Genotyping of human parvovirus B19 in clinical samples from Brazil and Paraguay using heteroduplex mobility assay, single-stranded conformation polymorphism and nucleotide sequencing

Mendonça, Marcos César Lima de; Ferreira, Ana Maria de Amorim; Bravo, Marta Santos; Oviedo, Elva; Bello, Maria Sônia Dal; Siqueira, Marilda Mendonça; Maceira, Juan Piñeiro; Hubinger, M. G. Von; Couceiro, José Nelson dos Santos Silva

doi:10.1590/s0074-02762011000400019

Cited by 10 publications

(10 citation statements)

References 12 publications

(19 reference statements)

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“…This is consistent with previous phylogenetic studies, which demonstrated a predominance of subgenotype 1A [Hübschen et al, 2009] over the other genotypes. Recent studies performed in Brazil (Midwest, South, and Southeast Brazil) and Paraguay also confirmed the high prevalence of genotype 1 [Mendonça et al, 2011].…”

Section: Discussionmentioning

confidence: 68%

Molecular and phylogenetic analyses of human Parvovirus B19 isolated from Brazilian patients with sickle cell disease and β‐thalassemia major and healthy blood donors

Slavov

Haddad

Silva-Pinto

et al. 2012

Journal of Medical Virology

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Human Parvovirus B19 (B19V) is a recognized cause of life-threatening conditions among patients with hemoglobinopathies. This study investigates B19V infection in patients with sickle cell disease and β-thalassemia using different experimental approaches. A total of 183 individuals (144 with sickle cell disease and 39 with β-thalassemia major) and 100 healthy blood donors were examined for B19V using anti-B19V IgG enzyme immunoassay, quantitative PCR, DNA sequencing, and phylogenetic analysis. Viremia was documented in 18.6% of patients and 1% of donors, and was generally characterized by low viral load (VL); however, acute infections were also observed. Anti-B19V IgG was detected in 65.9% of patients with sickle cell disease and in 60% of donors, whereas the patients with thalassemia exhibited relatively low seroreactivity. The seroprevalence varied among the different age groups. In patients, it progressively increased with age, whereas in donors it reached a plateau. Based on partial NS1 fragments, all isolates detected were classified as subgenotype 1A with a tendency to elicit genetically complex infections. Interestingly, quasispecies occurred in the plasma of not only patients but also donors with even higher heterogeneity. The partial NS1 sequence examined did not exhibit positive selection. Quantitation of B19V with a conservative probe is a technically and practically useful approach. The extensive spread of B19V subgenotype 1A in patients and donors and its recent introduction into the countryside of the São Paulo State, Brazil were demonstrated; however, it is difficult to establish a relationship between viral sequences and the clinical outcomes of the infection.

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Section: Discussionmentioning

confidence: 68%

Molecular and phylogenetic analyses of human Parvovirus B19 isolated from Brazilian patients with sickle cell disease and β‐thalassemia major and healthy blood donors

Slavov

Haddad

Silva-Pinto

et al. 2012

Journal of Medical Virology

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“…In other fields, the HMA is also a reliable method for identifying mutations, insertions and deletions in genes of interest. Indeed, this assay has been successfully used in epidemiological assays and in searches for linkage in infections, as well as in studies of polymorphism, genotyping, HIV-1 subtyping, phylogenetic analysis, natural HIV-1 recombination, viral genetic heterogeneity and diversity (Heyndrickx et al , 2000; White et al , 2000; Agwale et al , 2001; Mehta et al , 2010; Huang et al , 2011; Mendonça et al , 2011; Manigart et al , 2012). …”

Section: Discussionmentioning

confidence: 99%

Use of the heteroduplex mobility assay and cell sorting to select genome sequences of the CCR5 gene in HEK 293T cells edited by transcription activator-like effector nucleases

et al. 2014

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Engineered nucleases such as zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) are one of the most promising tools for modifying genomes. These site-specific enzymes cause double-strand breaks that allow gene disruption or gene insertion, thereby facilitating genetic manipulation. The major problem associated with this approach is the labor-intensive procedures required to screen and confirm the cellular modification by nucleases. In this work, we produced a TALEN that targets the human CCR5 gene and developed a heteroduplex mobility assay for HEK 293T cells to select positive colonies for sequencing. This approach provides a useful tool for the quick detection and easy assessment of nuclease activity.

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“…Currently genotype 1 is the genotype circulating most widely in Europe and worldwide, while genotype 2 is only found rarely. Genotype 3 is considered to be most prevalent in Africa but was also detected recently in Europe, the Americas and Asia [Servant et al, 2002; Candotti et al, 2004; Hokynar et al, 2004; Cohen et al, 2006; Sanabani et al, 2006; Toan et al, 2006; Hubschen et al, 2009; Ke et al, 2011; Mendonca et al, 2011; Opaleye et al, 2011].…”

Section: Introductionmentioning

confidence: 98%

Human parvovirus B19 surveillance in patients with rash and fever from Belarus

Yermalovich

Hübschen

Semeiko

et al. 2012

Journal of Medical Virology

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Human parvovirus B19 (B19V) infection in immunocompetent patients usually has a mild clinical course, but during pregnancy it can cause serious and even fatal complications in the fetus. The most common clinical presentation of B19V infection is erythema infectiosum and in this case laboratory confirmation is required for differentiation from other exanthematous diseases. Measles and rubella negative sera collected in Belarus between 2005 and 2008 from 906 patients with a rash and fever were screened for B19V infection by ELISA. More than 35% of the samples (322/906) were positive for B19V. The proportion ranged from 10.1% in 2008 to 53.2% in 2006 when an outbreak took place in Minsk city. All B19V outbreaks and cluster cases occurred during the winter-spring period, but sporadic cases were recorded basically throughout the year. The majority of the cases (56.5%) occurred among the 2 till 10 year old children, and 27.3% of the cases were observed in adults between 19 and 53 years. All 104 B19V strains sequenced in the NS1/VP1u region belonged to genotype 1 with a maximal genetic distance of 1.75%. The two phylogenetic clusters reflected the geographic origins of the viruses within the country. Forty-two unique nucleotide mutations as compared to sequences downloaded from GenBank were found in the VP1u and NS1 regions; most of these changes were nonsynonymous. This report highlights the importance of B19V infection in patients with a rash and fever in Belarus.

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Genotyping of human parvovirus B19 in clinical samples from Brazil and Paraguay using heteroduplex mobility assay, single-stranded conformation polymorphism and nucleotide sequencing

Cited by 10 publications

References 12 publications

Molecular and phylogenetic analyses of human Parvovirus B19 isolated from Brazilian patients with sickle cell disease and β‐thalassemia major and healthy blood donors

Molecular and phylogenetic analyses of human Parvovirus B19 isolated from Brazilian patients with sickle cell disease and β‐thalassemia major and healthy blood donors

Use of the heteroduplex mobility assay and cell sorting to select genome sequences of the CCR5 gene in HEK 293T cells edited by transcription activator-like effector nucleases

Human parvovirus B19 surveillance in patients with rash and fever from Belarus

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