Quantification of human polyomaviruses MCPyV and HPyV6 in malignant and non-malignant skin lesions

Abstract Background: Human Polyomaviruses such as MCPyV and HPyV6 are frequently found as part of healthy skin microbiota and have been associated with Merkel cell carcinoma (MCC), pruritic and dyskeratotic dermatoses, respectively. Their presence in other types of skin conditions varies greatly depending on lesion type and population. Objectives: To analyse comparatively the presence of MCPyV and HPyV6 in nonmelanoma skin cancers and healthy skin. Methods: The authors utilized qPCR techniques to quantify these pathogens in NMSC, premalignant diseases, and healthy skin of 87 patients. Results: MCPyV was detected in over 40% of samples, while HPyV6 was in 9.6%. MCPyV load was higher in squamous cell carcinomas (SCC) compared to basal cell carcinomas (BCC) (p = 0.016) and HPyV6 showed a higher percentage of infected cells in areas of low solar exposure as well as normal skin (p = 0.012). A fair agreement (kappa = 0.301) was found between MCPyV detection in lesions and their respective perilesional skin, indicating a random process of local dissemination of the virus. Study limitations: The lack of a larger sampling of different lesion types and protein expression analyses limits the correlation findings. Conclusions: This is the first report of HPyV6 detection in the healthy skin of a Brazilian population, but the role of both polyomaviruses in NMSC has yet to be demonstrated.

Quantification of human polyomaviruses MCPyV and HPyV6 in malignant and non-malignant skin lesions.

BACKGROUND: Human Polyomaviruses such as MCPyV and HPyV6 are frequently found as part of healthy skin microbiota and have been associated with Merkel cell carcinoma (MCC), pruritic and dyskeratotic dermatoses, respectively. Their presence in other types of skin conditions varies greatly depending on lesion type and population. OBJECTIVE: To analyse comparatively the presence of MCPyV and HPyV6 in nonmelanoma skin cancers and healthy skin. METHODS: The authors utilized qPCR techniques to quantify these pathogens in NMSC, premalignant diseases, and healthy skin of 87 patients. RESULTS: MCPyV was detected in over 40% of samples, while HPyV6 was in 9.6%. MCPyV load was higher in squamous cell carcinomas (SCC) compared to basal cell carcinomas (BCC) (p=0.016) and HPyV6 showed a higher percentage of infected cells in areas of low solar exposure as well as normal skin (p=0.012). A fair agreement (kappa=0.301) was found between MCPyV detection in lesions and their respective perilesional skin, indicating a random process of local dissemination of the virus. STUDY LIMITATIONS: The lack of a larger sampling of different lesion types and protein expression analyses limits the correlation findings. CONCLUSION: This is the first report of HPyV6 detection in the healthy skin of a Brazilian population, but the role of both polyomaviruses in NMSC has yet to be demonstrated.

Human pegivirus-1 infection in kidney transplant recipients: A single-center experience.

Kidney transplantation is the treatment of choice for patients with end-stage renal disease. In the posttransplant period, the induced immunosuppression leads to an increased risk of developing infectious diseases, a leading cause of death after kidney transplantation. Human pegivirus-1 (HPgV-1) is considered a nonpathogenic human virus and is highly frequent in individuals parenterally exposed, however, its impact on kidney transplantation outcome is poorly understood. Given the scarcity of epidemiological data for this infection on organ recipients in Brazil, we conducted a study in a single center for kidney transplantation in Rio de Janeiro, aiming to determine HPgV-1 prevalence and genotypic distribution. Serum samples from 61 renal recipients, followed up for the first year after transplantation, were evaluated for viral RNA and genotypes were determined by sequencing of the 5'-untranslated region. HPgV-1 RNA was detected in 36.1% (22/61) of patients. Genotype 2 was the most commonly found (80.9%), followed by genotypes 3 (9.5%), 1, and 5, in 4.8% each. Statistical comparisons did not reveal any significant impact of HPgV-1 in patient outcome. Further epidemiologic studies are needed to understand if immunosuppression may interfere in HPgV-1 persistence rates and if viremia might impact graft dysfunction rates in kidney recipients.

BK polyomavirus genotypes Ia and Ib1 exhibit different biological properties in renal transplant recipients.

BK polyomavirus (BKV) is an opportunist agent associated with nephropathy (BKVAN) in 1-10% of kidney transplant recipients. BKV is classified into genotypes or subgroups according to minor nucleotidic variations with unknown biological implications. Studies assessing the possible association between genotypes and the risk of BKVAN in kidney transplant patients have presented conflicting results. In these studies, genotype Ia, which is highly prevalent in Brazil, was less frequently found and, thus, comparative data on the biological properties of this genotype are lacking. In this study, BKV Ia and Ib1 genotypes were compared according to their viral load, genetic evolution (VP1 and NCCR) - in a cohort of renal transplant recipients. The patients infected with Ia (13/23; 56.5%) genotype exhibited higher viral loads in urine [>1.4 log over Ib1 (10/23; 43.5%); p=0.025]. In addition, genotype Ia was associated with diverse mutations at VP1 loops and sites under positive selection outside loops, which were totally absent in Ib1. Although the number of viremic patients was similar, the three patients who had BK nephropathy (BKVAN) were infected with Ia genotype. NCCR architecture (ww or rr) were not distinctive between Ia and Ib1 genotypes. Ia genotype, which is rare in other published BKV cohorts, presented some diverse biological properties in transplanted recipients in comparison to Ib1.

ASSOCIATION OF EPSTEIN-BARR VIRUS (EBV) BUT NOT HUMAN PAPILLOMAVIRUS (HPV) WITH GINGIVITIS AND/OR PERIODONTITIS IN TRANSPLANTED INDIVIDUALS.

The aim of this study was to investigate the association of EBV and HPV with gingivitis and/or periodontitis according to the immunologic status. To this end, 74 oral biopsies from transplanted and non-transplanted individuals with the abovementioned oral manifestations were submitted to a screening by PCR for both viruses. According to the results, EBV was strongly associated with gingivitis and/or periodontitis in transplanted individuals (p = 0.011) but not HPV (p = 0.766). EBV-HPV co-detections did not enhance the presence of tissue injury as well. Although a causal relationship was not investigated in this study, the higher frequency of these two oncoviruses in lesion tissues must be investigated in follow-up studies, especially among immunocompromised individuals.

Phylogenetic and structural analysis of merkel cell polyomavirus VP1 in Brazilian samples.

Our understanding of the phylogenetic and structural characteristics of the Merkel Cell Polyomavirus (MCPyV) is increasing but still scarce, especially in samples originating from South America. In order to investigate the properties of MCPyV circulating in the continent in more detail, MCPyV Viral Protein 1 (VP1) sequences from five basal cell carcinoma (BCC) and four saliva samples from Brazilian individuals were evaluated from the phylogenetic and structural standpoint, along with all complete MCPyV VP1 sequences available at Genbank database so far. The VP1 phylogenetic analysis confirmed the previously reported pattern of geographic distribution of MCPyV genotypes and the complexity of the South-American clade. The nine Brazilian samples were equally distributed in the South-American (3 saliva samples); North American/European (2 BCC and 1 saliva sample); and in the African clades (3 BCC). The classification of mutations according to the functional regions of VP1 protein revealed a differentiated pattern for South-American sequences, with higher number of mutations on the neutralizing epitope loops and lower on the region of C-terminus, responsible for capsid formation, when compared to other continents. In conclusion, the phylogenetic analysis showed that the distribution of Brazilian VP1 sequences agrees with the ethnic composition of the country, indicating that VP1 can be successfully used for MCPyV phylogenetic studies. Finally, the structural analysis suggests that some mutations could have impact on the protein folding, membrane binding or antibody escape, and therefore they should be further studied.

Genetic and structural analysis of polyomavirus BK T-antigens reveal a higher density of mutations at inter-domain and hexamerization regions, regardless the status of infection.

Polyomavirus BK (BKPyV) T-antigens (large and small tumor antigens, or Lt-ag and st-ag, respectively), control key aspects of viral replication and are able to regulate cell cycle, promoting cell proliferation. However, the structural effects of genetic mutations on T-antigens are poorly investigated. In this study, 214 sequences of T-antigens from individuals with different BKPyV infections (16 renal transplant with nephropathy; 78 asymptomatic renal transplant; 24 hematopoietic stem cell transplant with hemorrhagic cystitis; 96 healthy non-transplant), were analyzed from the genetic and structural standpoints. We found a high concentration of non-synonymous mutations at inter-domains and hexamerization regions of both proteins, being five of them under positive selection in the Lt-ag but none in the st-ag. The in silico analysis indicated that two mutations, located at positions 164 in the st-ag and 592 in the Lt-ag, would significantly affect the interaction with PP2A and p53 cell targets, respectively, although they were not associated to a specific clinical status. No mutations were detected on the J-domains or at the ATPase motif. In sum, the profile of the mutations found seem not to be associated to increased morbidity. This is the first work to analyze structural modifications on T-antigens in different BKPyV infections, and managed to map conserved and variable regions of the T-antigens, which will be helpful for the study of new antiviral drugs.

Human papillomavirus infections and renal transplantation: a review / Infecções causadas por papilomavírus humanos e transplante renal: uma revisão

The immune suppressive therapy in renal allograft recipients provides a favorable environment to the development of viral infections. Among them,human papillomavirus (HPV) infections are usually related to potential life-threatening mucocutaneous neoplasias. Data from clinical surveys suggestthat transplant recipients may have up to 5-fold increased risk of developing multiple malignancies due to the increased susceptibility to persistent HPV infection. High risk HPV induced oncogenesis is a multi-step process in which a persistent infection is the initiating causative event, though subsequentgenetic and epigenetic alterations may be necessary for malignant transformation. The main tumoral types associated with persistent HPV infection areanogenital, oral and skin cancers, common conditions in transplant recipients and responsible for substantial morbidity and mortality. Since prophylactic vaccines with high rates of efficacy have been approved for human population, studies to evaluate its immunogenicity and efficacy should be considered forlong-term survivors after allogeneic transplantation. Hence, we conducted an extensive revision published data for the last 10 years regarding the theme.To achieve our objectives, we searched in diverse data basis such as Lilacs, ScIELO, Medline, Scopus. We concluded that, concerning the increase in thepopulation of transplant recipients as well as in the incidence of HPV associated diseases, measures for prevention and control are necessary, and includecapaciting human resources and the use of last generation methodologies of diagnosis and prophylaxis.

A terapia imunossupressora em pacientes receptores de transplante renal fornece um ambiente favorável ao desenvolvimento de infecções virais. Dentreestas infecções, aquelas causadas pelos papilomavírus humanos (HPV) são geralmente associadas a neoplasias mucocutâneas que podem ameaçar asobrevida pós-transplante. Pesquisas clínicas sugerem que receptores de transplante podem apresentar um risco até cinco vezes maior de desenvolverem quadros de doenças malignas múltiplas, devido à maior frequência da persistência do HPV. A oncogênese induzida por HPV de alto risco é um processo demúltiplos estágios, no qual a infecção persistente é o evento fundamental, apesar de alterações genéticas e epigenéticas adicionais serem necessárias para a transformação maligna. Os principais tipos tumorais relacionados à infecção persistente por HPV são os cânceres anogenitais, orais e de pele, comunsem receptores de transplante e responsáveis por grande morbidade e mortalidade. Uma vez que vacinas profiláticas de alta eficácia contra a infecçãopelo HPV foram aprovadas para uso na população humana, estudos para avaliar a imunogenicidade e eficácia destas vacinas em imunossuprimidos são recomendáveis. Assim, objetivamos fazer extensa revisão sobre o tema. Para tal, pesquisamos o assunto nas bases de dados Lilacs, ScIELO, Medline,Scopus nos últimos 10 anos. Concluímos que, com o aumento na população de receptores de transplantes e a crescente incidência das doenças associadasao HPV, medidas de prevenção e controle se fazem necessárias e englobam desde a formação de profissionais capacitados até a aplicação de metodologias de diagnóstico e profilaxia, de última geração.

Acute respiratory viral infections in children in Rio de Janeiro and Teresópolis, Brazil / Infecções respiratórias agudas causadas por vírus em crianças do Rio de Janeiro e de Teresópolis, Brasil

The frequency of viral pathogens causing respiratory infections in children in the cities of Rio de Janeiro and Teresópolis was investigated. Nasal swabs from children with acute respiratory illnesses were collected between March 2006 and October 2007. Specimens were tested for viral detection by conventional (RT)-PCR and/or real time PCR. Of the 205 nasal swabs tested, 64 (31.2%) were positive for at least one of the viral pathogens. Single infections were detected in 56 samples, 50 of those were caused by RNA viruses: 33 samples tested positive for rhinovirus, five for influenza A, five for metapneumovirus, four for coronavirus and, three for respiratory syncytial virus. For the DNA viruses, five samples were positive for bocavirus and one for adenovirus. Co-infections with these viruses were detected in eight samples. Our data demonstrate a high frequency of viral respiratory infections, emphasizing the need for a more accurate diagnosis particularly for the emerging respiratory viruses. The fact that the emerging respiratory viruses were present in 9.2% of the tested samples suggests that these viruses could be important respiratory pathogens in the country.

Neste estudo foi investigada a frequência de patógenos virais causando infecção em crianças nas cidades do Rio de Janeiro e Teresópolis. Foram coletados 205 swabs nasais de crianças com infecção aguda do trato respiratório no período de março de 2006 a outubro de 2007. Os espécimes foram testados para detecção de vírus através de (RT)-PCR e/ou PCR em tempo real. Dentre as 205 amostras testadas, 64 (31,2%) foram positivas para pelo menos um vírus. Infecções causadas por um único agente viral foram detectadas em 56 amostras, 50 das quais eram causadas por vírus de RNA: 33 amostras foram positivas para rinovírus, cinco amostras foram positivas para influenza A, cinco amostras foram positivas para metapneumovírus, quatro amostras foram positivas para coronavírus e três amostras foram positivas para vírus respiratório sincicial. Para os vírus de DNA foram detectadas cinco amostras positivas para bocavírus humano e uma amostra positiva para adenovírus. Foram identificados oito casos de co-infecção. Nossos dados demonstram frequência elevada de infecções respiratórias virais, enfatizando a necessidade de um diagnóstico mais acurado destes patógenos, principalmente os vírus considerados emergentes. O fato de alguns vírus respiratórios emergentes terem sido detectados em 9,2% das amostras testadas sugere que estes vírus podem ser patógenos respiratórios importantes no país.

Acute respiratory viral infections in children in Rio de Janeiro and Teresópolis, Brazil.

The frequency of viral pathogens causing respiratory infections in children in the cities of Rio de Janeiro and Teresópolis was investigated. Nasal swabs from children with acute respiratory illnesses were collected between March 2006 and October 2007. Specimens were tested for viral detection by conventional (RT)-PCR and/or real time PCR. Of the 205 nasal swabs tested, 64 (31.2%) were positive for at least one of the viral pathogens. Single infections were detected in 56 samples, 50 of those were caused by RNA viruses: 33 samples tested positive for rhinovirus, five for influenza A, five for metapneumovirus, four for coronavirus and, three for respiratory syncytial virus. For the DNA viruses, five samples were positive for bocavirus and one for adenovirus. Co-infections with these viruses were detected in eight samples. Our data demonstrate a high frequency of viral respiratory infections, emphasizing the need for a more accurate diagnosis particularly for the emerging respiratory viruses. The fact that the emerging respiratory viruses were present in 9.2% of the tested samples suggests that these viruses could be important respiratory pathogens in the country.

Differential evolution of Human Immunodeficiency Virus type 1 Protease and Reverse Transcriptase genes between HAART-failing and naïve-treated individuals.

We described differential selective pressures along codon sites of the RT and PR genes of HIV-1 from HAART-failing and naïve-treated individuals, through the comparison of the ratio of non-synonymous mutations (d(N)) to synonymous mutations (d(S)) substitution per site. Resistance-associated mutations were found in 1/71 (1.4%) and 109/117 (93.1%) samples from naïve-treated and HAART-failing individuals, respectively, although most of positively selected codons represented polymorphisms in positions 123, 211, 245, 297 in RT and 37, 63 in PR of naïve-treated samples and positions 122, 123, 245, 272, 277, 286, 297 in RT and 10, 15, 20, 35, 37, 62, 63, 64, 71, 72, 77, 93 in PR of HAART-failing samples, except by ARV-resistance codons 74, 184, 215 in RT and 90 in PR exclusively found in HAART-failing group. The number and diversity of sites under selective pressure at populational level also increased in RT but not in PR of treated individuals. Our results demonstrated no evolution of drug-associated codons among untreated individuals, indicating unlikely transmission and adaptation of resistant HIV-1 strains in a free drug environment. Polymorphic sites observed exclusively in HAART-failing group, may contribute to HIV-1 escape and adaptation at individual and populational levels in a drug environment, although those mutually found in HIV-1 despite of previous exposure to ARV treatment, should not be considered accurate resistance markers.

Novel respiratory virus infections in children, Brazil.

Recently discovered respiratory viruses were detected in 19 (9.2%) of 205 nasal swab specimens from children in Brazil with respiratory illnesses. Five each were positive for human metapneumovirus (HMPV) alone and human bocavirus (HBoV) alone, 3 for human coronaviruses (HCoV-HKU1 or -NL63) alone, and 6 for more than 1 recently discovered virus.

Diagnóstico laboratorial da infeccão pelo vírus herpes simples (HSV) em pacientes transplantados e não-transplantados / Laboratorial diagnosis of herpes simplex virus infection (HSV) in transplanted and non-transplanted patients

INTRODUÇAO: O vírus herpes simples (HSV) é dividido em dois sorotipos (HSV-1 e HSV-2) responsáveis, respectivamente, pelos herpes labial e genital. Embora a infeccão pelo HSV tenha um curso rápido, esse agente está freqüentemente relacionado a complicacões no tratamento de pacientes imunocomprometidos, como indivíduos transplantados, na condicão de agente oportunista. OBJETIVOS: Comparar e avaliar o uso de três técnicas atuais para diagnóstico de HSV em pacientes transplantados e não-transplantados. MATERIAL E MÉTODOS: Oitenta e quatro amostras clínicas consecutivas provenientes de 47 indivíduos transplantados e 37 não-transplantados foram coletadas de junho de 2001 a julho de 2002, sendo, simultaneamente, submetidas a nested multiplex reacão em cadeia da polimerase (PCR) (nmPCR), multiplex PCR (mPCR) e isolamento viral (IV) em células vero. RESULTADOS: Das amostras, 33,3 por cento (28/84) foram positivas para o HSV por IV, 34,5 por cento(29/84) por mPCR e 42,8 por cento (36/84) por nmPCR. Pela técnica de imunofluorescência direta (IFD), 85,7 por cento (24/28) das amostras foram caracterizadas como HSV-1, 86,2 por cento (25/29) pelo mPCR e 88,9 por cento(32/36) pelo nmPCR. Foram caracterizadas como HSV-2 pelas três técnicas empregadas 4,8 por cento(4/84) das amostras. Não houve diferenca significante de deteccão entre as técnicas de diagnóstico do HSV (p = 0,38), embora o nmPCR tenha detectado mais amostras de pacientes transplantados (p = 0,05). CONCLUSAO: Apesar do desempenho similar entre as técnicas, o nmPCR se mostrou ferramenta útil para pacientes transplantados ou para aqueles sob tratamento antiviral, onde é esperada baixa carga viral em suas amostras.