Genetic characterization of Cacipacoré virus from ticks collected in São Paulo State, Brazil.

Cacipacoré virus (CPCV) is a potential emerging virus classified in the genus Flavivirus, family Flaviviridae. In the present study, we present the genetic characterization of a CPCV isolated from ticks (Amblyomma cajennense) collected from a sick capybara (Hydrochoerus hydrochaeris) in São Paulo State, Brazil. The CPCV isolate shares the typical genomic organization of flaviviruses with 10,857 nucleotides in length and a single open reading frame of 10,284 nucleotides encoding a polyprotein of 3,427 amino acids. Phylogenetic analysis revealed that CPCV is unique, as a potentially tick-borne virus, in the Japanese encephalitis virus serogroup.

Development and optimization of a sensitive TaqMan® real-time PCR with synthetic homologous extrinsic control for quantitation of Human cytomegalovirus viral load.

Human cytomegalovirus (Human herpesvirus 5, HCMV) causes frequent asymptomatic infections in the general population. However, in immunosuppressed patients or congenitally infected infants, HCMV is related to high morbidity and mortality. In such cases, a rapid viral detection is crucial for monitoring the clinical outcome and the antiviral treatment. In this study, we optimized a sensitive biplex TaqMan® real-time PCR for the simultaneous detection and differentiation of a partial HCMV UL97 sequence and homologous extrinsic control (HEC) in the same tube. HEC was represented by a plasmid containing a modified HCMV sequence retaining the original primer binding sites, while the probe sequence was substituted by a phylogenetically divergent one (chloroplast CF0 subunit plant gene). It was estimated that the optimal HEC concentration, which did not influence the HCMV amplification is 1,000 copies/reaction. The optimized TaqMan® PCR demonstrated high analytical sensitivity (6.97 copies/reaction, CI = 95%) and specificity (100%). Moreover, the reaction showed adequate precision (repeatability, CV = 0.03; reproducibility, CV = 0.0027) and robustness (no carry-over or cross-contamination). The diagnostic sensitivity (100%) and specificity (97.8%) were adequate for the clinical application of the molecular platform. The optimized TaqMan® real-time PCR is suitable for HCMV detection and quantitation in predisposed patients and monitoring of the applied antiviral therapy. J. Med. Virol. 88:1604-1612, 2016. © 2016 Wiley Periodicals, Inc.

Polymorphisms in human leukocyte antigens, human platelet antigens, and cytokine genes in hantavirus cardiopulmonary syndrome patients from Ribeirão Preto, Brazil.

Hantavirus cardiopulmonary syndrome is a severe human disease associated with hantavirus infection. The clinical course of illness varies greatly among individuals, possibly due to viral and immunological elements and the influence of host genetic factors on clinical outcome. As the magnitude of immune activation has been associated with disease severity, polymorphisms in genes involved in the immune response that may affect the development of this syndrome were investigated. Polymorphisms in the TGF-ß, IL-10, IL-6, and IFN-γ genes, human leukocyte antigens (HLA), and human platelet alloantigens (HPA) genes were investigated in 122 patients with Araraquara virus infection from Ribeirão Preto, Brazil. Patients were divided into two groups: hantavirus cardiopulmonary syndrome (HCPS group; n = 26) and hantavirus seropositive only (n = 96). The frequencies of HLA alleles, cytokines and platelet antigen genotypes were evaluated in both groups and compared to a control group. The data demonstrated no significant influence of the HLA alleles, HPA, IL-6, and IL-10 genotypes on susceptibility to hantavirus infection. However, the hantavirus seropositive group presented a significantly higher frequency of a polymorphism associated with a high IFN-γ production than the HCPS group. In addition, a genotype associated with high TGF-ß production was found more frequently in individuals infected with hantavirus than in the control group. This phenotype was associated with a less intense thrombocytopenia in the HCPS group and may be protective against the most severe form of hantavirus disease. More studies are required to quantify further the influence of the high TGF-ß producer phenotype on the outcome of hantavirus infection.

Association of -308G/A polymorphism in the tumor necrosis factor-alpha gene promoter with susceptibility to development of hantavirus cardiopulmonary syndrome in the Ribeirão Preto region, Brazil.

Activation of the immune response in hantavirus cardiopulmonary syndrome (HCPS) leads to a high TNF production, probably contributing to the disease. The polymorphic TNF2 allele (TNF -308G/A) has been associated with increased cytokine production. We investigated the association of the TNF2 allele with the outcome of hantavirus infection in Brazilian patients. A total of 122 hantavirus-exposed individuals (26 presenting HCPS and 96 only hantavirus seroconversion) were studied. The TNF2 allele was more frequently found in HCPS patients than in individuals with positive serology for hantavirus but without a history of HCPS illness, suggesting that the TNF2 allele could represent a risk factor for developing HCPS.

Evaluation of an enzyme-linked immunosorbent assay based on Araraquara virus recombinant nucleocapsid protein.

Laboratory diagnosis of hantavirus cardiopulmonary syndrome (HCPS) in Brazil has been performed mostly by detection of IgM antibodies to recombinant antigen purified from Sin Nombre virus and Andes virus (ANDV). Recently, a recombinant nucleocapsid (rN) protein of Araraquara virus (ARAV), a Brazilian hantavirus, was obtained in Escherichia coli. To evaluate ARAV rN as antigen for antibody detection, serum samples from 30 patients from Argentina seropositive for hantavirus were tested. All samples were positive for IgG and IgM by enzyme-linked immunosorbent assay (ELISA) using either ARAV rN or ANDV rN antigens. In Brazil, six of 60 serum samples from patients with suspected HCPS (10%) were positive for IgM by ELISA using ARAV rN antigen and 7 were positive using ANDV rN antigen. For results obtained with 90 serum samples analyzed by IgM ELISA with ANDV rN antigen, the sensitivity of the IgM ELISA using ARAV rN antigen was 97.2%, the specificity was 100%, the positive predictive value was 100%, and the negative predictive value was 98.1%. The results show that ARAV rN is a suitable antigen for diagnosis of hantavirus infection in Brazil and Argentina.