Molecular detection and characterization of human enteroviruses in Korean surface water

In this study, the genetic epidemiology of enteroviruses (EVs) in Korean surface water was evaluated by conducting phylogenetic analyses of the nucleotide sequences of the 5' non-coding region (5' NCR), which was determined by RT-PCR analysis of total culturable virus assay-positive samples. The results showed that the nucleotide sequences of the EVs could be classified into 4 genetic clusters, and that the predominant presence of Korea EVs were very similar to echoviruses type 30. Interestingly, two nucleotide sequences were very similar to those of coxsackievirus type B1 isolated from aseptic meningitis patients in Seoul, Korea, implying the possibility of a common source for the viruses circulated in water systems and humans. In addition, 3 nucleotide sequences clustered strongly with the nucleotide sequences from China or Japan, and one fell into the same cluster as echovirus type 11 from Taiwan, which suggests that EVs in Asia may have evolved in a region-specific manner. Taken together, the results of this study revealed that EVs from Korea surface waters could be genetically classified as coxsackieviruses or echoviruses, and that they evolved in Asia in a region-specific manner.     

Infection by echoviruses 1 and 8 depends on the alpha 2 subunit of human VLA-2.

Anti-VLA-2 antibodies protected HeLa cells from infection by echoviruses 1 and 8 but not from infection by other echovirus serotypes. Echoviruses 1 and 8 bound to and infected nonpermissive hamster cells transfected with the alpha 2 subunit of human VLA-2. These results indicate that the human alpha 2 subunit is critical for infection by echoviruses 1 and 8 but that other echovirus serotypes must bind receptors other than VLA-2.     

Sequence analysis of the downstream 5' nontranslated region of seven echoviruses with different neurovirulence phenotypes.

The downstream 5' nontranslated regions of seven echoviruses with different neurovirulent phenotypes were amplified and sequenced. Neurovirulent echovirus serotypes 4, 6, 9, 11, and 30 were identical to the putative poliovirus in 18S rRNA binding sequence and the flanking conserved sequences. Less neurovirulent echoviruses, serotypes 2 and 12, exhibited variations within these regions.     

Optimal conditions for plaque assay of echoviruses in human embryonic lung fibroblast cells

The present paper gives optimal conditions for plaque assays tn human embryonic lung fibroblast cells with 45 echoviruses including 11 fresh isolates, covering 31 serotypes. All the viruses tested except echovirus type 23 produced plaques at a high titer in the same cells by the methods described in this communication. Plaque formations by a number of strains were markedly enhanced in size or number or both by the addition of polycation.     

Inactivation of murine norovirus, feline calicivirus and echovirus 12 as surrogates for human norovirus (NoV) and coliphage (F+) MS2 by ultraviolet light (254 nm) and the effect of cell association on UV inactivation

Aims: To determine inactivation profiles of three human norovirus (NoV) surrogate viruses and coliphage MS2 by ultraviolet (UV) irradiation and the protective effect of cell association on UV inactivation. Methods and Results: The inactivation rate for cell‐free virus or intracellular echovirus 12 was determined by exposure to 254‐nm UV light at fluence up to 100 mJ cm^?2. The infectivity of murine norovirus (MNV), feline calicivirus (FCV) and echovirus 12 was determined by cell culture infectivity in susceptible host cell lines, and MS2 infectivity was plaque assayed on Escherichia coli host cells. The UV fluencies to achieve 4‐log₁₀ inactivation were 25, 29, 30 and 70 (mJ cm^?2) for cell‐free FCV, MNV, echovirus 12 and MS2, respectively. However, a UV fluence of 85 mJ cm^?2 was needed to inactivate intracellular echovirus 12 by 4 log₁₀. Conclusions: Murine norovirus and echoviruses 12 are more conservative surrogates than FCV to predict the UV inactivation response of human NoV. Intracellular echovirus 12 was 2·8‐fold more resistant to UV irradiation than cell‐free one. Significance and Impact of the Study: Variation in UV susceptibilities among NoV surrogate viruses and a likely protective effect of cell association on virus susceptibility to UV irradiation should be considered for effective control of human NoV in water.     

Poliovirus and echovirus survival in Tetrahymena pyriformis culture in vivo

Axenic cultures of Tetrahymena pyriformis, strain I MT IV, grown in a defined medium at room temperature, were used to study interactions of these protozoa with vaccination strain L Sc 2ab of poliovirus type 1, vaccination strain P 712 of poliovirus type 2 and with type 30 echovirus, strain 480/78. T. pyriformis cultures in media containing 10(3.0) TCD50/1 ml of type poliovirus, 10(3.0) TCD50/1 ml of type 2 poliovirus or 10(2.5) TCD50/1 ml echovirus 30 and in virus-free medium did not differ one from another in their growth and die-away kinetics during the 21 days of observation. Two-day T. pyriformis cultures were infected with poliovirus 1 (initial concentration 10(3.2) TCD50/1 ml), and poliovirus 2 and echovirus 30 (initial concentrations 10(3.0) TCD50/1 ml). Viruses were titrated in test tube cultures of BGM cells. The supernatant fluid, standardized sediment and samples of control virus suspension free of protozoa were titrated after 0, 2, 6, 10, 13, 18, 28 and 30 days. Most of the virus in culture was found associated with the sediment, both in the period of active growth and during the die-away phase of T. pyriformis protozoa. The virus in sediment was present at higher titres and its survival time was longer than in virus in liquid phase. Thirteen days after the first contact between T. pyriformis and virus the sediment and supernatant fluid of the old protozoan culture and the T. pyriformis-free control viral suspension were taken and used as inocula for new two-day T. pyriformis cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Survival of enteroviruses in rapid-infiltration basins during the land application of wastewater

The downward migration through soil of seeded poliovirus type 1 and echovirus type 1 and of naturally occurring enteroviruses during infiltration of sewage effluent through rapid-infiltration basins was investigated. After 5 days of flooding, the amount of seeded poliovirus type 1 that had migrated 5 to 10 cm downward through the soil profile was found to be 11% of that remaining at the initial burial depth. The amount of echovirus type 1 determined to have moved an equal distance was at least 100-fold less. Migration of naturally occurring enteroviruses during infiltration of sewage effluent through soil could not be measured with accuracy because of the possibility of virus survival from previous applications of effluent. The rate of inactivation for seeded poliovirus 1 and echovirus 1 buried in the infiltration basins ranged between 0.04 and 0.15 log10 units per day during the time when the basins were flooded. Inactivation of these same seeded virus types and of indigenous enterovirus populations in the infiltration basins during the drying portion of the sewage application cycle ranged between 0.11 and 0.52 log10 units per day. The rate of virus inactivation was dependent upon the rate of soil moisture loss. These results indicate that drying cycles during the land application of wastewater enhance virus inactivation in the soil.     

Survival of enteroviruses in rapid-infiltration basins during the land application of wastewater.

The downward migration through soil of seeded poliovirus type 1 and echovirus type 1 and of naturally occurring enteroviruses during infiltration of sewage effluent through rapid-infiltration basins was investigated. After 5 days of flooding, the amount of seeded poliovirus type 1 that had migrated 5 to 10 cm downward through the soil profile was found to be 11% of that remaining at the initial burial depth. The amount of echovirus type 1 determined to have moved an equal distance was at least 100-fold less. Migration of naturally occurring enteroviruses during infiltration of sewage effluent through soil could not be measured with accuracy because of the possibility of virus survival from previous applications of effluent. The rate of inactivation for seeded poliovirus 1 and echovirus 1 buried in the infiltration basins ranged between 0.04 and 0.15 log10 units per day during the time when the basins were flooded. Inactivation of these same seeded virus types and of indigenous enterovirus populations in the infiltration basins during the drying portion of the sewage application cycle ranged between 0.11 and 0.52 log10 units per day. The rate of virus inactivation was dependent upon the rate of soil moisture loss. These results indicate that drying cycles during the land application of wastewater enhance virus inactivation in the soil.     

Characterization of an echovirus type 30 variant isolated from patients with aseptic meningitis

An outbreak of echovirus type 30 infection associated with aseptic meningitis occurred among newborn babies in a hospital neonatal room at Fukui City in 1983. The isolated virus was identified as an antigenic variant of echovirus type 30 by cross-neutralization tests with antisera against the prototype Bastianni strain and the present isolate. Western blot analysis demonstrated that the antigenic determinants responsible for virus neutralization, some of which were type specific and others strain specific, were present on the capsid protein VP1. The current strain was more thermoresistant than the prototype, suggesting some alterations in virus structural proteins.     

Virion conformational forms and the complex inactivation kinetics of echovirus by chlorine in water

Aberrant inactivation kinetics were observed when monodispersed echovirus type 1 (Farouk) was inactivated with chlorine. An initial 1- to 2-log10-unit decrease in titer was followed by lag period, during which the titer stayed the same or increased, and this was followed by a final decline in titer. First-order kinetics were obtained with poliovirus type 1 under the same conditions. Isoelectric focusing studies of echovirus before chlorine treatment showed that the virus distributed into two pH-dependent and interconvertible isoelectric forms. After chlorine treatment all remaining virus infectivity was associated with a third pH-independent isoelectric form. The complex inactivation kinetics appeared to be due to shifts between these conformational forms during inactivation in certain ionic environments. Under certain conditions the conformational shifts resulted in substantial resistance of monodispersed echovirus to chlorine.     

Decay-accelerating factor CD55 is identified as the receptor for echovirus 7 using CELICS, a rapid immuno-focal cloning method.

Using an anti-receptor mAb that blocks the attachment of echovirus 7 and related viruses (echoviruses 13, 21, 29 and 33), we have isolated a complementary DNA clone that encodes the human decay-accelerating factor (CD55). Mouse cells transfected with the CD55 clone bind echovirus 7, and this binding is blocked by the anti-receptor mAb. The method used (CELICS) allows rapid and direct cloning of genes encoding cell surface receptors. It is based on episomal replication and high efficiency expression of complementary DNA clones in the vector pCDM8 in COS or WOP cells, in conjunction with a sensitive immuno-focal screen that uses antibody probes linked to beta-galactosidase. Receptor positive cells were identified by a colour change and isolated individually using a micromanipulator. DNA extracted from a small number of cells was then cloned directly in Escherichia coli.     

Virion conformational forms and the complex inactivation kinetics of echovirus by chlorine in water.

Aberrant inactivation kinetics were observed when monodispersed echovirus type 1 (Farouk) was inactivated with chlorine. An initial 1- to 2-log10-unit decrease in titer was followed by lag period, during which the titer stayed the same or increased, and this was followed by a final decline in titer. First-order kinetics were obtained with poliovirus type 1 under the same conditions. Isoelectric focusing studies of echovirus before chlorine treatment showed that the virus distributed into two pH-dependent and interconvertible isoelectric forms. After chlorine treatment all remaining virus infectivity was associated with a third pH-independent isoelectric form. The complex inactivation kinetics appeared to be due to shifts between these conformational forms during inactivation in certain ionic environments. Under certain conditions the conformational shifts resulted in substantial resistance of monodispersed echovirus to chlorine.     

Echovirus type 17 in the neonate

An outbreak of echovirus type 17 in a neonatal nursery with recovery of virus from the stools of the seven affected infants and the cerebrospinal fluid of one is described. Intensive nursing care and supportive therapy prevented a possible fatal outcome in all instances. A further case in another community is described in which a 5-week-old child succumbed to his illness and echovirus type 17 was isolated from autopsy tissues — lung, liver, kidney and spleen. This child had presumably received poor home care. The suggestion is made that while echovirus type 17 is not frequently associated with adult disease, it may exhibit affinity for infant tissues. This is thought to be the first time that this virus has been associated with clinical disease in a neonatal nursery and also the first account of its recovery from postmortem tissues.     

Influence of water quality on enteric virus concentration by microporous filter methods.

Four enteric viruses, poliovirus type 1, echovirus type 1, reovirus type 3, and simian adenovirus SV-11, were concentrated from seeded 1.3-liter volumes of raw, finished, and granular activated carbon-treated waters by adsorption to 47-mm-diameter (17 cm2), electropositive ( Virosorb 1MDS ) filters at pH 7.5 or electronegative ( Filterite ) filters at pH 3.5 with and without 5 mM added MgCl2, followed by elution with 0.3% beef extract in 50 mM glycine at pH 9.5. Removal of particulates from raw and finished waters by 0.2-micron prefiltration before virus addition and pH adjustment had little effect on virus concentration efficiencies. Soluble organic compounds reduced virus adsorption efficiencies from both raw and finished waters compared with granular activated carbon-treated water, but the extent of interference varied with virus type and adsorption conditions. For electropositive 1MDS filters, organic interference was similar with all virus types. For Filterite filters, organic interference was evident with poliovirus and echovirus, but could be overcome by adding MgCl2. Reovirus and SV-11 were not adversely affected by organics during adsorption to Filterite filters. Elution of reovirus and adenovirus was inefficient compared with that of poliovirus and echovirus. None of the three adsorption schemes ( 1MDS at pH 7.5 and Filterite with and without 5 mM MgCl2 at pH 3.5) could be judged superior for all viruses and water types tested.     

Influence of water quality on enteric virus concentration by microporous filter methods

Four enteric viruses, poliovirus type 1, echovirus type 1, reovirus type 3, and simian adenovirus SV-11, were concentrated from seeded 1.3-liter volumes of raw, finished, and granular activated carbon-treated waters by adsorption to 47-mm-diameter (17 cm2), electropositive ( Virosorb 1MDS ) filters at pH 7.5 or electronegative ( Filterite ) filters at pH 3.5 with and without 5 mM added MgCl2, followed by elution with 0.3% beef extract in 50 mM glycine at pH 9.5. Removal of particulates from raw and finished waters by 0.2-micron prefiltration before virus addition and pH adjustment had little effect on virus concentration efficiencies. Soluble organic compounds reduced virus adsorption efficiencies from both raw and finished waters compared with granular activated carbon-treated water, but the extent of interference varied with virus type and adsorption conditions. For electropositive 1MDS filters, organic interference was similar with all virus types. For Filterite filters, organic interference was evident with poliovirus and echovirus, but could be overcome by adding MgCl2. Reovirus and SV-11 were not adversely affected by organics during adsorption to Filterite filters. Elution of reovirus and adenovirus was inefficient compared with that of poliovirus and echovirus. None of the three adsorption schemes ( 1MDS at pH 7.5 and Filterite with and without 5 mM MgCl2 at pH 3.5) could be judged superior for all viruses and water types tested.     

Isolation of enteroviruses from water, suspended solids, and sediments from Galveston Bay: survival of poliovirus and rotavirus adsorbed to sediments.

The distribution and quantitation of enteroviruses among water, suspended solids, and compact sediments in a polluted estuary are described. Samples were collected sequentially from water, suspended solids, fluffy sediments (uppermost layer of bottom sediments), and compact sediment. A total of 103 samples were examined of which 27 (26%) were positive for virus. Polioviruses were recovered most often, followed by coxsackie B viruses and echoviruses 7 and 29. Virus was found most often attached to suspended solids: 72% of these samples were positive, whereas only 14% of water samples without solids yielded virus. Fluffy sediments yielded virus in 47% of the samples, whereas only 5% of compact bottom-sediment samples were positive. When associated with solids, poliovirus and rotavirus retained their infectious quality for 19 days. The same viruses remained infectious for only 9 days when freely suspended in seawater. Collection of suspended solids at ambient water pH appears to be very useful for the detection of virus; it has advantages over collecting and processing large volumes of water, with accompanying pH adjustment and salt addition for processing.     

Isolation of enteroviruses from water, suspended solids, and sediments from Galveston Bay: survival of poliovirus and rotavirus adsorbed to sediments

The distribution and quantitation of enteroviruses among water, suspended solids, and compact sediments in a polluted estuary are described. Samples were collected sequentially from water, suspended solids, fluffy sediments (uppermost layer of bottom sediments), and compact sediment. A total of 103 samples were examined of which 27 (26%) were positive for virus. Polioviruses were recovered most often, followed by coxsackie B viruses and echoviruses 7 and 29. Virus was found most often attached to suspended solids: 72% of these samples were positive, whereas only 14% of water samples without solids yielded virus. Fluffy sediments yielded virus in 47% of the samples, whereas only 5% of compact bottom-sediment samples were positive. When associated with solids, poliovirus and rotavirus retained their infectious quality for 19 days. The same viruses remained infectious for only 9 days when freely suspended in seawater. Collection of suspended solids at ambient water pH appears to be very useful for the detection of virus; it has advantages over collecting and processing large volumes of water, with accompanying pH adjustment and salt addition for processing.     

Comparative inactivation of enteroviruses and adenovirus 2 by UV light

The doses of UV irradiation necessary to inactivate selected enteric viruses on the U.S. Environmental Protection Agency Contaminant Candidate List were determined. Three-log reductions of echovirus 1, echovirus 11, coxsackievirus B3, coxsackievirus B5, poliovirus 1, and human adenovirus type 2 were effected by doses of 25, 20.5, 24.5, 27, 23, and 119 mW/cm(2), respectively. Human adenovirus type 2 is the most UV light-resistant enteric virus reported to date.     

Echovirus 1 replication, not only virus binding to its receptor, VLA-2, is required for the induction of cellular immediate-early genes.

Induction of immediate-early genes c-jun, junB, and c-fos was demonstrated during echovirus 1 infection in a human osteogenic sarcoma (HOS) cell line. Tenfold induction was seen at 10 h postinfection, corresponding approximately to the end of the first replication cycle of the virus. Echovirus 1 uses VLA-2 integrin as its cellular receptor, and ligand binding by integrin is known to trigger signal transduction pathways ultimately activating immediate-early genes. In the present study, however, VLA-2 binding alone was not sufficient to induce their expression; viral replication was needed. This conclusion was based on the observations that no stimulation of the immediate-early genes occurred in the MG-63 cell line where the virus attached only to VLA-2 but was not able to replicate and that induction of these genes was observed when the HOS cells were infected with echovirus type 7, known to use a different cellular receptor. Induction was not seen in the presence of the antiviral compound WIN 54954, which evidently inhibits the uncoating but not receptor binding of echovirus 1, suggesting that viral replication triggers the activation of the immediate-early genes. The induction of these genes may have a role in viral replication and in the pathogenesis of infection.     

Comparative Inactivation of Enteroviruses and Adenovirus 2 by UV Light

The doses of UV irradiation necessary to inactivate selected enteric viruses on the U.S. Environmental Protection Agency Contaminant Candidate List were determined. Three-log reductions of echovirus 1, echovirus 11, coxsackievirus B3, coxsackievirus B5, poliovirus 1, and human adenovirus type 2 were effected by doses of 25, 20.5, 24.5, 27, 23, and 119 mW/cm², respectively. Human adenovirus type 2 is the most UV light-resistant enteric virus reported to date.