Molecular epidemiology of KI and WU polyomaviruses in infants with acute respiratory disease and in adult hematopoietic stem cell transplant recipients

Polyomaviruses KI (KIPyV) and WU (WUPyV) were described recently in children with acute respiratory disease. The pathogenic potential of these human viruses has not been determined completely, but a correlation between immunosuppression and virus reactivation has been suggested. In the present study, the association between KI/WUPyV infection and immunosuppression was investigated using sequential nasopharyngeal aspirates from asymptomatic adult hematopoietic stem cell transplant recipients. In parallel, an investigation on the WU/KIPyV prevalence in children with acute respiratory disease was also carried out. Two of the 126 samples obtained from the 31 hematopoietic transplant recipients were positive for KIPyV (1 sample, 0.79%) and WUPyV (1 sample, 0.79%). Both samples were obtained 15 days after allogeneic transplantation and virus persistence was not observed in subsequent samples. In symptomatic children, 7 of the 486 nasopharyngeal aspirates were positive for WUPyV (1.4%) and 1 for KIPyV (0.2%). Single polyomavirus infection was detected in four patients, whereas the remaining patients were co‐infected with respiratory syncityal virus (three patients) or adenovirus (one patient). The results suggest that WU/KIPyVs have a limited circulation in Italy and a low pathogenic potential in young children. Brief and asymptomatic infection can occur in hematopoietic transplant recipients. J. Med. Virol. 82:153–156, 2010. © 2009 Wiley‐Liss, Inc.     

WU多瘤病毒衣壳蛋白原核表达及初步分析

目的 获得WU多瘤病毒重组衣壳蛋白,分析其抗原性,筛选具有诊断价值的抗原.方法 采用PCR技术扩增WU多瘤病毒衣壳蛋白VP1、VP2和VP3的基因片段,并将目的基因插入PGEX-20T表达载体,转化大肠杆菌BL21(DE3)后诱导表达.用免疫印迹法对表达蛋白进行鉴定和分析并用临床样本进行初步验证.结果 重组质粒在BL21(DE3)菌株中经IPTG诱导、SDS-PAGE和免疫印迹鉴定显示在相对分子质量(Mr) 69×103、63×103和56×103处出现清晰条带,重组质粒经双酶切鉴定,表达产物经GST标签鉴定均正确.16份呼吸道分泌物WU多瘤病毒核酸阳性血清标本和70份阴性标本免疫印迹分析表明,重组蛋白具有良好的抗原性.结论 成功构建了WU多瘤病毒衣壳蛋白VP1、VP2和VP3的表达载体,表达的重组蛋白具有良好的抗原性,为进一步研究提供了资料.

Abstract：

Objective To express the capsid proteins of WU polyomavirus(WUPyV) for research and find antigen for diagnostic value. Methods Coding sequences of capsid proteins of WU polyomavirus by PCR were cloned in prokaryotic expression vector PGEX-20T. Recombinant plasmids were transformed into E. coli BL21(DE3) and induced by IPTG for proteins expression. Recombinant proteins were identified by Western blot. Results SDS-PAGE proved that recombinant proteins showed three bands with molecular relative mass of 69×103, 63×103 and 56×103. The recombinant proteins were recognized by anti-GST McAb. The antigenicity was tested by Western blot using 16 WU polyomavirus positive and 70 negative sera. Conclusion Recombinant VP1, VP2 and VP3 expressed in E. coli can combine with WUPyV-Ab and have good antigenicity. They can be used for further research.     

Secondary lymphoid tissue as an important site for WU polyomavirus infection in immunocompetent children

The polyomaviruses KI and WU (KIPyV and WUPyV) have been identified in respiratory specimens from children with acute respiratory infections, which suggests the respiratory tract as a possible site of infection. However, the persistence of infection in the lymphoid system is unknown. Fresh samples (n = 211) of tonsils, adenoids, and peripheral blood mononuclear cells (PBMCs) from 83 immunocompetent children (mean age 4.8 years) were tested for amplification of the KIPyV VP1 and WUPyV VP2 genes. The known BK and JC polyomaviruses and the lymphotropic human herpesvirus (HHV)-6 were also investigated by quantitative real-time PCR and direct sequencing. In addition, 98 nasopharyngeal swabs collected from children (mean age 6.2 years) affected by seasonal influenza-like illness were tested. Of the lymphoid tissues, 34.9% were positive for WUPyV, 4.8% for BK virus, and 33.8% for HHV-6. KIPyV and JC virus were not detected in these specimens. None of the polyomaviruses were detected in PBMCs. Among the nasopharyngeal samples, the prevalence of WUPyV was 27.5%, although 70% of the positive samples were co-infected with at least one of the following respiratory viruses: influenza virus, adenovirus, and respiratory syncytial virus. Phylogenetic analysis revealed high sequence homology (99%) between lymphoid- and nasopharynx-derived WUPyV strains. These results suggest that the tonsils and adenoids of immunocompetent children are a reservoir for WUPyV infection; probably due to the respiratory route of transmission. In addition, the prevalence of WUPyV was high among the children, and the virus was identified more frequently in older children than during the first years of life.     

The human polyomaviruses KI and WU: virological background and clinical implications

In 2007, two novel polyomaviruses KI and WU were uncovered in the respiratory secretions of children with acute respiratory symptoms. Seroepidemiological studies showed that infection by these viruses is widespread in the human population. Following these findings, different biological specimens and body compartments have been screened by real‐time PCR in the attempt to establish a pathogenetic role for KI polyomavirus (KIPyV) and WU polyomavirus (WUPyV) in human diseases. Although both viruses have been found mainly in respiratory tract samples of immunocompromised patients, a clear causative link with the respiratory disease has not been established. Indeed, the lack of specific clinical or radiological findings, the frequent co‐detection with other respiratory pathogens, the detection in subjects without signs or symptoms of respiratory disease, and the variability of the viral loads measured did not allow drawing a definitive conclusion. Prospective studies carried out on a large sample size including both immunocompromised and immunocompetent patients with and without respiratory symptoms are needed. Standardized quantitative real‐time PCR methods, definition of a clear clinical cutoff value, timing in the collection of respiratory samples, are also crucial to understand the pathogenic role, if any, of KIPyV and WUPyV in human pathology.     

WU and KI polyomaviruses in respiratory,blood and urine samples from renal transplant patients

BackgroundIt is suggested that immunosuppression due to transplantation might be a risk for human polyomavirus KI (KIPyV) and WU (WUPyV) infection. Most of the publications report data about stem cell transplant patients, little is known about these virus infections in renal transplant patients.ObjectivesTo study the presence of KIPyV and WUPyV in upper respiratory, plasma and urine samples from renal transplant patients. To analyse clinical and personal data.Study design532 respiratory, 503 plasma and 464 urine samples were collected from 77 renal transplant patients. KIPyV and WUPyV were detected by nested and quantitative real-time PCR. Patient and clinical data from medical records were analyzed.ResultsKIPyV was detected in respiratory, plasma and urine samples from 14.3%, 3.9% and 4.1% of renal transplant patients. WUPyV was found in respiratory and plasma specimens from 9.1% and 5.3% of the patients. Significant association was revealed between the detection of KIPyV and WUPyV and the time of samples collection and the age of the patients. KIPyV was presented in respiratory and plasma sample at the same time. KIPyV was detected in plasma samples from two patients and in urine samples of three other patients providing also KIPyV positive respiratory samples at the same time. No clinical consequences of KIPyV or WUPyV infection were found.ConclusionAlthough no clinical consequences of KIPyV and WUPyV infections were found in renal transplant patients, it is suggested that renal transplantation might result in higher susceptibility or reactivation of these infection.     

WU polyomavirus infection among children in South China

This study aimed at investigating the prevalence and clinical characteristics of children with respiratory infection by WU polyomavirus (WUPyV) in Southern China. Nasopharyngeal aspirate samples were collected from 771 children with acute respiratory tract infection admitted to hospital and 82 samples from healthy subjects for routine examination at the outpatient service at the Second Affiliated Hospital of Shantou University, Medical College from July 2008 to June 2009. WUPyV was detected by the polymerase chain reaction (PCR) and DNA sequencing. All WUPyV‐positive specimens were characterized further for nine viruses causing common respiratory infections, including in?uenza A and B, respiratory syncytial virus (RSV), parainfluenza virus (PIV) 1 and 3, human metapneumovirus, human bocavirus, adenovirus, and rhinovirus by PCR or real time (RT)‐PCR. Fifteen out of 771 specimens from patients with acute respiratory tract infection, but none from healthy subjects, were positive for WUPyV and the positivity rate was 2%. Patients with WUPyV infection were between 2 and 48 months of age, and nine of the patients were male while six female. Four out of 15 patients were co‐infected with RSV, one with adenovirus or rhinovirus, respectively. Patients with WUPyV infection displayed predominantly cough, moderate fever, and wheezing, and were diagnosed with pneumonia (n = 8), bronchiolitis (n = 4), upper respiratory tract infections (n = 2) and bronchitis (n = 1). One patient developed encephalitis. Therefore, WUPyV infection can cause acute respiratory tract infection with atypical symptoms, including severe complications, in children. J. Med. Virol. 83:1440–1445, 2011. © 2011 Wiley‐Liss, Inc.     

我国急性呼吸道感染患儿中检测到KI和WU多瘤病毒

目的 了解多瘤病毒WU和KI在我国儿童急性呼吸道疾病中的感染情况.方法 采用PCR扩增的方法对2006年11月至2007年10月收集的急性呼吸道感染患儿的318份鼻咽抽吸物(NPA)标本进行了多瘤病毒WU和KI基因检测.结果 318份标本共检测出14份病毒核酸阳性标本,其中WUV 7份(2.2%),KIPyV 7份(2.2%).该14例基因检测阳性患儿临床均有上呼吸道感染或下呼吸道感染症状.结论 WUV和KIPyV可能也是儿童急性呼吸道感染中较为重要的一个病原,且与儿童上呼吸道感染和下呼吸道感染存在相关性.     

Prevalence of WU and KI polyomaviruses in plasma, urine, and respiratory samples from renal transplant patients

WU and KI polyomaviruses (WUPyV, KIPyV) have been detected in respiratory, blood, stool, and lymphoid tissue, but not in urine samples. PCR based detection revealed higher frequency in immunocompromised individuals. In this study the prevalence of WUPyV and KIPyV was analyzed in respiratory, urine, and blood samples from renal transplant patients compared with healthy individuals. WUPyV and KIPyV were detected by nested PCR. The PCR products were sequenced and viral DNA loads were determined by quantitative real-time PCR. WUPyV and KIPyV were found in plasma (3.6%; 7/195), urine (14%; 7/50), and respiratory samples (10%; 9/90) of renal transplant patients, but not in plasma (0/200) and urine (0/36) specimens from healthy blood donors. WUPyV and KIPyV were detected mainly early after renal transplantation and the viral loads were low. A higher prevalence of WUPyV was found in plasma and urine samples, KIPyV was found more frequently in respiratory samples from renal transplant patients. It is hypothesized that immunosuppression due to the transplantation may result in reactivation of these viruses or may establish greater susceptibility to infection with KIPyV and WUPyV.     

Identification of WU polyomavirus from pediatric patients with acute respiratory infections in Beijing, China

A novel polyomavirus (WU virus) has been identified in pediatric patients with acute respiratory tract infections (ARI), but its role as a respiratory pathogen has not yet been demonstrated. To investigate if WU virus is related to acute respiratory infections in infants and children in Beijing, specimens collected from 674 pediatric patients with ARI from April 2007 to May 2008 and from 202 children without ARI were used for this investigation. Common respiratory viruses were tested by virus isolation and/or antigen detection by indirect immunofluorescent assay followed by RT-PCR or PCR for other viruses associated with respiratory infections in specimens collected from patients with ARI before WU virus DNA was detected. WU virus DNA was detected by initial screening and secondary confirmation PCR for all specimens. The region encoding the VP2 gene of the virus was amplified from 17 WU-virus-positive clinical specimens, and sequence analysis was performed. Thirty-eight of 674 (5.6%) specimens from patients with ARI and 3 of 202 (1.5%) specimens from children without ARI yielded PCR products with the predicted molecular weight, using either screening or confirmation primer sets, indicating that these specimens were WU virus positive. However, more than 60% of the 38 WU-virus-positive specimens from patients with ARI were also positive for one or more respiratory viruses. The nucleotide and deduced amino acid sequences of the region encoding the VP2 gene from 17 Beijing WU viruses shared high homology (>98.5%) with sequences from GenBank and among themselves. The data indicated that WU virus in Beijing occurred 3.7 times more frequently in pediatric patients with ARI than in those without ARI (p < 0.05).     

Prevalence and molecular characterization of WU/KI polyomaviruses isolated from pediatric patients with respiratory disease in Thailand

WU and KI polyomaviruses represent novel viruses discovered in respiratory secretions from human patients with acute respiratory tract infection. However, the association between WU/KI polyomaviruses and human disease has remained unclear. In this study, the prevalence of these two novel viruses and occurrence of co-infection with other respiratory viruses were determined in Thai pediatric patients with respiratory disease. Previously described PCR assays were applied to detect WU/KI polyomaviruses as well as other respiratory viruses in 302 nasopharyngeal suction specimens collected from February 2006 through February 2007. The results revealed the anneal prevalence of WU and KI polyomaviruses in the Thai population was 6.29% and 1.99%, respectively. The frequency of co-detection of WU and KI polyomaviruses with other respiratory viral pathogens was 42.11% and 33.33%, respectively. Moreover, each of the two complete genome sequences of WU (CU_295 and CU_302) and KI (CU_255 and CU_258) polyomaviruses were genetically and phylogenetically characterized. Sequence analysis showed that they contained features common to those found in previous studies. However, there were several nucleotide variations within the non-coding regulatory regions and various non-synonymous mutations within the coding regions which may influence virulence and pathogenesis of these viruses. Nevertheless, it is still possible that these viruses are not the causative agents of clinical respiratory disease. Therefore, judging the association of WU/KI polyomavirus infections with a particular disease will be challenging and require more comprehensive case control investigations.     

DNA from BK virus and JC virus and from KI, WU, and MC polyomaviruses as well as from simian virus 40 is not detected in non-UV-light-associated primary malignant melanomas of mucous membranes

The single most important causative factor for malignant melanomas of the skin is UV radiation. However, this is not true for melanomas on body surfaces sheltered from the sun; thus, it is important to seek new causative factors of melanoma genesis. Human papillomaviruses and gammaherpesviruses are associated with human skin cancer; for example, human papillomavirus types 5 and 8 are associated with epidermodysplasia verruciformis, and human herpesvirus 8 is associated with Kaposi's sarcoma. Recently, a newly described human polyomavirus, Merkel cell polyomavirus (MCPyV), has been associated with Merkel cell carcinoma, an unusual form of neurotropic skin cancer. Moreover, melanocytes are of neuroepithelial origin. This background impelled us to investigate if human polyomavirus DNA could play a role in the development of extracutaneous melanomas. Sixty-four extracutaneous melanomas were initially collected and dissected. Of these, 38 could be successfully used for further testing for the presence of the five human polyomaviruses known so far—BK virus (BKV), JC virus (JCV), KI polyomavirus (KIPyV), WU polyomavirus (WUPyV), and MCPyV—and of simian virus 40 (SV40). No polyomavirus DNA could be detected in any of the samples tested by use of a nested PCR detecting BKV, JCV, and SV40; a newly designed PCR detecting KIPyV and WUPyV; or a newly designed PCR for MCPyV. We conclude that since no human polyomavirus DNA was detected in primary malignant melanomas on non-sun-exposed body surfaces, these polyomaviruses presumably are not major factors for the development of extracutaneous melanomas.     

Merkel cell polyomavirus DNA in immunocompetent and immunocompromised patients with respiratory disease

Merkel cell polyomavirus (MCPyV) was identified originally in association with a rare but aggressive skin cancer, Merkel cell carcinoma. The virus has since been found in the respiratory tract of some patients with respiratory disease. However, the role of MCPyV in the causation of respiratory disease has not been established. To determine the prevalence of MCPyV in 305 respiratory samples from immunocompetent and immunocompromised patients and evaluate their contribution to respiratory diseases, specimens were screened for MCPyV using single, multiplex, or real-time PCR; co-infection with other viruses was examined. Of the 305 samples tested, 10 (3.27%) were positive for MCPyV. The virus was found in two groups of patients: in 6 (2%) nasopharyngeal aspirate samples from children aged 26 days to 7 months who were immunocompetent; and in 4 (1.3%) of nasopharyngeal aspirate samples taken from patients aged 41 to 69 years who were severely immunosuppressed from leukemia or transplant therapy. Both groups had upper or lower respiratory tract infection. Co-infections with other viruses were found in 30% of the MCPyV positive samples. The data present a pattern of infection similar to that seen with the polyomaviruses JC and BK in which the virus is acquired during childhood, probably by the respiratory route. The viruses then establish latency and become reactivated in the event of immunosuppression.     

实时荧光定量PCR检测儿童下呼吸道感染的WU多瘤病毒

目的 建立并应用检测儿童下呼吸道感染WU多瘤病毒(WU polyomavirus)的实时荧光定量PCR( real-time fluorescent quantitative PCR,FQ-PCR)方法.方法 选择WU多瘤病毒的VP2基因作为检测的目标基因,设计FQ-PCR引物和检测探针,以重组质粒为标准品建立标准曲线,并对该方法的特异性、灵敏度、重复性进行评价;应用该方法对温州医学院附属温岭医院收集的临床下呼吸道感染患儿的痰液、咽拭子846份及血清标本846份进行定量检测.结果 本研究建立FQ-PCR检测方法,质粒标准曲线的方差系数为0.998,灵敏度可达到50拷贝;应用该方法检测700份痰液标本,检测到7例阳性标本,146份咽试子标本中未检测到阳性标本,总阳性率为1.00％ (7/700),846份血清标本未检测到阳性标本.结论 本研究建立的FQ-PCR方法可以特异、快速、灵敏地对儿童下呼吸道感染的WU多瘤病毒进行定量检测;痰液标本较咽拭子或血清标本更适用于WU多瘤病毒感染的核酸检测.     

Polyomaviruses KI and WU in children with respiratory tract infection

The polyomaviruses KI (KIPyV) and WU (WUPyV) have recently been discovered in specimens from patients with respiratory tract infections. To analyze the frequency and clinical impact in a cohort of pediatric patients in a German University Children’s Hospital. Nasopharyngeal aspirates or bronchoalveolar lavage specimens of 229 children with acute respiratory tract infection were screened for KIPyV and WUPyV using polymerase chain reaction-based methods. KIPyV was detected in 2 (0.9%) and WUPyV in 1 (0.4%) patients, without co-infections with other respiratory viruses but with co-detection of CMV, EBV and HHV 6 in one immunocompromised patient. Only a very small proportion (1.3%) of positive samples for KIPyV and WUPyV was documented in this study; the clinical relevance of these viruses remains unclear and requires further evaluation.     

Seroepidemiology of WU polyomavirus among children exposed perinatally to HIV-1

WU polyomavirus (WUPyV), a new member of the genus wukipolyomavirus in the family Polyomaviridae, has been detected in serum and tissues of individuals infected with HIV. However, the epidemiology of WUPyV among children exposed perinatally to HIV‐1 is unknown. To investigate the epidemiology of WUPyV in children exposed to and infected perinatally with HIV, serum samples from 150 children exposed to HIV and 114 children infected with HIV were screened for IgG antibodies to WUPyV. A subset was screened for IgM antibodies to WUPyV. Both antibody detection assays were performed using a recombinant WUPyV VP1‐based ELISA. The overall seroprevalence of WUPyV IgG was 76.3% in children infected with HIV and 62% in children exposed perinatally to HIV. In the group of children infected with HIV, the prevalence of WUPyV IgG antibody reached its peak in 2–3 year olds (90.9%). In children 0–5 months of age, IgG seroprevalence was lower in those children exposed to HIV compared to children infected with HIV (43.1% vs.75%, P = 0.047). However, the seroprevalence of WUPyV IgM antibody was higher in children exposed to HIV compared to infants infected with HIV (27.4% vs. 8.3%, P = 0.044). WUPyV infection is acquired in early childhood in the majority of children born to mothers infected with HIV. The implication of this infection and the specific clinical syndrome that it produces, if any, remain to be defined. J. Med. Virol. 84:188–193, 2012. © 2011 Wiley Periodicals, Inc.     

Age-related pattern of KI and WU polyomavirus infection

BACKGROUND: The role of two recently identified polyomaviruses, KI and WU, in the causation of respiratory disease has not been established. OBJECTIVES: To determine the prevalence of KI and WU viruses (KIV and WUV) in 371 respiratory samples and evaluate their contribution to respiratory disease. STUDY DESIGN: Specimens were screened for KIV and WUV using single, multiplex or real time PCR; co-infection with other respiratory viruses was evaluated. RESULTS: Of the 371 samples analysed, 10 (2.70%) were positive for KIV and 4 (1.08%) were positive for WUV yielding an overall case prevalence of KIV and WUV infection of 3.77%. KIV and WUV were identified in patients aged <15 years (11 patients) with upper or lower respiratory tract infection and >45 years (3 patients) with upper respiratory tract infection. Co-infections were found in 5 (50%) and 3 (75%) of the KIV and WUV positive samples, respectively. CONCLUSIONS: This study supports previous conclusions that KIV and WUV detection in the respiratory tract may be coincidental and reflect reactivation of latent or persistent infection with these viruses. The age distribution of KIV and WUV infection in this study mirrors that found for the other human polyomaviruses, BK and JC.     

Detection of BK virus DNA in nasopharyngeal aspirates from children with respiratory infections but not in saliva from immunodeficient and immunocompetent adult patients.

Our understanding of important stages in the pathogenesis of the human polyomavirus BK virus (BKV) and JC virus (JCV) infections is limited. In this context, nasopharyngeal aspirates from 201 children with respiratory diseases and saliva from 60 human immunodeficiency virus type 1-infected adults and 10 healthy adult controls were collected and analyzed for the presence of BKV and JCV DNA by PCR. Neither BKV nor JCV DNA was detected in the saliva specimens. We demonstrated BKV DNA, but no infectious BKV, in 2 of 201 nasopharyngeal aspirates. Each sample contained one unique rearranged noncoding control region variant of BKV. The results indicate that (i) BKV and JCV are not regularly associated with respiratory infections in children requiring hospitalization, (ii) nasopharyngeal cells are not an important site for primary replication of human polyomavirus BKV and JCV, and (iii) the salivary glands and oropharyngeal cells seem not to be involved in BKV and JCV persistence. We propose that for the polyomaviruses BKV and JCV the alimentary tract should be considered as a portal of entrance to the human organism.     

Complete coding sequences and phylogenetic analysis of Human Bocavirus (HBoV)

Human Bocavirus (HBoV) is a novel virus which can cause respiratory tract disease in infants or children. Recently, the prevalence of this virus has been studied worldwide. In this study, 18 of 252 (7.14%) nasopharyngeal suctions from infants or children between 1 month and 9 years of age with respiratory tract illness were HBoV-positive by PCR. Three positive samples were selected for sequencing the entire coding sequences using a new conserved set of primers. The results showed that the most conserved regions of HBoV are the NS1 and NP1 genes, whereas VP1 and VP2 showed frequent variations. However, the complete coding sequences showed that the variations did not depend on the origin of virus found. The complete coding sequences determined in this study can resolve the problem of an HBoV detection method, which can be advantageously implemented in laboratory detection.     

Human Coronavirus-NL63 infections in Korean children, 2004-2006.

BACKGROUND: Human coronavirus-NL63 (HCoV-NL63) has been isolated from children with respiratory tract infections and its prevalence in Korea has not been reported. OBJECTIVES: This study was designed to investigate the presence and the clinical features of HCoV-NL63 during two winter seasons. STUDY DESIGN: During April 2004-April 2006, nasopharyngeal specimens from children hospitalized with acute respiratory disease were tested for common respiratory viruses, including RSV, influenza A, influenza B, parainfluenza viruses, and adenovirus by IFA. hMPV infection was excluded by nested RT-PCR using primers for F-gene. To detect HCoV-NL63, previously described nested PCR assays for 1a and 1b were used. PCR products of the 1a gene for HCoV-NL63 were sequenced. RESULTS: Out of 872 nasopharyngeal aspirate from children aged under 16 years, 14 (1.7%) were positive for HCoV-NL63. Most of the patients had croup (64.2%) or bronchiolitis (21.4%). The peak prevalence was found in November (28.5%). Most were collected between November 2004 and February 2005. CONCLUSIONS: HCoV-NL63 may be one of the causative agents of acute respiratory tract infection, especially croup.