Human Disease Virus Research Articles

Whole Exome Sequencing Analysis in Chronic Lymphoproliferative Disorder of NK Cells (CLPD-NK) Patients Fails to Detect Significant Viral Load

Background. Chronic Lymphoproliferative Disorder of Natural Killer cells (CLPD-NK) is a rare disease characterized by chronic NK cell lymphocytosis, typically equipped with a restricted pattern of activating killer immunoglobulin like receptors. Several lines of evidence point to a putative role of viral agents in the pathogenetic mechanisms accounting for NK cell proliferation. A role of Epstein Barr Virus (EBV) has been claimed in the aggressive form of NK cell proliferation [i.e. Aggressive NK Cell Leukemia (Kawa Ha et al, JCI 1989)]. At variance, in the indolent CLPD-NK, EBV DNA was seldom detected by PCR (Loughran TP Jr et al, Blood 1993), whereas we provided serologic evidence for previously experienced viral infections in the majority of CLPD-NK patients (Zambello R et al, Leukemia 1995). However, the role of infectious agents in the pathogenesis of chronic antigen stimulation of NK cells in CLPD-NK patients is still elusive. Methods. Several studies showed that whole exome sequencing (WES), as well as RNA-seq, can identify pathogens, particularly viruses, in cancer clones enabling the definition of possible integration sites in the host genome or their presence as episomal forms. In the present study, we searched viral sequences in WES data of a cohort of 10 CLPD-NK patients. High-depth (average 51 M reads) WES data of highly purified (>97% purity) leukemic NK clone and autologous granulocytes, as control, from CLPD-NK patients were analyzed for pathogen discovery. Three different methods for virus detection were used: i) Centrifuge (Kim et al, Genome Research 2016), a metagenomic tool performing a wide search against GenBank viral and bacterial sequences, which has been recently used to detect EBV load in ANKL and NK T cell Lymphoma (NKTCL) tumor clones (Dufva et al. Nat Comm 2018); ii) VirusSeq (Chen et al., Bioinformatics 2013), a software to identify viruses and eventually their integration sites, grounding on its custom and relatively wide viral sequence database; and iii) ViFi (Nguyen et al. Nucleic Acids Res, 2018), which can find viral integration in the human genome, with an analysis focused on a narrow set of viruses (EBV, HPV, HBV) with well-established oncogenic potential. Results. First, as positive control two datasets were used: RNA-seq data of an HBV positive sample with known site of integration (provided by VirusSeq) and WES data of three NKTCL tumor and control clones with high, moderate or low EBV load in the tumor samples previously detected by Dufva et al. (Dufva O et al, Nat Comm 2018), publicly available at NCBI SRA. HBV presence was detected in the positive sample by all the three methods and our reanalysis of NKTCL samples with Centrifuge confirmed published results, detecting EBV in all tumoral samples and in none of the matched control samples. Next, analysis of samples from the 10 CLPD-NK patients with the three methods did not identify a significant viral load neither in tumor nor in control specimens. Neither VirusSeq nor ViFi found viruses in CLPD-NK samples, whereas, according to Centrifuge analysis, the human disease virus with the highest load in most patients was Varicella Zoster (Human alphaherpesvirus 3) but with low read count and, most relevant, without specificity for the cancer clone. Conclusions. Our very thorough WES analysis did not identify a significant and meaningful viral load in CLPD-NK samples, neither in leukemic clones nor in control cell population. Although not ruling out that a viral infection might play a role as starting event for inciting NK cell proliferation, our data do not support a role of viral infections in maintaining the proliferation in CLPD-NK. Disclosures Zambello: Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Computational identification, characterization and validation of potential antigenic peptide vaccines from hrHPVs E6 proteins using immunoinformatics and computational systems biology approaches

High-risk human papillomaviruses (hrHPVs) are the most prevalent viruses in human diseases including cervical cancers. Expression of E6 protein has already been reported in cervical cancer cases, excluding normal tissues. Continuous expression of E6 protein is making it ideal to develop therapeutic vaccines against hrHPVs infection and cervical cancer. Therefore, we carried out a meta-analysis of multiple hrHPVs to predict the most potential prophylactic peptide vaccines. In this study, immunoinformatics approach was employed to predict antigenic epitopes of hrHPVs E6 proteins restricted to 12 Human HLAs to aid the development of peptide vaccines against hrHPVs. Conformational B-cell and CTL epitopes were predicted for hrHPVs E6 proteins using ElliPro and NetCTL. The potential of the predicted peptides were tested and validated by using systems biology approach considering experimental concentration. We also investigated the binding interactions of the antigenic CTL epitopes by using docking. The stability of the resulting peptide-MHC I complexes was further studied by molecular dynamics simulations. The simulation results highlighted the regions from 46–62 and 65–76 that could be the first choice for the development of prophylactic peptide vaccines against hrHPVs. To overcome the worldwide distribution, the predicted epitopes restricted to different HLAs could cover most of the vaccination and would help to explore the possibility of these epitopes for adaptive immunotherapy against HPVs infections.

Human Protoparvoviruses.

Next-generation sequencing and metagenomics have revolutionized the discovery of novel viruses. In recent years, three novel protoparvoviruses have been discovered in fecal samples of humans: bufavirus (BuV) in 2012, tusavirus (TuV) in 2014, and cutavirus (CuV) in 2016. BuV has since been studied the most, disclosing three genotypes that also represent serotypes. Besides one nasal sample, BuV DNA has been found exclusively in diarrheal feces, but not in non-diarrheal feces, suggesting a causal relationship. According to both geno- and seroprevalences, BuV appears to be the most common of the three novel protoparvoviruses, whereas TuV DNA has been found in only a single fecal sample, with antibody detection being equally rare. Moreover, the TuV sequence is closer to those of non-human protoparvoviruses, and so the evidence of TuV being a human virus is thus far insufficient. Interestingly, besides in feces, CuV has also been detected in skin biopsies of patients with cutaneous T-cell lymphoma and a patient with melanoma, while all other skin samples have tested PCR negative. Even if preliminary disease associations exist, the full etiological roles of these viruses in human disease are yet to be resolved.

Novel human DNA viruses and their putative associations with human diseases

In this review, we described human small DNA viruses discovered on the cusp of the 20th and 21st centuries as a result of cutting-edge technologies established in molecular biology. The problems of obtaining an evidence of the etiological role of new viruses in human diseases have been considered.

Calibrated real-time polymerase chain reaction for specific quantitation of HHV-6A and HHV-6B in clinical samples

The recent classification of human herpesvirus 6 (HHV-6) A and B, previously considered as two variants of the same virus, as two distinct herpesvirus species, emphasizes the need to develop and standardize specific methods for their detection and quantitation for clinical use. The development of two highly sensitive calibrated real-time PCR to quantify HHV-6A and -6B variants in clinical specimen is described. Both assays displayed the same wide linear dynamic range from 100 to 106 copies of viral DNA in a single reaction and sensitivity of one copy/reaction. These systems allow for HHV-6A/B DNA load quantitation in different types of clinical specimens: blood or tissue cells when combined with the CCR5 assay; cell-free samples (plasma or other biological fluids) in combination with the calibrator technology. Due to the absence of cross-amplification and cross-hybridization, these methods detect minute amounts of one viral species even in the presence of a large excess of the other, allowing a specific quantitation of both viruses in the case of mixed infections. The new qPCR methods provide sensitive and specific tool for monitoring HHV-6A/B DNA load in clinical samples, facilitating the study of these viruses in human diseases.

HexaPrime: A novel method for detection of coronaviruses

Despite intense efforts to develop novel and better tools to identify known viruses and to discover new viruses, establishing etiological roles for viruses in human disease is challenging. In large part, this may be attributed to the high variability of viral species and the difficulties in developing broad-spectrum, yet specific, diagnostic assays. To overcome this problem, a novel method for the detection of viruses is described in the current manuscript. The technique relies on the addition of synthetic oligonucleotides to both termini of RNA fragments in a sequence-dependent manner during first- and second-strand DNA synthesis; these oligonucleotides are used subsequently for amplification of the viral nucleic acids of interest. The recognition of the target sequence by the oligonucleotides is mediated by short (6-8 nt) conserved regions, which facilitates development of broad-spectrum assays. The method has been tested for coronaviruses, although it may be also adopted for other RNA viruses.

Parvoviruses PARV4 and PARV5 and Hepatitis C Virus

To the Editor: Parvoviruses are small, nonenveloped DNA viruses that infect both vertebrate and invertebrate hosts. Until recently, parvovirus B19 and adeno-associated viruses, which belong to the genera Erythrovirus and Dependovirus, respectively, were the only known members of the family Parvoviridae that infected humans (1). However, 2 recent publications have identified 2 distinct, novel parvoviruses in humans by using the DNase sequence–independent single-primer amplification technique and a related method (2,3). The first of these viruses, termed PARV4, was observed in a patient with symptoms of acute viral infection syndrome after high-risk behavior for infection with HIV-1, although the patient was subsequently confirmed as negative for HIV-1 (2). The second parvovirus was identified in respiratory samples from children with lower respiratory tract infections and termed human bocavirus (3). Parvovirus B19 is a frequent contaminant of plasma pools that are used in the manufacture of blood products, which results in high viral loads in pools and viral transmission in recipients of clotting factors (4). We identified PARV4 in such pools (5), albeit at a lower frequency and titer than parvovirus B19, when parvovirus B19 was not excluded by screening with nucleic acid amplification techniques. Sequence analysis identified a second genotype of PARV4, which we have termed PARV5, that shares ≈92% nucleotide identity with PARV4 (5). PARV4 was originally identified in a plasma sample from a homeless, injection drug user with fatigue, night sweats, pharyngitis, neck stiffness, vomiting, diarrhea, arthralgia, and confusion (2). This person was coinfected with hepatitis B virus. In this study, we looked retrospectively for PARV4 and PARV5 in blood samples from a similar cohort of persons, many of whom were known to be infected with hepatitis C virus (HCV) (as determined by the presence of both HCV RNA and antibodies to HCV), and some of whom were intravenous drug users (IVDUs) (6). Blood samples were collected from 26 cadavers in London and the surrounding area as part of a study to investigate the inhibition of nucleic acid amplification techniques for bloodborne viruses in tissue samples (6). The cohort was composed of 10 HCV RNA–positive IVDUs, 8 HCV RNA–positive non-IVDUs, 4 HCV RNA–negative IVDUs, and 4 HCV RNA–negative non-IVDUs (Table). Nucleic acid was extracted as previously described (4) by using the MagNA Pure LC instrument (Roche, Basel, Switzerland). PCR was performed with primers specific for the second open reading frame (ORF2) in the PARV4 genome (2), which is homologous to the VP1 capsid of parvovirus B19. Primers PVORF2F (5′-AGGAGCAGCAAACAAACTCAGAC-3′) and PVORF2R (5′-TCCTTCATCGCGGCTGTCACTAA-3′) amplify a 268-bp region of ORF2 (nucleotides 2710–2977, GenBank accession no. {type:entrez-nucleotide,attrs:{text:AY622943,term_id:52854178,term_text:AY622943}}AY622943). The PCRs were performed and analyzed as previously described (5). The assay is highly specific (no cross-reactivity with parvovirus B19) and sensitive (detects 5–10 copies of PARV4 virus DNA per reaction). Table Analysis of 26 cadavers for parvoviruses PARV4 and PARV5* PCR products were cloned, sequenced, and compared with the prototype PARV4. Two blood samples were positive for PARV4, and a third sample was positive for PARV5, with 99%–100% nucleotide identity. These positive samples were from HCV RNA–positive IVDUs (Table). The titer of PARV4 and PARV5 DNA in the positive samples was low and did not exceed >700 copies/mL of plasma, as determined by using a consensus TaqMan assay (J. Fryer, unpub. data). None of the other blood samples tested was positive for PARV4 and PARV5, including those for persons who were HCV RNA negative and not IVDUs. In our previous study (5) of >130 fractionation pools (composed of thousands of units from screened healthy donors) for PARV4, the only positive pools were from North America and no European pools were positive for PARV4 or PARV5. These viruses may be present in such pools but diluted to undetectable levels. In the present study, PARV4 and PARV5 have been identified in blood samples obtained from persons from the United Kingdom. For parvovirus B19, there is evidence of persistent virus infection, at low levels, in bone marrow of previously exposed persons (7) and in plasma of immunocompromised and immunocompetent persons (8,9). There is also evidence for the lifelong persistence of parvovirus B19 (genotypes 1 and 2) in tissues such as skin and synovia (10). PARV4 and PARV5 virus genomes share only limited homology with parvovirus B19 (<30% amino acid similarity). Although they have been detected in blood and plasma, nothing is known about the role of these viruses in human disease or their ability to persist in infected persons, healthy or otherwise. Further studies will be required to determine the prevalence of PARV4 and PARV5 in healthy persons compared with its prevalence in those with chronic infections and at high risk, such as IVDUs, and to investigate the nature of persistence of these novel viruses.

Human T lymphotropic viruses (HTLVs): Clinical and pathogenic properties

The human T lymphotropic viruses type I (HTLV-I) and type II (HTLV-II) are closely related retroviruses which are associated with the development of lymphoproliferative disorders. HTLV-I has a preferential tropism for CD4+ T lymphocytes and is associatedwith adult T cell leukaemia/lymphoma (ATLL) an aggressive of CD4 T cell malignancy. HTLV-II has a preferential tropism for CD8+ T lymphocytes, and while the role of this virus in human disease remains unclear, infection has been associated with benign CD8+ T lymphoproliferative disorders. In vitro studies have demonstrated that the virus regulatory protein Tax plays a central role in cell transformation and proliferation, and that this appears to be due to the ability of the protein to disregulate both cell cycle controls and the transcription of a number of cellular genes. To further investigate this we have been investigating the properties of HTLV-I and HTLV-II Tax proteins both in vitro and in vivo. These studies will be described. Of note, the latter have allowed the development of an animal model of ATLL which will allow further investigation of the oncogenic properties of the HTLV Tax proteins.

Détection, quantification et analyse des génomes viraux dans les infections à herpèsvirus humains 6 et 7 (HHV-6, HHV-7)

The detection, quantification and genetic analysis of viral genomes have become a common practice in clinical virology. This is exemplified by the study of human herpesviruses 6 and 7. The detection of viral DNA has led to implicate theses viruses in human diseases far beyond the evidence obtained by conventional approaches such as virus culture and serology. This is specially the case for immunocompromised subjects. The quantitation of DNA, in particular by means of real-time PCR, is now susceptible to differenciate reactivation from latency, predict the occurrence of clinical events, and monitor the efficacy of antiviral therapy. Genetic analyses have permitted the characterization of variant-specific polymorphisms and mutations responsible for resistance to antivirals. Despite some limitations that users have to be aware of, the study of viral genomes is an outstanding progress in medical virology.

The role of the Epstein-barr virus in human disease

The role of the Epstein-barr virus in human disease

The Role of the Epstein-Barr virus in human disease.

EBV is a B lymphotropic virus that is associated with a range of human malignancies. Although for many of these tumours the association has long been established, unraveling the precise role of EBV in disease pathogenesis has been more difficult. This review summarizes current knowledge concerning the association between EBV and human cancers and illustrates how an increasing appreciation of patterns of latent gene expression and latent gene function in different cell environments is already helping towards a better understanding of both the natural history of infection in normal individuals and how EBV contributes to malignant transformation. Finally, therapeutic strategies that target EBV in tumours are discussed.

Increasing evidence for involvement of SV40 in human cancer.

SV40, a small DNA virus, is known to possess strong oncogenic potential. Millions of people were exposed to SV40 as an unknown contaminant of some early poliovaccines. This article briefly summarizes the increasing evidence of the association of SV40 with certain types of human cancer, including mesotheliomas and brain tumors. Unanswered questions pertaining to the pathogenesis of human infections by SV40 and the functional role of the virus in tumor development are noted. It is concluded that SV40 should be considered a candidate human tumor virus and that vigorous efforts to clarify the role of the virus in human disease should be supported.

Mosquito-borne viruses and epidemic polyarthritis.

The two most common mosquito-borne viruses associated with epidemic polyarthritis and polyarticular disease are Ross River and Barmah Forest viruses, accounting for about 90% and 10%, respectively, of serologically confirmed cases. Occasional cases of polyarticular disease in Australia have been associated with infection by other mosquito-borne arboviruses, but the role of these viruses in human disease remains to be confirmed.

Human T cell leukemia/lymphoma virus, type II (HTLV-II): emergence of an important newly recognized pathogen

In the 10 year period since its discovery, human T cell leukemia virus type II (HTLV-II) has emerged as a significant human pathogen. Infection has been shown to be endemic in a large number of native Indian populations throughout the Americas, and high rates of infection have also been demonstrated in intravenous drug abusers (IVDAs) in both the United States and Europe. While the role of the virus in human disease remains to be clearly defined, there is accumulating evidence that it may be associated with neurological, and perhaps, rare lymphoproliferative disorders. This review summarizes the current status of our understanding of HTLV-II infections, and will emphasize important aspects of the epidemiology of infection, and the molecular and biological properties of the virus. In addition, the salient features of the clinical disorders known to be associated with infection are described.

Human T-Cell Lymphotropic Viruses in Human Diseases

Human T-Cell Lymphotropic Viruses in Human Diseases

Role of Herpes Viruses in Human Diseases

Role of Herpes Viruses in Human Diseases

Respiratory illness in prematures and children. Illness caused by parainfluenza type 3 virus.

Since the first report by Chanock et al.,¹in 1958, on the association of hemadsorption viruses with respiratory illness in childhood, several investigators in this country and abroad^2-13have recovered hemadsorption viruses from the respiratory tracts of children and adults affected by respiratory infections of variable severity. Until recently, the hemadsorption viruses included 2 strains, and it has been proposed that these agents be included in the group termed "parainfluenza viruses."¹⁴A third agent has been isolated from an adult and several children and it has been proposed that it be called "parainfluenza Type 4."¹⁵ Although the role of parainfluenza viruses in human disease has not been completely established, there is good evidence that these viruses should be considered as responsible agents of some of the nonbacterial respiratory infections occurring in infants and children.^16,17 During the past winter we have had the opportunity of studying

The Role of Coxsackie and ECHO Viruses in Human Disease

The Role of Coxsackie and ECHO Viruses in Human Disease

REVIEW ARTICLE

CURRENT opinion is divided as to the role of the encephalomyocarditis (EMC) group of viruses in human disease. In the serologic survey of Tarahumara Indians reported elsewhere in this issue it has been shown that antibody neutralizing significant amounts (namely, 20, 80, 1000, and 6000 LD50, respectively) of the AMS strain of EMC virus was found in 4 of the 56 serum specimens studied. This finding serves to focus attention again on the scattered reports suggesting the EMC virus as an etiologic agent in human disease. History of the Discovery of EMC Viruses Jungeblut and Sanders isolated from cotton rats inoculated with Yale-SK strain of poliomyelitis virus, a virus different from that which they had inoculated. They designated this new agent "Columbia-SK." Later Jungeblut and Dalldorf isolated an agent they called MM-virus from hamsters inoculated with central nervous system (CNS) tissue of a fatal case of undiagnosed paralysis suspected of being poliomyelitis. Many workers have assumed that these first isolations were of viruses latent in the laboratory rodents. Helwig and Schmidt obtained encephalomyocarditis (EMC) virus from a captive chimpanzee with myocarditis and hydrothorax in Florida; and Dick, Best, Haddow, and Smithburn obtained so-called Mengo encephalomyelitis virus in Uganda from a paralyzed rhesus monkey, later from mosquitoes (Taeniorrhynchus fuscopennatus), and from a wild mongoose caught near the monkey compound in Entebbe, Uganda. Accidental infection of a laboratory worker in Entebbe with Mengo virus demonstrated the pathogenicity of this agent for man. The worker developed an encephalitic illness and the virus was isolated from his serum.

The importance of Coxsackie viruses in human disease, particularly herpangina and epidemic pleurodynia.

The importance of Coxsackie viruses in human disease, particularly herpangina and epidemic pleurodynia.