Genetic variation within the premembrane coding region of dengue viruses from the Yucatan peninsula of Mexico

Single-strand conformation polymorphism (SSCP) and sequence analyses were used to characterize genetic polymorphisms and phylogenetic relationships, respectively, among dengue (DEN) viruses isolated between 1980 and 1997 from Yucatan, Mexico and surrounding states. Amplified cDNAs from the premembrane (prM) coding region of the DEN viruses were characterized by SSCP. There were six distinct haplotypes of DEN-1 viruses, four haplotypes of DEN-2, four haplotypes of DEN-3, and eight haplotypes of DEN-4. The diversity index for DEN-3 isolates was significantly lower than that of the other serotypes, probably reflecting the recent introduction of this viral serotype into Mexico. The SSCP was a sensitive (84.5%) and specific (95.5%) technique for identifying nucleotide substitutions. Sequence analyses provided insight into the phylogenetic relationships of the DEN strains isolated in Yucatan. One DEN-2 isolate from 1996 was demonstrated to cluster with viruses of the Sri Lanka genotype, none of which have been detected before in the Americas.     

Differential susceptibility of Aedes aegypti to infection by the American and Southeast Asian genotypes of dengue type 2 virus

Outbreaks of dengue hemorrhagic fever have coincided with the introduction of the Southeast (SE) Asian genotype of dengue type 2 virus in the Western Hemisphere. This introduced genotype appears to be rapidly displacing the indigenous, American genotype of dengue 2 virus throughout the region. These field observations raise the possibility that the SE Asian genotype of dengue 2 is better adapted for vector transmission than its American counterpart. To evaluate this hypothesis, we compared the ability of viral strains of the SE Asian and American genotypes to infect, replicate, and disseminate within vector mosquitoes (Aedes aegypti). Viral strains of the SE Asian genotype tended to infect and disseminate more efficiently in mosquitoes than did variants of the American genotype. These differences, however, were observed solely in field-derived mosquitoes, whereas viral infection rates were virtually identical in the laboratory-adapted Rockefeller colony of Ae. aegypti. Our findings could provide a physiological basis for the contrasting patterns of dengue virus genotype transmission and spread. Such an understanding of functional differences between viral strains and genotypes may ultimately improve surveillance and intervention strategies.     

American trypanosomiasis in dogs from an urban and rural area of Yucatan, Mexico

American trypanosomiasis in dogs is reported from South of the United States to Argentina and Chile. It is transmitted through the contact of dogs with reduviid insects when they feed; reduviid insects are well established in the southern state of Yucatan, Mexico. However, there are no reports available about trypanosomiasis prevalence in dogs of urban and rural areas. A cross-sectional study was performed in 345 stray dogs, 102 from a rural community of Yucatan (Tunkas) and 243 from Merida, capital city of the Yucatan state. Serum samples were obtained for detection of immunoglobulin G antibodies against Trypanosoma cruzi by indirect immunofluorescence assay and Western blot. DNA was extracted from whole blood of urban dogs for polymerase chain reaction (PCR) assay. Prevalence was determined and the risk to become infected in both urban and rural dogs was evaluated using a 2 x 2 contingency table. In addition to the effect of body condition score (BCS), the age and sex of dogs were also evaluated to determine the risk of infection. Antibody prevalence against T. cruzi in rural areas was 9.8%, whereas in urban dogs was 14.4%. When PCR results were included, prevalence in dogs from the urban area increased to 17.3%. PCR allowed the detection of active asymptomatic acute-phase disease. The risk to become infected was not different between urban and rural areas, suggesting that the vector is well adapted both to rural areas and urban sprawling. Dogs with poor BCS tended to have a higher probability of seroreactivity to T. cruzi proteins than dogs with regular or good BCS (p = 0.05).     

Aedes aegypti vectorial capacity is determined by the infecting genotype of dengue virus

Dengue viruses causing severe, hemorrhagic disease have displaced less virulent strains in the Americas during the past three decades. The American (AM) genotype of dengue serotype 2 has been endemic in the Western Hemisphere and South Pacific, causing outbreaks of dengue fever (DF), but has not been linked to dengue hemorrhagic fever (DHF). The Southeast Asian (SEA) genotype of dengue was introduced into this hemisphere in 1981, has caused outbreaks with numerous cases of DHF, and has displaced the AM genotype in several countries. We investigated the effect of viral genotype on the potential for transmission by infecting Aedes aegypti mosquitoes collected in South Texas with six viruses, representing these two genotypes. Viral replication in the midgut was significantly higher in SEA-infected mosquitoes, and virus-specific proteins could be detected in salivary glands 7 days earlier in SEA- than AM-infected mosquitoes. This much earlier appearance of dengue virus in salivary glands resulted in an estimated 2- to 65-fold increase in the vectorial capacity of these mosquitoes for the viruses that can cause DHF. This may be one of the mechanisms through which more virulent flaviviruses spread and displace others globally.     

Local Evolution of Pyrethroid Resistance Offsets Gene Flow Among Aedes aegypti Collections in Yucatan State,Mexico

The mosquito Aedes aegypti is the major vector of the four serotypes of dengue virus (DENV1–4). Previous studies have shown that Ae. aegypti in Mexico have a high effective migration rate and that gene flow occurs among populations that are up to 150 km apart. Since 2000, pyrethroids have been widely used for suppression of Ae. aegypti in cities in Mexico. In Yucatan State in particular, pyrethroids have been applied in and around dengue case households creating an opportunity for local selection and evolution of resistance. Herein, we test for evidence of local adaptation by comparing patterns of variation among 27 Ae. aegypti collections at 13 single nucleotide polymorphisms (SNPs): two in the voltage-gated sodium channel gene para known to confer knockdown resistance, three in detoxification genes previously associated with pyrethroid resistance, and eight in putatively neutral loci. The SNPs in para varied greatly in frequency among collections, whereas SNPs at the remaining 11 loci showed little variation supporting previous evidence for extensive local gene flow. Among Ae. aegypti in Yucatan State, Mexico, local adaptation to pyrethroids appears to offset the homogenizing effects of gene flow.     

Variation in vector competence for dengue 2 virus among 24 collections of Aedes aegypti from Mexico and the United States

Aedes aegypti from 24 collections in Mexico and the United States were challenged orally with dengue 2 virus JAM1409 (DEN-2 JAM1409). The vector competence (VC) of the populations ranged from 24% to 83%. Mosquito populations from the Yucatan exhibited greater VC than those from other areas of Mexico. The presence or absence of a midgut infection barrier (MIB) and a midgut escape barrier (MEB) was determined for mosquitoes in each population. The percentage of mosquitoes exhibiting an MIB ranged from 14% to 59%, and those exhibiting an MEB ranged from 4% to 43% in the collections. The MIB and MEB were not completely independent as determined by regression analysis. Midgut infection rates were dose dependent.     

Role of interleukin-28B polymorphisms in the treatment of hepatitis C virus genotype 2 infection in Asian patients

Genome-wide association studies have linked single nucleotide polymorphisms (SNPs) near the interleukin-28B gene to the hepatitis C virus genotype 1 (HCV-1) response to peginterferon/ribavirin treatment. We aimed to explore the impact on the treatment outcomes of Asian HCV-2 patients. We determined rs8105790, rs8099917, rs4803219, and rs10853728 to be candidate SNPs in 482 Asian HCV-2 patients treated with the standard of care. Because the first three SNPs were in very strong linkage disequilibrium with one another (r2 = 0.94-0.96), rs8099917 and rs10853728 were selected for an analysis of their influence on the achievement of rapid virological response [RVR; seronegativity for hepatitis C virus (HCV) RNA in treatment week 4] and sustained virological response (SVR; seronegativity for HCV RNA throughout 24 weeks of posttreatment follow-up). The rs10853728 genotype did not predict RVR or SVR in HCV-2 patients. However, patients with the rs8099917 TT genotype, in comparison with patients with GT/GG genotypes, had a significantly higher rate of achieving RVR (85.2% versus 72.0%, P = 0.017) but did have not a significantly higher rate of achieving SVR (89.4% versus 86.0%). Multivariate analysis revealed that a baseline HCV viral load <400,000 IU/mL was the strongest predictor of RVR [odds ratio (OR) = 4.27, 95% confidence interval (CI) = 2.31-7.87, P < 0.001], and this was followed by advanced liver fibrosis (OR = 0.28, 95% CI = 0.15-0.53, P < 0.001), the carriage of the rs8099917 TT genotype (OR = 3.10, 95% CI = 1.34-7.21, P = 0.008), and the pretreatment level of aspartate aminotransferase (OR = 0.996, 95% CI = 0.99-1.00, P = 0.04). Nevertheless, the achievement of RVR was the single predictor of SVR with an OR of 19.37 (95% CI = 8.89-42.23, P < 0.001), whereas the rs8099917 genotypes played no role in achieving SVR with or without RVR. CONCLUSION: The rs8099917 TT genotype is significantly independently predictive of RVR, which is the single best predictor of SVR, in Asian HCV-2 patients.     

Classifying genotype F of hepatitis B virus into F1 and F2 subtypes

AIM: To explore the propriety of providing hepatitis B virus (HBV) genotypes F and H with two distinct genotypes. METHODS: Eleven HBV isolates of genotype F (HBV/F) were recovered from patients living in San Francisco, Japan, Panama, and Venezuela, and their full-length sequences were determined. Phylogenetic analysis was carried out among them along with HBV isolates previously reported. RESULTS: Seven of them clustered with reported HBV/F isolates in the phylogenetic tree constructed on the entire genomic sequence. The remaining four flocked on another branch along with three HBV isolates formerly reported as genotype H. These seven HBV isolates, including the four in this study and the three reported, had a sequence divergence of 7.3-9.5% from the other HBV/F isolates, and differed by > 13.7% from HBV isolates of the other six genotypes (A-E and G). Based on a marked genomic divergence, falling just short of > 8% separating the seven genotypes, these seven HBV/F isolates were classified into F2 subtype and the former seven into F1 subtype provisionally. In a pairwise comparison of the S-gene sequences among the 7 HBV/F2 isolates and against 47 HBV/F1 isolates as well as 136 representing the other six genotypes (A-E and G), two clusters separated by distinct genetic distances emerged. CONCLUSION: Based on these analyses, classifying HBV/F isolates into two subtypes (F1 and F2) would be more appropriate than providing them with two distinct genotypes (F and H).     

Classifying genotype F of hepatitis B virus into F1 and F2 subtypes

AIM: To explore the propriety of providing hepatitis B virus(HBV) genotypes F and H with two distinct genotypes.METHODS: Eleven HBV isolates of genotype F (HBV/F)were recovered from patients living in San Francisco,Japan, Panama, and Venezuela, and their full-length sequences were determined. Phylogenetic analysis was carried out among them along with HBV isolates previously reported.RESULTS: Seven of them clustered with reported HBV/F isolates in the phylogenetic tree constructed on the entire genomic sequence. The remaining four flocked on another branch along with three HBV isolates formerly reported as genotype H. These seven HBV isolates, including the four in this study and the three reported, had a sequence divergence of 7.3-9.5% from the other HBV/F isolates,and differed by ＞ 13.7% from HBV isolates of the other six genotypes (A-E and G). Based on a marked genomic divergence, falling just short of ＞8% separating the seven genotypes, these seven HBV/F isolates were classified into F2 subtype and the former seven into F1 subtype provisionally. In a pairwise comparison of the S-gene sequences among the 7 HBV/F2 isolates and against 47HBV/F1 isolates as well as 136 representing the other six genotypes (A-E and G), two clusters separated by distinct genetic distances emerged.CONCLUSION: Based on these analyses, classifying HBV/F isolates into two subtypes (F1 and F2) would be more appropriate than providing them with two distinct genotypes (F and H).     

Antibodies to West Nile virus in asymptomatic mammals, birds, and reptiles in the Yucatan Peninsula of Mexico

Surveillance for evidence of West Nile virus (WNV) infection in taxonomically diverse vertebrates was conducted in the Yucatan Peninsula of Mexico in 2003 and 2004. Sera from 144 horses on Cozumel Island, Quintana Roo State, 415 vertebrates (257 birds, 52 mammals, and 106 reptiles) belonging to 61 species from the Merida Zoo, Yucatan State, and 7 farmed crocodiles in Ciudad del Carmen, Campeche State were assayed for antibodies to flaviviruses. Ninety (62%) horses on Cozumel Island had epitope-blocking enzyme-linked immunosorbent assay (ELISA) antibodies to flaviviruses, of which 75 (52%) were seropositive for WNV by plaque reduction neutralization test (PRNT). Blocking ELISA antibodies to flaviviruses also were detected in 13 (3%) animals in the Merida Zoo, including 7 birds and 2 mammals (a jaguar and coyote) seropositive for WNV by PRNT. Six (86%) crocodiles in Campeche State had PRNT-confirmed WNV infections. All animals were healthy at the time of serum collections and none had a history of WNV-like illness.     

Molecular analysis of hepatitis B virus isolates in Mexico: Predominant circulation of hepatitis B virus genotype H

AIM:To determine the genotypes in Mexican hepatitis B virus (HBV) isolates and characterize their precore and core promoter mutations.METHODS: Forty-nine HBV isolates of Mexico obtained from sera of 15 hepatitis patients, 6 hemodialysis patients, 20 men seeking HIV testing, and 8 AIDS patients were analyzed. HBV isolates were amplified by PCR,and genotyped by line probe assay (INNO-LiPA HBV Genotyping; INNOGENETICS N V, Ghent, Belgium).HBV genotype confirmation was performed by DNA sequencing part of the sAg region. Precore and core promoter mutation characterization was performed by line probe assay (INNO-LiPA HBV PreCore; INNOGENETICS N.V.; Ghent, Belgium).RESULTS: Overall, HBV genotype H was found in 37(75.5%) out of the 49 isolates studied. HBV genotypes G, A, and D were found in 5 (10.2%), 4 (8.2%), and 3(6.1%) isolates, respectively. HBV genotype H was predominant in isolates from hemodialysis patients (100%),hepatitis patients (80%), and men seeking HIV testing (75%), and accounted for half of infections in AIDS patients (50%). Six (12.2%) out of the 49 HBV isolates showed both wild type and mutant populations at precore codon 28. These mixed wild type and precore murant populations were observed in one HBV genotype A isolate and in all HBV genotype G isolates. A dual variant core promoter mutation was observed in 1 (2%) of the isolates, which was genotype H.CONCLUSION: HBV genotype H is highly predominant in HBV isolates of Mexico followed by genotypes G, A and D. A low frequency of precore and core promoter mutations is observed in HBV Mexican isolates.     

Molecular typing of dengue virus type 2 in Brazil

Strain typing is a critical tool for molecular epidemiological analysis and can provide important information about the spread of dengue viruses. Here, we performed a molecular characterization of DEN-2 viruses isolated in Brazil during 1990-2000 from geographically and temporally distinct areas in order to investigate the genetic distribution of this serotype circulating in the country. Restriction site-specific polymerase chain reaction (RSS)-PCR presented the same pattern for all 52 Brazilian samples, showing the circulation of just one DEN-2 variant. Phylogenetic analysis using progressive pairwise alignments from 240-nucleotide sequences of the E/NS1 junction in 15 isolates showed that they belong to genotype III (Jamaica genotype).     

Absence of leukocytes permissive to dengue 2 virus in the acute phase of dengue hemorrhagic fever.

Patients with primary dengue infection developed dengue 2 virus (D2V) permissive peripheral blood leukocytes (PBL) 2--3 weeks after infection. PBL from healthy individuals with dengue antibody were permissive to D2V in vitro, suggesting that immunologically mediated in vitro D2V permissiveness persists for a relatively long time after recovery from dengue infection. However, PBL obtained from second infection dengue hemorrhagic fever patients did not support D2V growth during the acute phase of illness but did so during convalescence. Leukocytes from dengue-immune patients with typhoid fever or non-dengue viral illness were permissive throughout both acute and convalescent phases of illness although there was tendency for increased permissiveness during convalescence. Acute phase PBL from DHF patients synthesized and secreted dengue neutralizing antibody in culture. Absence of D2V replication in these cultures was strongly, but not completely, correlated with antibody production. Other immunological mechanisms, in addition to antibody, may be operating in vitro or in vivo during acute phase dengue hemorrhagic fever to alter the permissiveness of PBL to D2V infection.     

Molecular analysis of hepatitis B virus isolates in Mexico： Predominant circulation of hepatitis B virus genotype H

INTRODUCTIONHepatitis B virus(HBV)is an important cause of morbidity and mortality worldwide.It is estimated that2billion people are infected with HBV and350million individuals suffer from chronic HBV infection in the world[1,2].Chronic HBV infection may …