Removal of indigenous coliphages and enteric viruses during riverbank filtration from highly polluted river water in Delhi (India).

Emerging countries frequently afflicted by waterborne diseases require safe and cost-efficient production of drinking water, a task that is becoming more challenging as many rivers carry a high degree of pollution. A study was conducted on the banks of the Yamuna River, Delhi, India, to ascertain if riverbank filtration (RBF) can significantly improve the quality of the highly polluted surface water in terms of virus removal (coliphages, enteric viruses). Human adenoviruses and noroviruses, both present in the Yamuna River in the range of 10(5) genomes/100 mL, were undetectable after 50 m infiltration and approximately 119 days of underground passage. Indigenous somatic coliphages, used as surrogates of human pathogenic viruses, underwent approximately 5 log10 removal after only 3.8 m of RBF. The initial removal after 1 m was 3.3 log10, and the removal between 1 and 2.4 m and between 2.4 and 3.8 m was 0.7 log10 each. RBF is therefore an excellent candidate to improve the water situation in emerging countries with respect to virus removal.

[Detection of viruses in raw water as a basic tool for risk assessment]. / Nachweis von Viren im Rohwasser als Grundlage einer Risikoabschätzung.

Human pathogenic viruses may end up in surface waters by fecal contamination. However, the German drinking water ordinance requests that pathogens in drinking water should not be present in concentrations constituting a potential danger to human health. Since many viruses do have a very low dose of infection, they have to be sufficiently eliminated in the process of drinking water purification. Waterborne virus outbreaks in Europe, over the last few decades, were mostly linked to noncompliance with the generally accepted codes of practice for drinking water production. The aimed level of protection of drinking water supplies in Germany, however, exceeds prevention of outbreaks by even protecting against sporadic virus infections. Documentation of such a high level of protection is not achieved by end product control alone but requires a process analysis with risk assessment. To do such an analysis, information regarding the presence of viruses in the raw water used for drinking water production, as well as data of virus elimination rates during purification processes, are of major importance. This paper presents suggestions for implementation of such a risk assessment, focusing on the evaluation of raw water quality.

Molecular detection of cosaviruses in a patient with acute flaccid paralysis and in sewage samples in Germany.

Cosaviruses (CoSV) were first identified in stool samples collected from non-polio acute flaccid paralysis (AFP) cases and their healthy contacts in Pakistan in 2003. The clinical importance of CoSV remains unclear as data on epidemiology are scarce and no routine diagnostic testing is done. In this study, we characterized human CoSV (HCoSV) in a child with non-polio AFP and in sewage samples collected in Berlin, Germany. Using unbiased high-throughput sequencing and specific PCR, we characterized a HCoSV-D in stool samples of a three-year-old child hospitalized in Germany with non-polio AFP and travel history to Pakistan. The shedding pattern and absence of other relevant pathogens suggests that HCoSV-D may have been involved in the genesis of AFP. The HCoSV-RNA concentration was high, with 2.57 × 106 copies per mL fecal/suspension, decreasing in follow-up samples. To investigate the possibility of local circulation of HCoSV, we screened Berlin sewage samples collected between 2013 and 2018. Molecular testing of sewage samples has shown the presence of CoSV in several parts of the world, but until now not in Germany. Of our sewage samples, 54.3 % were positive for CoSV, with up to three viral species identified in samples. Phylogenetically, the German sequences clustered intermixed with sequences obtained globally. Together, these findings emphasize the need for further clinical, epidemiological, environmental, pathogenicity and phylogenetic studies of HCoSV.

The soluble form of two N-terminal domains of the poliovirus receptor is sufficient for blocking viral infection.

By means of deleting a C-terminal portion of the open reading frame of the poliovirus receptor cDNA, and by vaccinia virus-mediated overexpression we have produced a protein corresponding to the first two N-terminal Ig-like domains of the poliovirus receptor. This protein that lacked the third Ig-like domain, the transmembrane region and most of the intracellular C-terminal tail was detected in the medium of vaccinia virus infected cells. The properties of the truncated PVR cDNA were further characterized by in vitro translation and modification. The molecular weight of the unmodified protein was found to be 27 kDa; translation in the presence of dog pancreas microsomes led to an increase in molecular weights which we attribute to N-glycosylation. Upon incubation with poliovirus at 37 degrees C, the vaccinia-virus generated protein specifically reduced infectivity of poliovirus. Sucrose gradients of poliovirus particles derived after incubation with the protein showed the induction of a slower sedimenting particle (135S). Our experiments suggest that the two N-terminal domains of the poliovirus receptor in soluble form are sufficient for the conversion of poliovirus into a non-infectious particle.

Chinese hamster ovary cells are non-permissive towards infection with coxsackievirus B3 despite functional virus-receptor interactions.

Viral infection is a complex process which includes binding and interaction of the virus with specific cell surface receptors, uptake and uncoating of the virus, and finally replication. Chinese hamster ovary (CHO) cells are non-permissive towards infection with coxsackieviruses of group B (CVB), although they do express a putative CVB-specific receptor protein. In order to localize the block of infection in CHO cells, these cells were tested for binding of radiolabelled CVB3, receptor-mediated transformation of virions into A-particles, and replication of the viral genome. Binding of CVB3 to CHO cells was found to be comparable to the binding of this virus to permissive cell lines. Detergent-solubilized membrane proteins of CHO cells were tested in virus overlay protein-binding assays (VOPBAs) and shown to express a 100 kDa CVB-binding membrane protein similar to the CVB receptor protein which we recently described for permissive HeLa cells. Incubation of CVB3 with intact CHO cells resulted in transformation of cell-bound virions into non-infectious A-particles (deprived of capsid protein VP4), demonstrating the functional activity of the CVB receptor protein on CHO hamster cells. Transfection of recombinant CVB3 cDNA or viral RNA into CHO cells resulted in the production of infectious CVB3 virions, implying that the failure of CVB to infect CHO cells is not caused by a defect in virus replication but results from a block in the uptake of virus particles into the cell after the initial steps of virus-receptor interactions.

Mechanisms and consequences of enterovirus persistence in cardiac myocytes and cells of the immune system.

In humans and experimental murine models enteroviruses, and in particular coxsackieviruses of group B (CVB), may induce chronic myocarditis associated with a persistent type of heart muscle infection. Persistent myocardial infection has been characterized by restricted viral replication and gene expression, which is capable of sustaining chronic inflammation. Altered replication and transcription of the virus, in addition to an immune response insufficient to recognize and clear infected cells entirely, are essential mechanisms for initiation and maintenance of persistent heart muscle infection. Viral cytotoxicity was found to be crucial for organ pathology both during acute and persistent infection, indicating that enterovirus myocarditis is a virus-induced rather than an immune-mediated disease. Notably, resistance to the development of persistent heart muscle infection is not linked to the H-2 haplotype of the host. In addition to persistently infected myocytes, detection of the replicative minus-strand RNA intermediate provided evidence for virus replication in lymphoid cells of the spleen, predominantly in splenic B lymphocytes, during the course of the disease. Whereas viral RNA was also detected in certain CD4+ helper T cells and Mac1+ macrophages, no enteroviral genomes were identified in CD8+ T cells. Detection of infected activated B lymphocytes both in heart tissue of CVB3-infected immunocompetent mice and syngenic SCID mice receiving splenocytes from CVB3-infected donors support the concept that B cell traffic may contribute to maintenance of chronic disease. Dissection of the diversity of viral and host-specific determinants in susceptible and resistant hosts will allow us to define the protective mechanisms that mediate resistance to the development of life-threatening acute and chronic enterovirus myocarditis.

[Pathogenesis, diagnosis and therapy of heart disease caused by enteroviruses (molecular biological study)]. / K patogenezu, diagnostike i terapii porazheniia serdtsa, vyzvannogo énterovirusami (molekuliarno-biologicheskoe issledovanie).

Molecular hybridization studies have demonstrated that human enteroviruses, including group B coxsackieviruses (CVB), are detectable in myocardial tissue of patients with acute and chronic myocarditis. As well, such infections are observed in some patients with end-stage dilated cardiomyopathy indicating the possibility of persistent heart muscle infection. Enterovirus persistence in the human heart is supported by the discovery in various murine models of chronic myocarditis, demonstrating that coxsackievirus B3 (CVB3), typically a cytolytic virus, is capable of evading immunological surveillance in a host-dependent manner. Currently attention is focused on the analysis of molecular mechanisms of virus persistence, the characterization of viral and host factors and their impact in determining the natural course of myocardial enterovirus infections. The evidence for a causal linkage of enterovirus infection with heart muscle diseases has emerged therapeutic implications. From the view of a virologist, immunosuppressive treatment of patients revealing enterovirus infection in the myocardium with steroids is clearly contraindicated. The evaluation of potent antiviral agents, such as interferons, in established in vitro and in vivo model systems of enterovirus infection is expected to contribute significantly to new therapeutic strategies in human enteroviral heart disease.

Evidence for a mutual influence of idiotype-expressing donor cells and recipient lymphocytes in neonatal hosts.

Nonirradiated recipients do not permit activation of transferred syngeneic lymphocytes. This transplantation barrier gives us insight into the mechanism of the regulatory circuits of the donor's and host's immune network. The present report demonstrates that this barrier depends on the state of differentiation of cells from both donor and host. We measured the expression of anti-alpha (1-3)dextran (Dex) correlated idiotypes by cells from responder BALB/c animals (allotype IgHa, Dex+, IdDex+) in nonresponder (IgHb, Dex-, IdDex-) congeneic mice. Adult IgHb recipients did not permit activation of either adult spleen or bone marrow cells (isogeneic barrier). Neonatal recipients showed functional tolerance towards adult spleen cells. By contrast, neonates neither permitted activation of adult B cell-depleted bone marrow cells, nor of neonatal immature spleen cells. The results show that the immature system of the neonate is permissive towards mature (adult) congeneic lymphocytes, but not towards immature cells from congeneic neonates or adult bone marrow. It appears that mature adult cell populations are dominant in the immature neonatal host. However, the time required by transferred immature cells to differentiate in the neonatal recipient concomitantly allow the latter to gain maturity and functionally reject the grafted cells.

xid mice fail to express an anti-dextran immune response but carry alpha(1-3)dextran-specific lymphocytes in their potential repertoire.

Even after repeated immunizations with alpha(1-3)dextran (Dex) male (CBA/N x BALB/c)F1 mice fail to produce specific antibodies whereas nondefective female littermates express an idiotype-positive anti-Dex immune response. This failure of xid mice to express serum anti-Dex immunoglobulins is not only limited to immunization with the thymus-independent (TI-2) antigen Dex, since immunization with anti-idiotypic antibodies against Dex-specific idiotypes does not overcome this defect. When xid lymphocytes are cultured in the presence of mitogens, in vitro anti-Dex responses are markedly reduced. We show here that alpha(1-3)Dex-specific hybridomas can be established by fusion of splenic lymphocytes from xid mice to Sp2/0 myeloma cells. Therefore, these mice do carry the potential to generate B cells specific for Dex. All hybridoma antibodies were found to be of IgM isotype, bearing the lambda light chain typical for alpha(1-3)Dex-specific antibodies. Whereas monoclonal anti-Dex antibodies obtained from spleen cell hybridomas from female littermates showed variable idiotope patterns, hybridoma proteins from the immune-defective NBF1-xid mouse expressed only a limited pattern of Dex-specific idiotopes, suggesting that these hybridomas derived from a common precursor.

Tyrosine phosphorylation events during coxsackievirus B3 replication.

In order to study cellular and viral determinants of pathogenicity, interactions between coxsackievirus B3 (CVB3) replication and cellular protein tyrosine phosphorylation were investigated. During CVB3 infection of HeLa cells, distinct proteins become phosphorylated on tyrosine residues, as detected by the use of antiphosphotyrosine Western blotting. Two proteins of 48 and 200 kDa showed enhanced tyrosine phosphorylation 4 to 5 h postinfection (p.i.), although virus-induced inhibition of cellular protein synthesis had already occurred 3 to 4 h p.i. Subcellular fractionation experiments revealed distinct localization of tyrosine-phosphorylated proteins of 48 and 200 kDa in the cytosol and membrane fractions of infected cells, respectively. In addition, in Vero cells infected with CVB3, echovirus (EV)11, or EV12, increased tyrosine phosphorylation of a 200-kDa protein was detected 6 h p.i. Herbimycin A, a specific inhibitor of Src-like protein tyrosine kinases, was shown to inhibit virus-induced tyrosine phosphorylations and to reduce the production of progeny virions. In contrast, in cells treated with the inhibitors staurosporine and calphostin C, the synthesis of progeny virions was not affected. Immunoprecipitation experiments suggested that the tyrosine-phosphorylated 200-kDa protein in CVB3-infected cells is of cellular origin. In summary, these investigations have begun to unravel the effect of CVB3 as well as EV11 and EV12 replication on cellular tyrosine phosphorylation and support the importance of tyrosine phosphorylation events for effective virus replication. Such cellular phosphorylation events triggered in the course of enterovirus infection may enhance virus replication.

B cell dependence of the congeneic barrier towards idiotype-carrying cells.

Immune cells transferred into nonirradiated animals of the same genotype face a barrier which severely affects their capacity to function in the host. We studied this phenomenon in allotype-congeneic animals. When anti-dextran immune cells of the responder strain (BALB/c-Igha) are injected into an allotype-congeneic host (BALB-Ighb) the grafted cells are suppressed to give an idiotype-positive response. This congeneic barrier of thymus-independent immune cells is lost in mice carrying an X-linked B cell defect: when idiotype-positive immune cells from responder (CBA/N X BALB/c)F1 mice are transplanted into congeneic nonresponder animals (CBA/N X BALB-Ighb)F1, female recipients are nonpermissive towards grafted cells whereas B cell-defective male littermates allow donor cells to develop an idiotype-positive anti-dextran response. These results show that the congeneic barrier towards dextran-immune cells is related to the maturation stage of B cells in the recipient. Since B cell-defective (CBA/N X BALB/c)F1 animals do not display an anti-idiotypic response in contrast to intact littermates and because CB16-KN mice are nonpermissive towards CB8-K lymphocytes, differing in VH genes only, we suggest that the "isogeneic barrier" depends on a mechanism recognizing VH structures.

Poliovirus can enter and infect mammalian cells by way of an intercellular adhesion molecule 1 pathway.

Mouse fibroblast cell lines were transfected with truncated forms of the human poliovirus receptor (PVR) cDNA and tested for the expression of functional receptors for poliovirus. Several receptor constructs, all containing the coding region of the first 143 amino acids of PVR, were able to render mouse cells susceptible to poliovirus infection. A deletion of 65 amino acids in the first extracellular domain of PVR prevented virus attachment and infection. These data suggest that domain 1 is necessary and sufficient for the virus-receptor interaction. A PVR/intercellular adhesion molecule 1 hybrid receptor, expressing the PVR variable domain on a truncated receptor molecule for human rhinovirus 14, was shown to be a functional receptor for poliovirus. This observation indicates that, subsequent to attachment to the PVR-binding domain, poliovirus can use the same pathway as the major receptor group rhinoviruses to enter cells.

A chimeric poliovirus/CD4 receptor confers susceptibility to poliovirus on mouse cells.

The human poliovirus receptor consists of three extracellular immunoglobulinlike domains, a transmembrane domain, and an intracytoplasmic domain. The amino-terminal variable-type domain (V domain) of the human poliovirus receptor is necessary and sufficient for its function as a viral receptor (H.-C. Selinka, A. Zibert, and E. Wimmer, Proc. Natl. Acad. Sci. USA 88:3598-3602, 1991). In this paper, data are presented showing that transfer of the putative poliovirus receptor-binding domain to a truncated receptor for the human immunodeficiency virus results in a functional receptor for poliovirus. After expression in mouse cells, this chimeric protein confers susceptibility to poliovirus. Thus, unlike human immunodeficiency virus, poliovirus can enter mouse cells by way of a truncated CD4 receptor if the specific binding domain for poliovirus is provided.

Detection of human papillomavirus type 16 E6/E7 oncogene transcripts in dysplastic and nondysplastic cervical scrapes by nested RT-PCR.

Infections with high-risk human papillomaviruses (e.g., HPV-16) play an important role in the development of cervical intraepithelial neoplasia (CIN) and invasive cervical cancer (IC). Continued expression of the viral E6 and E7 genes is believed to be a key factor for oncogenic transformation of infected cells. Two spliced transcripts of the E6/E7 oncogenes, termed E6*I and E6*II, can be detected by reverse transcriptase polymerase chain reaction (RT-PCR). To increase the sensitivity of detecting E6/E7 transcripts in cervical scrapes we took advantage of a nested RT-PCR (nRT-PCR) protocol. In a series of 30 HPV-16-positive patients with histologic diagnoses ranging from nondysplastic epithelium to IC, the application of nRT-PCR significantly improved the detection of E6/E7 transcripts compared to conventional RT-PCR. The prevalence of E6/E7 spliced transcripts correlated with lesion severity and the nRT-PCR protocol allowed detection of these transcripts even in nondysplastic epithelium and CIN I lesions. Therefore, in epidemiologic follow-up studies, detection of E6/E7 transcripts by nRT-PCR should prove to be a useful diagnostic tool for risk evaluations regarding the development of CIN and its progression to cervical cancer, especially in high-risk HPV-type-infected patients with CIN 0 and CIN I.

Coxsackie B virus and its interaction with permissive host cells.

BACKGROUND: Observations in humans and the results of experiments on laboratory animals have provided evidence that coxsackieviruses of group B (CVB) are major etiologic agents of acute and chronic enterovirus myocarditis and various other virus-induced diseases. OBJECTIVE: This minireview briefly summarizes the investigations to elucidate various molecular mechanisms for the induction and maintenance of persistent CVB infections. With regard to the recent findings that CVB may use several different receptor proteins, this article focuses on virus-host cell interactions and the potential impact of these interactions for enteroviral replication. STUDY DESIGN: The interaction of CVB with specific cell surface proteins was analyzed in cultured cell lines and murine tissues at the level of virus attachment and virus internalization. As example for the interaction of CVB with intracellular proteins, the state of p21rasGTPase-activating protein (RasGAP) was investigated in mock-infected and CVB3-infected HeLa cells. RESULTS AND CONCLUSIONS: The experiments to elucidate the virus receptor interactions revealed the necessity to differentiate between CVB attachment proteins and proteins involved in virus internalization. Since more than one protein may be required to initiate the uptake of CVB into permissive host cells, a model of the putative interaction of these proteins within a multimeric receptor complex is proposed. It is further tempting to speculate that the presence of multiple attachment proteins may influence the tissue tropism of CVB as well as pathogenicity.

Vaccinia virus-mediated expression and identification of the human poliovirus receptor.

We have used cDNA encoding the cellular receptor for poliovirus (PVR) to prepare polyclonal antisera against beta-galactosidase PVR fusion proteins. One of these antisera allowed identification of a glycoprotein doublet band of about 67 kDa in membrane preparations of HeLa cells and in a PVR cosmid-bearing mouse cell line. In vitro translation of PVR-specific transcripts gave rise to a protein of 46 kDa; the product had a molecular weight of 67 kDa when microsomal membranes were added to the cell-free extract. Overexpression of PVR cDNA in mouse L-cells by means of a recombinant vaccinia virus led to the synthesis of a glycoprotein having a molecular weight identical to that of the glycosylated in vitro product. The vaccinia virus-mediated protein was also recognized by a monoclonal antibody that blocks poliovirus infection. Its biological activity was demonstrated by poliovirus binding and infectivity assays. The data show that PVR is a glycoprotein of 67 kDa and that this protein is sufficient to confer poliovirus susceptibility to mouse cells.

Visualization of enteroviral replication in myocardial tissue by ultrastructural in situ hybridization: identification of target cells and cytopathic effects.

In humans as well as in various murine models, enteroviruses are capable of inducing a severe acute and chronic myocarditis, which is characterized by myocytotoxic alterations and interstitial mononuclear infiltrates. With regard to the pathogenesis of enteroviral myocarditis, coxsackievirus B3 (CVB3)-infected immunocompetent A.CA/SnJ (H-2f) mice were used as a model to trace viral plus- and minus-strand RNA during acute and chronic organ infection by ultrastructural in situ hybridization techniques. For electron microscopic detection of enteroviral RNA in myocardial tissue, a pre-embedding hybridization technique was developed and optimized for excellent conservation of structural integrity and RNA retention. Herein, we demonstrate how the virus gains access to the myocardium during viremia involving infection of the capillary endothelial cells. In myocytes, viral replication was found to be closely associated with the generation of vesicular regions and lysis of myofibrils, resulting in complete destruction of the internal architecture of the cell. In the course of acute infection, the direct cell-to-cell spread of the virus from one myocyte to the other was found to be related with filaments of the cytoskeleton. The observation of prominent cytopathic alterations in close spatial association with viral replication before the development of the reactive cellular immune response strongly implies that the loss of host cell integrity is a direct consequence of acute viral replication. In addition to myocytes, non-heart muscle cells were found to be infected during acute as well as chronic disease. Viral replication observed in myocardial fibroblasts and immune cells such as B lymphocytes proved to be associated with minor cytopathic effects. The technique of electron microscopic in situ hybridization established for the detection of viral RNA within myocardial tissue provides a powerful tool for the elucidation of molecular and structural interrelationships in organ pathology.

Human papillomavirus type 16-associated primary squamous cell carcinoma of the rectum.

Primary squamous cell carcinoma (SCC) of the colorectum is an extremely rare malignancy of unknown etiology and pathogenesis. We describe an 87-year-old man with primary SCC of the rectum. Routine histology demonstrated a squamous metaplasia-dysplasia sequence of the rectal mucosa with subsequent malignant transformation. Molecular biologic analysis using polymerase chain reaction (PCR) and in situ hybridization revealed the presence of human papillomavirus type 16 (HPV-16) DNA within metaplastic, dysplastic, and SCC lesions and in tumor-free rectal mucosa. Moreover, nested reverse-transcription PCR showed transcriptional activity of the viral E6/E7 oncogenes in tumor tissue and tumor-free rectal mucosa. By contrast, 4 typical adenocarcinomas of the rectum and their adjacent normal mucosa were found to be negative for HPV by nested PCR. In line with the well-established concept of HPV-associated anogenital carcinogenesis, our results strongly suggest an etiologic role of HPV-16 in the pathogenesis of the metaplasia-dysplasia-SCC sequence in the case described.

Molecular pathology and structural features of enteroviral replication. Toward understanding the pathogenesis of viral heart disease.

Enteroviruses of the Picornaviridae and primarily coxsackieviruses of group B (CVB) can be detected in humans and various experimental murine models of acute myocarditis and chronic heart muscle diseases indicating enterovirus persistence in the myocardium. Persistent myocardial infection is characterized by restricted viral replication and gene expression in myocytes capable of sustaining chronic inflammation. Viral cytotoxicity was found to be crucial for organ pathology both during acute and persistent infection. In-situ hybridization experiments at the cellular and subcellular level have demonstrated that virus replication is associated with severe structural changes of the cardiomyocyte cytoarchitecture at any stage of the disease. In tissue culture experiments and transgenic mice, it was shown that restricted replication and gene expression of the virus are capable of inducing myocytopathic effects. Investigations at the molecular level revealed that interference of coxsackievirus replication with the cellular metabolism is mediated by cleavage of host cell proteins by virus-encoded proteinases. Notably, there is also evidence that enteroviruses are able to activate specific cellular signal transduction pathways in the course of infection, thus promoting enteroviral replication. In summary, these data indicate that mutual influences of virus replication and subsequent modifications of the host cell metabolism are crucial for cardiac injury and dysfunction during acute and chronic disease.

DNA-induced structural changes in the papillomavirus capsid.

Human papillomavirus capsid assembly requires intercapsomeric disulfide bonds between molecules of the major capsid protein L1. Virions isolated from naturally occurring lesions have a higher degree of cross-linking than virus-like particles (VLPs), which have been generated in eukaryotic expression systems. Here we show that DNA encapsidation into VLPs leads to increased cross-linking between L1 molecules comparable to that seen in virions. A higher trypsin resistance, indicating a tighter association of capsomeres through DNA interaction, accompanies this structural change.