovirology concerns the origin of epidemic Venezuelan equine encephalitis (VEE) viruses; these viruses caused periodic, extensive epidemics/epizootics in the Americas from 1938-1973 (reaching the United States in 1971) but had recently been presumed extinct. We have documented the 1992 emergence of a new epidemic/epizootic VEE virus in Venezuela. Phylogenetic analysis of strains isolated during two outbreaks indicated that the new epidemic/epizootic virus(es) evolved recently from an enzootic VEE virus in northern South America. These results suggest continued emergence of epizootic VEE viruses; surveillance of enzootic viruses and routine vaccination of equines should therefore be resumed.