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1.
BMC Infect Dis ; 19(1): 738, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31438880

RESUMO

BACKGROUND: The technique most frequently used to genotype HCV is quantitative RT-PCR. This technique is unable to provide an accurate genotype/subtype for many samples; we decided to develop an in-house method with the goal of accurately identifying the genotype of all samples. As a Belgium National Centre of reference for hepatitis, we developed in-house sequencing not only for 5'UTR and core regions starting from VERSANT LiPA amplicons but also for NS5B regions. The sequencing of VERSANT LiPA amplicons might be useful for many laboratories worldwide using the VERSANT LiPA assay to overcome undetermined results. METHODS: 100 samples from Hepatitis C virus infected patients analysed by the VERSANT HCV Genotype 2.0 LiPA Assay covering frequent HCV types and subtypes were included in this study. NS5B, 5'UTR and Core home-made sequencing were then performed on these samples. The sequences obtained were compared with the HCV genomic BLAST bank. RESULTS: All the samples were characterised by the VERSANT LiPA assay (8 G1a, 17 G1b, 6 G2, 11 G3, 13 G4, and 10 G6). It was not possible to discriminate between G6 and G1 by the VERSANT LiPA assay for 8 samples and 27 had an undetermined genotype. Forty-one samples were sequenced for the three regions: NS5B, 5'UTR and Core. Twenty-three samples were sequenced for two regions: 5' UTR and Core and 36 samples were sequenced only for NS5B. Of the 100 samples included, 64 samples were analysed for 5'UTR and Core sequencing and 79 samples were analysed for NS5B sequencing. The global agreement between VERSANT LiPA assay and sequencing was greater than 95%. CONCLUSIONS: In this study, we describe a new, original method to confirm HCV genotypes of samples not discriminated by a commercial assay, using amplicons already obtained by the screening method, here the VERSANT LiPA assay. This method thus saves one step if a confirmation assay is needed and might be of usefulness for many laboratories worldwide performing VERSANT LiPA assay in particular.


Assuntos
Técnicas de Genotipagem/métodos , Hepacivirus/genética , Hepatite C/diagnóstico , Técnicas de Sonda Molecular , Kit de Reagentes para Diagnóstico , Análise de Sequência de RNA/métodos , Regiões 5' não Traduzidas , Sequência de Bases , Comércio , Genômica/métodos , Genótipo , Técnicas de Genotipagem/economia , Hepacivirus/isolamento & purificação , Hepatite C/virologia , Humanos , Técnicas de Sonda Molecular/economia , Filogenia , RNA Viral/análise , RNA Viral/isolamento & purificação , Kit de Reagentes para Diagnóstico/economia , Estudos Retrospectivos , Análise de Sequência de RNA/economia , Centros de Atenção Terciária
2.
Arch Virol ; 164(10): 2537-2543, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31309291

RESUMO

In May 2017, many free-ranging dorcas gazelles (Gazella dorcas) with suspected signs of peste des petits ruminants (PPR) were reported in Dinder National Park, South-Eastern Sudan. Peste des petits ruminants virus (PPRV) antigen and nucleic acid were detected in specimens from these gazelles using an immunocapture ELISA and a reverse transcription polymerase chain reaction (RT-PCR) assays. PPRV was also detected in four healthy semi-captive dorcas gazelles from two areas of Khartoum State. Phylogenetic analysis showed that these PPRV strains belonged to the lineage IV genotype. The present study demonstrates that gazelles are a potential wild small ruminant host for PPRV and may influence the epidemiology of PPR in the Sudan.


Assuntos
Antílopes/virologia , Reservatórios de Doenças , Peste dos Pequenos Ruminantes/virologia , Vírus da Peste dos Pequenos Ruminantes/isolamento & purificação , Animais , Antígenos Virais/isolamento & purificação , Ensaio de Imunoadsorção Enzimática , Genótipo , Vírus da Peste dos Pequenos Ruminantes/classificação , Vírus da Peste dos Pequenos Ruminantes/genética , Filogenia , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Gastropatias , Sudão
3.
BMC Infect Dis ; 19(1): 595, 2019 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-31288749

RESUMO

BACKGROUND: Noroviruses (NVs) are an important cause of acute gastroenteritis (AGE) worldwide. There are limited data on the prevalence and molecular characterization of NVs in children in Hohhot, China. METHODS: Between January 2012 and December 2017, 1863 stool samples were collected at Maternal and Child Health Hospital in Hohhot. All samples were screened for NVs by real-time reverse transcription polymerase chain reaction (real-time RT-PCR). RESULTS: NVs were detected in 24.15% of these inpatient cases, ranging from 12.78 to 32.92% in different years. NV was detected throughout the year, with a peak in winter. Based on sequence analysis of the partial VP1 gene, the 306 identified NV strains were divided into six genotypes: GII.3 (71.24%), GII.4 (23.53%), and GII.2, GII.5, GII.6, and GII.13 (total 5.23%). Based on further sequence analysis of the RNA-dependent RNA polymerase (RdRp), GII.P12/GII.3, GII.Pe/GII.4, and GII.P4/GII.4 were identified as predominant genotypes, accounting for 92.6% of genotyped strains. The median age of the children with NV infection was 8.0 (range 0-59) months. However, children infected with GII.3 were younger (median 7.0 months) than GII.4-positive patients (median 10.0 months). CONCLUSION: NV contributed greatly to AGE among hospitalized children in Hohhot in China. Continuous surveillance is important for understanding the local prevalence and characterization of NV.


Assuntos
Infecções por Caliciviridae/diagnóstico , Gastroenterite/diagnóstico , Norovirus/genética , Doença Aguda , Infecções por Caliciviridae/epidemiologia , Criança Hospitalizada , Pré-Escolar , China/epidemiologia , Fezes/virologia , Feminino , Gastroenterite/epidemiologia , Genótipo , Humanos , Lactente , Recém-Nascido , Masculino , Norovirus/classificação , Norovirus/isolamento & purificação , Filogenia , Prevalência , RNA Viral/isolamento & purificação , RNA Viral/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Estações do Ano
4.
Zhonghua Liu Xing Bing Xue Za Zhi ; 40(5): 580-584, 2019 May 10.
Artigo em Chinês | MEDLINE | ID: mdl-31177742

RESUMO

Objective: To analyze the change trend of HIV genetic subtypes and compare the first CD(4)(+)T cell counts of newly diagnosed HIV infected patients in Liuzhou from 1998 to 2012, and provide a reference for AIDS prevention and control. Methods: Newly diagnosed HIV-infected patients from 1998 to 2012 in Liuzhou were selected through national HIV/ADIS comprehensive response information management system. Their plasma samples were used for RNA gene extraction, amplification, sequencing and genotyping. Coharan-Armitage trend test was used to analyze the ratio trend of genetic subtypes and phylogenetic clusters of HIV and Wilcoxon Rank Sum Test was used to compare the first CD(4)(+)T cell counts (CD(4)) of the different subtype HIV infected patients. Results: A total of 1 877 newly diagnosed HIV infected patients were included in the study. From 1998 to 2012, the proportions of CRF01_AE and CRF01_AE (Cluster 1) increased from 78.4% (76/97) to 91.5% (1 441/1 574), from 63.9% (62/97) to 74.0% (1 164/1 574), and the proportion of CRF07_BC decreased from 17.5% (17/97) to 4.6% (72/1 574), respectively (Z=4.632, P<0.001; Z=2.455, P=0.014; Z=-5.943, P<0.001). The median and interquartile range of the first CD(4) of the patients infected with subtype CRF01_AE (Cluster 1), CRF01_AE (Cluster 2), CRF07_BC and CRF08_BC were 230 (83-375), 215 (48-351), 365 (254-503) and 334 (206-479) cell/µl, respectively. The first CD(4) levels of the patients infected with subtype CRF01_AE (Cluster 1) or CRF01_AE (Cluster 2) were significantly lower than those of CRF07_BC (Z=-4.795, P<0.001; Z=-4.238, P<0.001). Conclusion: The genetic subtypes of HIV were mainly CRF01_AE in newly diagnosed HIV-infected patients and this subtype proportion was in increase and the first CD(4) levels of the patients were low in Liuzhou during 1998 to 2012.


Assuntos
Infecções por HIV/diagnóstico , HIV-1/classificação , HIV-1/genética , RNA Viral/isolamento & purificação , Linfócitos T , Contagem de Células , China/epidemiologia , DNA Viral/genética , Genótipo , Infecções por HIV/sangue , Infecções por HIV/epidemiologia , Infecções por HIV/virologia , HIV-1/isolamento & purificação , Humanos , Filogenia , Reação em Cadeia da Polimerase , Análise de Sequência de DNA
5.
BMC Infect Dis ; 19(1): 566, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253127

RESUMO

BACKGROUND: To infer transmission direction of a HIV transmission chain is helpful not only in legal jurisdiction but also in precise intervention to prevent HIV spread. Recently, the direction of transmission is inferred by whether paraphyletic-monophyletic (PM) or a combination of paraphyletic and polyphyletic (PP) topologies is observed or not between the sequences of source and recipient in the phylogenetic tree. However, paraphyly between them often declines over time and may disappear between spouses due to bidirectional transmission after primary infection. In this study, our aim is to test the reliability of inferring HIV transmission direction between epidemiologically linked HIV-1 positive couples using whether or not paraphyly is observed in phylogenetic tree. METHODS: HIV quasi-species were sequenced using PCR product clones, and then Bayesian analysis of molecular sequences with MCMC was employed to construct phylogenetic relationship of env, gag, pol gene fragments of HIV-1 positive couples using BEAST software. RESULTS: Our results showed that all sequences of seven couples except pol sequences of couple 12 and 13 form their own monophyletic cluster in phylogenetic tree including the closest control sequences from GenBank or other studies on local samples, which are supported by significant Bayesian posterior probabilities more than 0.9932. Of seven couples, paraphyly is only observed in phylogenetic tree constructed with env and pol gene sequences of three couples and gag gene sequences of four couples. Paraphyly is not observed in half of HIV positive couples. Pol sequences of couple 13 is separated by Blast selected controls; pol sequences of couple 12 in phylogenetic tree is supported by a lower Bayesian posterior value. CONCLUSION: Paraphyly relationship between sequences of donator and recipient is only observed among partial HIV-1 positive couples with epidemiological link. Phylogenetic relationship is not always the same when various gene regions of HIV are used to conduct phylogenetic analysis. The combination of phylogenetic analysis based on various gene regions of HIV and enough epidemiology investigation is essential when inferring transmission direction of HIV in a transmission chain or in one couple. However, while observed paraphyly can be used to infer transmission direction in HIV-1 positive couple, no observed paraphyly cannot deny it.


Assuntos
Infecções por HIV/transmissão , HIV-1/genética , Quase-Espécies , Teorema de Bayes , Feminino , Infecções por HIV/epidemiologia , Infecções por HIV/virologia , HIV-1/classificação , Humanos , Masculino , Filogenia , Reação em Cadeia da Polimerase , RNA Viral/genética , RNA Viral/isolamento & purificação , RNA Viral/metabolismo , Comportamento Sexual , Produtos do Gene gag do Vírus da Imunodeficiência Humana/classificação , Produtos do Gene pol do Vírus da Imunodeficiência Humana/classificação
6.
BMC Vet Res ; 15(1): 214, 2019 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-31238913

RESUMO

BACKGROUND: Per- and polyfluoroalkyl substances (PFASs) are environmentally persistent and bioaccumulative chemicals. Immunomodulation is among the most concerning of toxic effects linked with PFAS exposure in mammalian models. However, no studies had yet shown this to be true in birds. Thus, we designed and conducted the first study to determine if PFASs could cause immunomodulation in birds. Secondly, we wanted to determine the effects on an avian host when exposed not only to immunomodulating chemicals, but also to a viral challenge. The aim, to determine if PFAS mediated immunmodulation functionally affects a pathogen challenge for a host. As innate immune system signalling pathways initiate crucial responses against a pathogen challenge, and are lesser studied than their adaptive counterparts, we focused on these pathways. To provide the first information on this, an in vitro experiment was designed and performed using chicken embryo fibroblasts exposed to perfluorooctane sulfonate (PFOS) (22 ppm) and immune markers characterised before and after being infected with gallid herpesvirus-2 (GaHV-2). RESULTS: The expression of two pro-inflammatory cytokines, namely interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-α), the nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NF-κB), and the anti-inflammatory cytokine interleukin 4 (IL-4) were investigated in various scenarios. These results showed that exposure to PFOS decreased immune gene expression in chicken fibroblasts from 36 h post-exposure. Next, it was shown that this decrease could be mitigated by infection with gallid herpesvirus-2, which increased gene expression back to the baseline/control levels. CONCLUSIONS: Not only is this the first study to provide the expected evidence that PFOS has immunomodulatory potential in birds, it also provides unexpected data that virus infections can mitigate this negative effect. Thereby, further research, including in vivo and in situ studies, on the impact of PFOS on host-virus interactions is now warranted, as it has been overlooked and might contribute to our understanding of recent disease outbreaks in wildlife. The mechanisms by which gallid herpesvirus mitigates immunomodulation were beyond the scope of this study, but are now of interest for future study.


Assuntos
Ácidos Alcanossulfônicos/toxicidade , Fluorcarbonetos/toxicidade , Herpesvirus Galináceo 2/imunologia , Imunomodulação/efeitos dos fármacos , Doença de Marek/imunologia , Viroses/veterinária , Ácidos Alcanossulfônicos/imunologia , Animais , Linhagem Celular , Embrião de Galinha , DNA Complementar , Fibroblastos/efeitos dos fármacos , Fluorcarbonetos/imunologia , Expressão Gênica/efeitos dos fármacos , Imunomodulação/genética , Mediadores da Inflamação/metabolismo , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Transdução de Sinais/efeitos dos fármacos , Viroses/imunologia
7.
Virol J ; 16(1): 76, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31159808

RESUMO

BACKGROUND: Stratified mucin-producing intraepithelial lesion (SMILE) is a rare precursor lesion in the uterine cervix that is considered a variant of adenocarcinoma in situ (AIS). Although human papillomavirus (HPV) is thought to be related to the development of SMILE, there is little information available on the detection of HPV integrated into the lesion. CASE PRESENTATION: A 30-year-old female underwent a routine uterine cervical cancer screening, and her Pap smear indicated the possible existence of atypical glandular cells. A cervical biopsy with endocervical curettage was performed. The histopathological analysis showed that she had SMILE and high-grade squamous intraepithelial lesion (HSIL) on her cervix. The lesion was found to be positive for HPV genotypes 52 and 68 by multiplex PCR. In situ hybridization with HPV RNA probes revealed that these HPV types were involved in the onset of HSIL and SMILE, respectively. CONCLUSIONS: Rare, high-risk HPV genotypes may contribute to the development of SMILE, and their detection can be useful for preventing the progression to carcinoma and ensuring adequate patient management.


Assuntos
Neoplasia Intraepitelial Cervical/virologia , Mucinas/biossíntese , Papillomaviridae/genética , Infecções por Papillomavirus/diagnóstico , RNA Viral/isolamento & purificação , Lesões Intraepiteliais Escamosas Cervicais/virologia , Neoplasia Intraepitelial Cervical/patologia , Colposcopia , Detecção Precoce de Câncer , Feminino , Humanos , Hibridização in Situ Fluorescente , Reação em Cadeia da Polimerase Multiplex , Papillomaviridae/isolamento & purificação , Sondas RNA , Lesões Intraepiteliais Escamosas Cervicais/patologia , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologia
8.
BMC Infect Dis ; 19(1): 562, 2019 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-31248372

RESUMO

BACKGROUND: The proportion of older HIV-1 infected people in China has increased rapidly in recent years. Elucidation of the transmission characteristics of this high-risk population subgroup is helpful for the development of tailored interventions. METHODS: A phylogenetic analysis was performed that uses available HIV-1 pol sequences amplified with nested RT-PCR from plasma samples of all newly diagnosed participants spanning from October 2017 to September 2018 in Fuyang, Anhui Province. Transmission clusters were identified as two or more sequences that shared a corresponding node with an aLRT-SH value ≥90 in the maximum-likelihood phylogenetic tree and had an overall mean genetic distance of ≤1.5%. A local transmission cluster was defined as a cluster that had more than 80% of its sequences from Fuyang. The role of older people in local HIV-1 transmission was determined using an integration of molecular and demographic data. RESULTS: Of 362 available sequences, 14 subtypes, and 28 local transmission clusters were identified. It was found that the proportion of older people in the local transmission cluster (69/77, 89.61%) was much higher than that of younger people (46/114, 40.35%) (χ2 test, P < 0.001). In the pretreatment drug resistance analysis, the proportion of sequences with PDRMs in the local transmission cluster was not significantly different between the older people group (57.14%, 4/7) and non-old-aged group (11.11%, 1/9) (Fisher's exact test, P > 0.05). CONCLUSION: By combining phylogenetic analyses with demographic data, more detailed information was provided about the local transmission structure in Fuyang. These findings suggested that older people play an important role in local transmission, and more tailored interventions for this population subgroup are urgently needed.


Assuntos
Infecções por HIV/diagnóstico , HIV-1/classificação , Adulto , Idoso , Antirretrovirais/uso terapêutico , China/epidemiologia , Farmacorresistência Viral/genética , Feminino , Genótipo , Infecções por HIV/tratamento farmacológico , Infecções por HIV/epidemiologia , Infecções por HIV/transmissão , HIV-1/genética , HIV-1/isolamento & purificação , Humanos , Masculino , Pessoa de Meia-Idade , Filogenia , Reação em Cadeia da Polimerase , RNA Viral/isolamento & purificação , RNA Viral/metabolismo
9.
BMC Vet Res ; 15(1): 168, 2019 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-31126297

RESUMO

BACKGROUND: Seneca Valley virus (SVV) has emerged in multiple countries in recent years. SVV infection can cause vesicular lesions clinically indistinguishable from those caused by other vesicular disease viruses, such as foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV), vesicular stomatitis virus (VSV), and vesicular exanthema of swine virus (VESV). Sensitive and specific RT-PCR assays for the SVV detection is necessary for differential diagnosis. Real-time RT-PCR (rRT-PCR) has been used for the detection of many RNA viruses. The insulated isothermal PCR (iiPCR) on a portable POCKIT™ device is user friendly for on-site pathogen detection. In the present study, SVV rRT-PCR and RT-iiPCR were developed and validated. RESULTS: Neither the SVV rRT-PCR nor the RT-iiPCR cross-reacted with any of the vesicular disease viruses (20 FMDV, two SVDV, six VSV, and two VESV strains), classical swine fever virus (four strains), and 15 other common swine viruses. Analytical sensitivities of the SVV rRT-PCR and RT-iiPCR were determined using serial dilutions of in vitro transcribed RNA as well as viral RNA extracted from a historical SVV isolate and a contemporary SVV isolate. Diagnostic performances were further evaluated using 125 swine samples by two approaches. First, nucleic acids were extracted from the 125 samples using the MagMAX™ kit and then tested by both RT-PCR methods. One sample was negative by the rRT-PCR but positive by the RT-iiPCR, resulting in a 99.20% agreement (124/125; 95% CI: 96.59-100%, κ = 0.98). Second, the 125 samples were tested by the taco™ mini extraction/RT-iiPCR and by the MagMAX™ extraction/rRT-PCR system in parallel. Two samples were positive by the MagMAX™/rRT-PCR system but negative by the taco™ mini/RT-iiPCR system, resulting in a 98.40% agreement (123/125; 95% CI: 95.39-100%, κ = 0.97). The two samples with discrepant results had relatively high CT values. CONCLUSIONS: The SVV rRT-PCR and RT-iiPCR developed in this study are very sensitive and specific and have comparable diagnostic performances for SVV RNA detection. The SVV rRT-PCR can be adopted for SVV detection in laboratories. The SVV RT-iiPCR in a simple field-deployable system could serve as a tool to help diagnose vesicular diseases in swine at points of need.


Assuntos
Picornaviridae/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Doenças dos Suínos/virologia , Animais , Variação Genética , Picornaviridae/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sensibilidade e Especificidade , Suínos , Doenças dos Suínos/diagnóstico
10.
BMC Infect Dis ; 19(1): 479, 2019 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-31142265

RESUMO

BACKGROUND: The increasing availability of high-throughput sequencing data provides researchers with unprecedented opportunities to investigate viral genetic elements in host genomes that contribute to virus-linked cancers. Almost all of the available computational tools for secondary analysis of sequencing data detect viral infection or genome integration events. However, viral oncogenes expression is likely of importance in carcinoma. We therefore developed a new software, DisV-HPV16, for the evaluation of HPV16 oncogenes expression. RESULTS: HPV16 virus and viral oncogenes expression was detected more rapidly using DisV-HPV16 compared to other software. DisV-HPV16 was proved highly convenient for detecting candidate virus after modification of the reference file. The accuracy of DisV-HPV16 was empirically confirmed in laboratory experiments. DisV-HPV16 exhibited greater reliability than other software. CONCLUSIONS: DisV-HPV16 is a new, dependable software to detect virus and viral oncogenes expression through analysis of RNA sequencing data. Use of DisV-HPV16 can yield deeper, more comprehensive insights into virus infection status and viral and host cell gene expression.


Assuntos
Papillomavirus Humano 16/genética , Proteínas Oncogênicas Virais/genética , Software , Sequência de Bases , Linhagem Celular Tumoral , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala , Papillomavirus Humano 16/isolamento & purificação , Humanos , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/virologia , RNA Viral/química , RNA Viral/isolamento & purificação , RNA Viral/metabolismo , Reprodutibilidade dos Testes , Análise de Sequência de RNA
11.
J Craniofac Surg ; 30(3): e259-e262, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31048622

RESUMO

Lymphomas of the oral cavity are rare and the most frequent type is diffuse large B-cell lymphoma (DLBCL). Epstein-Barr virus (EBV) is known to be associated with the development of different lymphomas. In 2008, the World Health Organization provisionally included the EBV-positive DLBCL of the elderly in the classification of hematopoietic and lymphoid tumors as a lymphoma occurring in older individuals without any known immunodeficiency. However, it has since been recognized that this entity may occur in younger individuals and present similar clinical parameters in both age groups. As a result, the 2017 revision has declined the term elderly and modified it to EBV-positive DLBCL, not otherwise specified (NOS). In this report, we describe a rare case of EBV-positive DLBCL, NOS, presenting as a painless swelling in the oral cavity. This entity shows a more aggressive clinical course than EBV-negative DLBCL, and other lymphoproliferative disorders should be considered in the differential diagnosis.


Assuntos
Herpesvirus Humano 4 , Linfoma Difuso de Grandes Células B/diagnóstico , Linfoma Difuso de Grandes Células B/virologia , Neoplasias Bucais/diagnóstico , Neoplasias Bucais/virologia , Infecções por Vírus Epstein-Barr , Evolução Fatal , Feminino , Humanos , Pessoa de Meia-Idade , RNA Viral/isolamento & purificação
12.
BMC Infect Dis ; 19(1): 370, 2019 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-31046684

RESUMO

BACKGROUND: Several inactivated enterovirus-A71 (EV-A71) vaccines are currently licensed in China; however, the development of additional EV-A71 vaccines is ongoing, necessitating extensive analysis of the molecular epidemiology of the virus worldwide. Until 2012, laboratory confirmation of EV-A71 for hand, foot, and mouth disease (HFMD) and other associated diseases had not occurred in the Philippines. Because EV-A71 has been linked with cases of acute flaccid paralysis (AFP), AFP surveillance is one strategy for documenting its possible circulation in the country. To expand current knowledge on EV-A71, molecular epidemiologic analysis and genetic characterization of EV-A71 isolates were performed in this study. METHODS: A retrospective study was performed to identify and characterize nonpolio enteroviruses (NPEVs) associated with AFP in the Philippines, and nine samples were found to be EV-A71-positive. Following characterization of these EV-A71 isolates, the complete viral protein 1 (VP1) gene was targeted for phylogenetic analysis. RESULTS: Nine EV-A71 isolates detected in 2000 (n = 2), 2002 (n = 4), 2005 (n = 2), and 2010 (n = 1) were characterized using molecular methods. Genomic regions spanning the complete VP1 region were amplified and sequenced using specific primers. Phylogenetic analysis of the full-length VP1 region identified all nine EV-A71 Philippine isolates as belonging to the genogroup C lineage, specifically the C2 cluster. The result indicated a genetic linkage with several strains isolated in Japan and Taiwan, suggesting that strains in the C2 cluster identified in the Asia-Pacific region were circulating in the Philippines. CONCLUSION: The study presents the genetic analysis of EV-A71 in the Philippines. Despite some limitations, the study provides additional genetic data on the circulating EV-A71 strains in the Asia-Pacific region, in which information on EV-A71 molecular epidemiology is incomplete. Considering that EV-A71 has a significant public health impact in the region, knowledge of its circulation in each country is important, especially for formulating vaccines covering a wide variety of strains.


Assuntos
Infecções por Enterovirus/diagnóstico , Paralisia/diagnóstico , Doença Aguda , Animais , Proteínas do Capsídeo/genética , Criança , Pré-Escolar , Enterovirus Humano A/classificação , Enterovirus Humano A/genética , Enterovirus Humano A/isolamento & purificação , Infecções por Enterovirus/virologia , Fezes/virologia , Febre Aftosa/diagnóstico , Febre Aftosa/virologia , Genótipo , Humanos , Lactente , Paralisia/virologia , Filipinas , Filogenia , RNA Viral/isolamento & purificação , RNA Viral/metabolismo , Estudos Retrospectivos
13.
Nat Commun ; 10(1): 2098, 2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068585

RESUMO

Hepatitis D virus (HDV) doesn't encode envelope proteins for packaging of its ribonucleoprotein (RNP) and typically relies on the surface glycoproteins (GPs) from hepatitis B virus (HBV) for virion assembly, envelopment and cellular transmission. HDV RNA genome can efficiently replicate in different tissues and species, raising the possibility that it evolved, and/or is still able to transmit, independently of HBV. Here we show that alternative, HBV-unrelated viruses can act as helper viruses for HDV. In vitro, envelope GPs from several virus genera, including vesiculovirus, flavivirus and hepacivirus, can package HDV RNPs, allowing efficient egress of HDV particles in the extracellular milieu of co-infected cells and subsequent entry into cells expressing the relevant receptors. Furthermore, HCV can propagate HDV infection in the liver of co-infected humanized mice for several months. Further work is necessary to evaluate whether HDV is currently transmitted by HBV-unrelated viruses in humans.


Assuntos
Coinfecção/transmissão , Hepatite D/transmissão , Vírus Delta da Hepatite/fisiologia , Montagem de Vírus , Animais , Linhagem Celular Tumoral , Coinfecção/virologia , Flavivirus/metabolismo , Hepacivirus/metabolismo , Hepacivirus/patogenicidade , Hepatite D/virologia , Vírus Delta da Hepatite/isolamento & purificação , Vírus Delta da Hepatite/patogenicidade , Hepatócitos/transplante , Hepatócitos/virologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Cultura Primária de Células , RNA Viral/isolamento & purificação , Ribonucleoproteínas/metabolismo , Vesiculovirus/metabolismo , Proteínas do Envelope Viral/metabolismo , Vírion/metabolismo
14.
Emerg Microbes Infect ; 8(1): 662-674, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31084471

RESUMO

Influenza defective interfering (DI) particles are replication-incompetent viruses carrying large internal deletion in the genome. The loss of essential genetic information causes abortive viral replication, which can be rescued by co-infection with a helper virus that possesses an intact genome. Despite reports of DI particles present in seasonal influenza A H1N1 infections, their existence in human infections by the avian influenza A viruses, such as H7N9, has not been studied. Here we report the ubiquitous presence of DI-RNAs in nasopharyngeal aspirates of H7N9-infected patients. Single Molecule Real Time (SMRT) sequencing was first applied and long-read sequencing analysis showed that a variety of H7N9 DI-RNA species were present in the patient samples and human bronchial epithelial cells. In several abundantly expressed DI-RNA species, long overlapping sequences have been identified around at the breakpoint region and the other side of deleted region. Influenza DI-RNA is known as a defective viral RNA with single large internal deletion. Beneficial to the long-read property of SMRT sequencing, double and triple internal deletions were identified in half of the DI-RNA species. In addition, we examined the expression of DI-RNAs in mice infected with sublethal dose of H7N9 virus at different time points. Interestingly, DI-RNAs were abundantly expressed as early as day 2 post-infection. Taken together, we reveal the diversity and characteristics of DI-RNAs found in H7N9-infected patients, cells and animals. Further investigations on this overwhelming generation of DI-RNA may provide important insights into the understanding of H7N9 viral replication and pathogenesis.


Assuntos
Vírus Defeituosos/genética , Subtipo H7N9 do Vírus da Influenza A/crescimento & desenvolvimento , Influenza Humana/patologia , Influenza Humana/virologia , RNA Viral/genética , Análise de Sequência de DNA , Animais , Brônquios/virologia , Vírus Defeituosos/isolamento & purificação , Modelos Animais de Doenças , Células Epiteliais/virologia , Genoma Viral , Humanos , Camundongos , Nasofaringe/patologia , Nasofaringe/virologia , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , RNA Viral/isolamento & purificação , Deleção de Sequência
15.
Ticks Tick Borne Dis ; 10(4): 868-874, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31047827

RESUMO

In Switzerland, tick-borne encephalitis (TBE) is a notifiable human disease with an average of 210 cases per year in the last 10 years (2008-2017). A national surveillance conducted in 2009 reported a prevalence of 0.46% for tick-borne encephalitis virus (TBEV) detected in ticks, which is in accordance with the prevalences found in Europe from 0.1%-5%. The Canton of Ticino in the southern part of Switzerland, geographically separated from the rest of the national territory by the Alps, is considered a non-endemic region, as no autochthonous clinical cases and no TBEV presence in ticks have ever been reported. In order to understand the epidemiological situation in Ticino, we conducted a large study investigating the TBEV presence in field-collected Ixodes ricinus ticks and in goat and human sera. Goats and sheep were considered as sentinel hosts showing persistence of antibodies also after 28 months in the absence of symptoms; this longevity supports the data validity to characterize an area with the TBEV status. The goat sera collection was composed of a total of 662 samples from 37 flocks. The total seroprevalence was 14.6%. 39 (40%) of the 97 SNT-positive samples showed an antibody titer ≥ 1:120 which indicates recent infection and consequently the probable presence of active foci among the pastures frequented by the goats belonging to 10 flocks. In total, 51 owners participated in the study and all were TBEV antibody-free. A total of 12'052 I. ricinus ticks (nymphs and adults) were collected and 1'371 pools were tested using quantitative real-time RT-PCR. Only one positive pool was reported with a prevalence of 0.35%. Metagenomic analysis revealed that the TBEV strain isolated from the ticks collected in Ticino is closely related to 2 strains coming from the Canton of Valais (99.1% and 98.7% identity, respectively), a neighbouring region of the Canton of Ticino. These two Cantons are close together but separated by high mountains (Alps) and we hypothesize that infected ticks were transported by wild animals from Valais into the Valle Maggia in Ticino where we found positive ticks. In conclusion, our data show for the first time the presence of TBEV in ticks and the related sero-reactivity in goats, confirming the presence of TBEV in the environment of the Canton of Ticino. Further surveillance studies will have to be conducted to follow the persistence of TBEV in this region.


Assuntos
Anticorpos Antivirais/sangue , Vírus da Encefalite Transmitidos por Carrapatos/isolamento & purificação , Encefalite Transmitida por Carrapatos/veterinária , Cabras/virologia , Ixodes/virologia , Infestações por Carrapato/veterinária , Animais , Vírus da Encefalite Transmitidos por Carrapatos/genética , Encefalite Transmitida por Carrapatos/epidemiologia , Cabras/parasitologia , Humanos , Metagenômica , Ninfa/virologia , Prevalência , RNA Viral/sangue , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Estações do Ano , Análise de Sequência de DNA , Estudos Soroepidemiológicos , Suíça/epidemiologia , Infestações por Carrapato/epidemiologia
16.
BMC Vet Res ; 15(1): 156, 2019 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-31109324

RESUMO

BACKGROUND: Bovine respiratory syncytial virus (BRSV) is an important respiratory pathogen worldwide, detrimentally affecting the economy and animal welfare. To prevent and control BRSV infection, further knowledge on virus shedding and transmission potential in individual animals is required. This study aimed to detect viral RNA and infective virions during BRSV infection to evaluate duration of the transmission period and correlation with clinical signs of disease. The outcome of BRSV re-exposure on calves, their housing environment and effect of introduction of sentinel calves was also investigated. A live animal experiment including 10 calves was conducted over 61 days. Initially, two calves were inoculated with a non-passaged BRSV field isolate. Two days later, six naïve calves (EG: Exposed group) were introduced for commingling and four weeks later, another two naïve calves (SG: Sentinel group) were introduced. Seven weeks after commingling, EG animals were re-inoculated. Clinical examination was performed daily. Nasal swabs were collected regularly and analysed for viral RNA by RT-ddPCR, while virus isolation was performed in cell culture. BRSV serology was performed with ELISA. RESULTS: All the EG calves seroconverted and showed clinical signs of respiratory disease. Viral RNA was detected from days 1-27 after exposure, while the infective virus was isolated on day 6 and 13. On day 19, all animals were seropositive and virus could not be isolated. Total clinical score for respiratory signs corresponded well with the shedding of viral RNA. The SG animals, introduced 27 days after exposure, remained negative for BRSV RNA and stayed seronegative throughout the study. Inoculation of the EG calves seven weeks after primary infection did not lead to new shedding of viral RNA or clinical signs of disease. CONCLUSION: Viral RNA was detected in nasal swabs from the calves up to four weeks after exposure. The detection and amount of viral RNA corresponded well with the degree of respiratory signs. The calves were shedding infective virions for a considerable shorter period, and naïve calves introduced after four weeks were not infected. Infected calves were protected from reinfection for at least seven weeks. This knowledge is useful to prevent spread of BRSV.


Assuntos
Doenças dos Bovinos/transmissão , Infecções por Vírus Respiratório Sincicial/veterinária , Vírus Sincicial Respiratório Bovino/fisiologia , Animais , Anticorpos Antivirais/sangue , Bovinos , Doenças dos Bovinos/patologia , Doenças dos Bovinos/virologia , Cavidade Nasal/virologia , RNA Viral/isolamento & purificação , Infecções por Vírus Respiratório Sincicial/patologia , Infecções por Vírus Respiratório Sincicial/transmissão , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Bovino/isolamento & purificação , Fatores de Tempo , Eliminação de Partículas Virais
17.
Nat Commun ; 10(1): 2300, 2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-31127091

RESUMO

Single-stranded circular RNAs (circRNAs), generated through 'backsplicing', occur more extensively than initially anticipated. The possible functions of the vast majority of circRNAs remain unknown. Virus-derived circRNAs have recently been described in gamma-herpesviruses. We report that oncogenic human papillomaviruses (HPVs) generate circRNAs, some of which encompass the E7 oncogene (circE7). HPV16 circE7 is detectable by both inverse RT-PCR and northern blotting of HPV16-transformed cells. CircE7 is N6-methyladenosine (m6A) modified, preferentially localized to the cytoplasm, associated with polysomes, and translated to produce E7 oncoprotein. Specific disruption of circE7 in CaSki cervical carcinoma cells reduces E7 protein levels and inhibits cancer cell growth both in vitro and in tumor xenografts. CircE7 is present in TCGA RNA-Seq data from HPV-positive cancers and in cell lines with only episomal HPVs. These results provide evidence that virus-derived, protein-encoding circular RNAs are biologically functional and linked to the transforming properties of some HPV.


Assuntos
Transformação Celular Neoplásica/patologia , Interações Hospedeiro-Patógeno/genética , RNA Viral/metabolismo , RNA/metabolismo , Neoplasias do Colo do Útero/virologia , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Conjuntos de Dados como Assunto , Feminino , Técnicas de Silenciamento de Genes , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/patogenicidade , Humanos , Camundongos , Camundongos Endogâmicos NOD , Proteínas E7 de Papillomavirus/genética , Polirribossomos/genética , Polirribossomos/metabolismo , RNA/genética , RNA/isolamento & purificação , RNA Interferente Pequeno/metabolismo , RNA Viral/genética , RNA Viral/isolamento & purificação , Análise de Sequência de RNA , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Emerg Infect Dis ; 25(5): 951-954, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31002058

RESUMO

We detected Zika virus RNA in rectal swab samples from 10 patients by using real-time reverse transcription PCR, and we isolated the virus from 1 patient. The longest interval from symptom onset to detection was 14 days. These findings are applicable to diagnosis and infection prevention recommendations.


Assuntos
Reto/virologia , Infecção por Zika virus/virologia , Zika virus/isolamento & purificação , Adulto , Feminino , Humanos , Masculino , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Adulto Jovem , Zika virus/genética , Infecção por Zika virus/sangue , Infecção por Zika virus/urina
19.
J Dermatol ; 46(5): 409-412, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30932227

RESUMO

A case of severe fever with thrombocytopenia syndrome (SFTS) in which a skin biopsy from the tick-bite region was analyzed is reported. The patient was a 72-year-old woman who developed fever and thrombocytopenia after a tick bite. SFTS was diagnosed from polymerase chain reaction (PCR) analysis of a blood sample. Histopathological analysis of a skin biopsy specimen from the tick-bite region showed CD20-positive perivascular and interstitial immunoblastic cells, which were positive to anti-SFTS virus (SFTSV) nucleoprotein antibody. In addition, SFTSV RNA was detected by real-time PCR from this biopsy specimen. Moreover, hemophagocytosis was also found in the tick-bite region. To the best of our knowledge, this is the first report to analyze the details of the tick-bite region of skin in SFTS, and the first to detect virus-infected cells in the skin. The present findings may help elucidate the mechanisms of entry of SFTSV.


Assuntos
Coagulação Intravascular Disseminada/virologia , Febre por Flebótomos/virologia , Phlebovirus/isolamento & purificação , Trombocitopenia/virologia , Picadas de Carrapatos/patologia , Idoso , Biópsia , Coagulação Intravascular Disseminada/sangue , Coagulação Intravascular Disseminada/diagnóstico , Evolução Fatal , Feminino , Humanos , Febre por Flebótomos/sangue , Febre por Flebótomos/diagnóstico , Phlebovirus/genética , RNA Viral/isolamento & purificação , Pele/patologia , Pele/virologia , Síndrome , Trombocitopenia/sangue , Trombocitopenia/diagnóstico , Picadas de Carrapatos/sangue , Picadas de Carrapatos/complicações , Picadas de Carrapatos/virologia
20.
Int J Mol Sci ; 20(8)2019 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-31010160

RESUMO

West Nile virus (WNV), a mosquito-borne arbovirus, remains a major global health concern. In this study, we optimized PCR methods then assessed serially-collected whole blood (WB), urine (UR), saliva, and semen specimens from a large cohort of WNV-positive participants to evaluate the natural history of infection and persistent shedding of WNV RNA. Viral RNA extraction protocols for frozen WB and UR specimens were optimized and validated through spiking experiments to maximize recovery of viral RNA from archived specimens and to assess the degradation of WNV RNA in stored UR specimens. The resultant procedures were used in conjunction with PCR detection to identify WNV-positive specimens and to quantify their viral loads. A total of 59 of 352 WB, 10 of 38 UR, and 2 of 34 saliva specimens tested positive for WNV RNA. Although a single semen specimen was positive 22 days post onset, we could not definitively confirm the presence of WNV RNA in the remaining specimens. WNV RNA-positive UR specimens exhibited profound loss of viral RNA during storage, highlighting the need for optimal preservation pre-storage. This study provides optimized methods for WNV RNA detection among different fluid types and offers alternative options for diagnostic testing during the acute stages of WNV.


Assuntos
Líquidos Corporais/virologia , Reação em Cadeia da Polimerase/métodos , Febre do Nilo Ocidental/virologia , Vírus do Nilo Ocidental/isolamento & purificação , Estudos de Coortes , Humanos , Masculino , RNA Viral/isolamento & purificação , Saliva/virologia , Sêmen/virologia , Febre do Nilo Ocidental/sangue , Febre do Nilo Ocidental/urina
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