Identification of enteroviruses by hemagglutination-inhibition

Kern, Jerome (Pacific Research Section, Honolulu, Hawaii), and Leon Rosen. Identification of enteroviruses by hemagglutination-inhibition. J. Bacteriol. 91:1936-1942. 1966.-Approximately 40% of a group of 906 enterovirus isolates cytopathic for monkey cell cultures were found to possess hemagglutinins for human erythrocytes when tested at temperatures of 4 and 37 C and at pH 5.8 and 7.3. The hemagglutinating isolates could be classified by relatively simple techniques into 18 serotypes. Four of these serotypes, echovirus type 24 and coxsackievirus B types 1, 5, and 6, had not previously been known to include hemagglutinating strains. One serotype, Toluca-3, represented a previously unrecognized enterovirus, and two other serotypes may also represent previously unrecognized enteroviruses.     

Infections du système nerveux central à entérovirus: 223 cas vus à un h?pital pédiatrique entre 1973 et 1981.

Between 1973 and 1981, 223 patients were seen at hôpital Sainte-Justine in Montreal for enteroviral infection of the nervous system. In 161 the cause was documented by isolation of an enterovirus from the cerebrospinal fluid (CSF). The viruses most frequently isolated were echovirus 11 (36 isolates), echovirus 30 (24), coxsackievirus B2 (23), coxsackievirus B3 (19), echovirus 6 (18), coxsackievirus B5 (16), coxsackievirus A9 (15), echovirus 9 (13), echovirus 7 (12) and coxsackievirus B1 (11). Aseptic meningitis was diagnosed in 200 cases and encephalitis in 12. The remaining 11 patients presented with the features of septicemia or with convulsions. In 33 patients an enterovirus was isolated from the CSF in the absence of pleocytosis. Polymorphonuclear cell predominance was noted in the initial CSF sample in 95 cases; it was persistent in 11. There were five mixed infections; in three cases two viruses were isolated from the same CSF sample. Two patients died: one, a child with hypogammaglobulinemia, had fatal polioencephalitis; the other, a 6-month-old infant brought to the emergency room in unexplained cardiopulmonary arrest, had echovirus 6 meningitis. Of the 172 patients admitted to hospital 96 received parenteral antibiotic therapy. The impact of enteroviral infections of the central nervous system on hospital resources could be substantially reduced if a rapid, sensitive and specific laboratory method of diagnosing these infections were available.     

Susceptibility of human bone marrow cells and hematopoietic cell lines to coxsackievirus B3 infection.

Viremia is commonly observed in association with enterovirus infections, and during this phase viruses can be transmitted to secondary target organs in the body. It is not known, however, whether blood cells play a role in the pathogenesis of enterovirus infection supporting virus replication. Our earlier work (T. Vuorinen, R. Vainionpää, H. Kettinen, and T. Hyypiä, Blood 84:823-829, 1994) demonstrated that coxsackievirus B3 is able to replicate in representatives of B- and T-cell lines but not in a monocytic cell line or peripheral blood mononuclear cells, indicating that virus replication may depend on the differentiation and maturation stages of the cells. Therefore, we have broaden our studies and analyzed the susceptibility of granulocyte-macrophage CFU and hematopoietic cell lines with various differentiation and maturation stages to coxsackievirus B3 infection. Virus replication was detected in B- and T-cell lines with no direct correlation to the maturation stage. Granulocyte-macrophage CFU were also able to support virus multiplication.     

Use of monoclonal antibody blends in the identification of enteroviral aseptic meningitis

Monoclonal antibody pools directed against group B coxsackievirus and echovirus antigens (Chemicon, Temecula, California) were evaluated as tools in the identification of enteroviral aseptic meningitis. Cerebrospinal fluid (CSF) and serial dilutions of stock coxsackievirus were inoculated into tube and shell vial Rhesus monkey kidney (RhMk) cell cultures. Positive cellular fluorescence was observed only in conjunction with cytopathic effect (CPE). The time from inoculation to CPE was similar with both tube and shell vial cultures.Direct CSF testing failed to reliably identify positive specimens as fluorescent debris, and a lack of available cells hindered results.Viral components of each antibody blend demonstrated positive cellular fluorescence when appropriately stained. False-positive fluorescence was not observed when cells, infected with other CSF viruses, were stained with these reagents.The findings suggest a role for these reagents (available both as blends and type-specific reagents) in the culture confirmation and identification of many common enteroviral serotypes associated with aseptic meningitis.     

Specific cell-surface alteration by enteroviruses as reflected by viral-attachment interference

Crowell, Richard L. (Hahnemann Medical College, Philadelphia, Pa.). Specific cell-surface alteration by enteroviruses as reflected by viral-attachment interference. J. Bacteriol. 91:198-204. 1966.-Exposure of HeLa cells to high levels of coxsackievirus B3 produced cells which were refractory to attachment of coxsackievirus B1, whereas poliovirus T2 attached normally. Under similar conditions, poliovirus T2 was found to interfere with the attachment of poliovirus T1 to HeLa cells without affecting the attachment rate of coxsackievirus B3. The data confirm earlier findings that the receptor sites on HeLa cells, which bind members of group B coxsackieviruses, are distinct from those for polioviruses. Quantitatively, coxsackieviruses B1 and B3 were found to be mutually exclusive in the attachment interference assay to suggest that they compete for the same receptors on the HeLa cell surface. The finding that input multiplicities of B3 virus which exceeded 500 saturated the homologous viral receptors of HeLa cells was unexpected, but was consistent with the results of interference assays. Excessive amounts of input virus did not, however, inhibit eclipse of homologous cell-associated virus. Attachment interference between enteroviruses occurred even though the interfering virus was eclipsed prior to addition of challenge virus. The finding that enterovirus attachment interference was reversible with acid pH suggested that attachment and eclipse of enterovirus does not result in a permanent alteration of the cell membrane and that these events occur at the cell surface.     

Enterovirus Recovery with Vegetable Floc

A lettuce floc was prepared and used for recovering enterovirus from an aqueous suspension. The method is simple, and the adsorption of coxsackievirus B5, echovirus 7, and poliovirus 1 is quantitative. The virus-floc complex may be removed from aqueous suspension by low-speed centrifugation and dissolved at an alkaline pH in a small volume of water; virus is then available for assay on cultured cells. Flocs from some other green vegetables also possess the property of virus adsorption     

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.     

Echovirus 22 is an atypical enterovirus

Although echovirus 22 (EV22) is classified as an enterovirus in the family Picornaviridae, it is atypical of the enterovirus paradigm, typified by the polioviruses and the coxsackie B viruses. cDNA reverse transcribed from coxsackievirus B3 (CVB3) RNA does not hybridize to genomic RNA of EV22, and conversely, cDNA made to EV22 does not hybridize to CVB3 genomic RNA or to molecular clones of CVB3 or poliovirus type 1. EV22 cDNA does not hybridize to viral RNA of encephalomyocarditis virus or to a molecular clone of Theiler's murine encephalomyelitis virus, members of the cardiovirus genus. The genomic RNA of EV22 cannot be detected by the polymerase chain reaction using generic enteroviral primers. EV22 does not shut off host cell protein synthesis, and the RNA of EV22 is efficiently translated in vitro in rabbit reticulocyte lysates. Murine enterovirus-immune T cells recognize and proliferate against EV22 as an antigen in vitro, demonstrating that EV22 shares an epitope(s) common to enteroviruses but not found among other picornaviruses.     

Elimination of some enteroviruses in the excrements of experimentally infected rats (Rattus norvegicus) and gulls (Larus ridibundus)

Young rats of both sexes, weight 150-170 g, the first laboratory progeny of captured wild parent pairs, were used throughout this experiment. Rats in two experimental groups comprising a total of 34 animals were infected orally with type 2 poliovirus vaccine strain given in each group at doses of 500, 5000 or 50,000 TCD50. In the first experiment, the presence of poliovirus in rat excrements was detectable irregularly till day 13, in the second experiment till day 2 after infection. Small quantities of virus were also detectable from the colon and cecum wall, exceptionally from the mesenteric lymph node. The third experiment included 8 rats orally infected with 5,000 TCD50 of echovirus 30; at the lower dose of virus all excrement samples were culture-negative, at the higher dose the positive virus recovery was recorded in 3 animals one day after infection. Analogous experiments in the fifth group of rats orally infected with 5,000 TCD50 or 50,000 TCD50 of enterovirus 71 yielded much the same results; organs of further 6 animals infected intranasally with 5,000 TCD50 of this virus were culture-negative and no virus-related changes could be histologically demonstrated in these animals. The second part of this study included the experiments conducted on 17 young Larus gulls bred in the laboratory from eggs collected in a colony of free living birds. Groups of these gulls were orally infected with 500 or 5,000 TCD50 of one of the following viruses: type 1 poliovirus vaccine strain, type 3 poliovirus vaccine strain, echovirus 30, enterovirus 71 and coxsackievirus B4. All samples of gull excrements collected till day 7 or 20 postinfection were culture-negative. These results suggest that wild rats may play some role in the spread of human enteroviruses in the environment, but no such role could be demonstrated in the Larus gull.     

Cleavage of RasGAP and phosphorylation of mitogen-activated protein kinase in the course of coxsackievirus B3 replication

Recently, we reported on tyrosine phosphorylation of distinct cellular proteins in the course of enterovirus infections (M. Huber, H.-C. Selinka, and R. Kandolf, J. Virol. 71:595-600, 1997). These phosphorylation events were mediated by Src-like kinases and were shown to be necessary for effective virus replication. That study is now extended by examination of the interaction of the adapter protein Sam68, a cellular target of Src-like kinases which has been shown to interact with the poliovirus 3D polypeptide, with cellular signaling proteins as well as the function of the latter during infection. Here, we report that the RNA-binding and protein-binding protein Sam68 associates with the p21(ras) GTPase-activating protein RasGAP. Remarkably, RasGAP is cleaved during infections with different strains of coxsackievirus B3 as well as with echovirus 11 and echovirus 12, yielding a 104-kDa protein fragment. This cleavage event, which cannot be prevented by the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, may promote the activation of the Ras pathway, as shown by the activating dual phosphorylation of the mitogen-activated protein kinases Erk-1 and Erk-2 in the late phase of infection. Moreover, downstream targets of the mitogen-activated protein kinases, i.e., the p21(ras) exchange factor Sos-1 and cytoplasmic phospholipase A2, are phosphorylated with parallel time courses during infection. Activation or inhibition of cellular signaling pathways may play a general role in regulating effective enterovirus replication and pathogenesis, and the results of this study begin to unravel the molecular cross talk between enterovirus infection and key cellular signaling networks.     

Murine cell-mediated immune response recognizes an enterovirus group-specific antigen(s).

Splenocytes taken from mice inoculated with coxsackievirus B3 (CVB3) (Nancy) developed an in vitro proliferative response against CVB3 antigen. This response could not be detected earlier than 8 days postinoculation but could be detected up to 28 days after exposure to CB3. CVB3-sensitized splenocytes responded not only to the CVB3 antigen but to other enteroviruses as well. This response was found to be enterovirus specific in that no response was detected to a non-enteroviral picornavirus, encephalomyocarditis virus, or to an unrelated influenza virus. The generation of a splenocyte population capable of responding to an enterovirus group antigen(s) was not limited to inoculation of mice with CVB3, as similar responses were generated when mice were inoculated with CVB2. Cell subset depletions revealed that the major cell type responding to the enterovirus group antigen(s) was the CD4+ T cell. Current evidence suggests that the group antigen(s) resides in the structural proteins of the virus, since spleen cells from mice inoculated with a UV-inactivated, highly purified preparation of CVB3 virions also responded in vitro against enteroviral antigens.     

Pathogenesis of murine enterovirus myocarditis: virus dissemination and immune cell targets.

In order to identify organ and cellular targets of persistent enterovirus infection in vivo, immunocompetent mice (SWR/J, H-2q) were inoculated intraperitoneally with coxsackievirus B3 (CVB3). By use of in situ hybridization for the detection of enteroviral RNA, we show that CVB3 is capable of inducing a multiorgan disease. During acute infection, viral RNA was visualized at high levels in the heart muscle, pancreas, spleen, and lymph nodes and at comparably low levels in the central nervous system, thymus, lung, and liver. At later stages of the disease, the presence of enteroviral RNA was found to be restricted to the myocardium, spleen, and lymph nodes. To characterize infected lymphoid cells during the course of the disease, enteroviral RNA and cell-specific surface antigens were visualized simultaneously in situ in spleen tissue sections. In acute infection, the majority of infected spleen cells, which are located primarily at the periphery of lymph follicles, were found to express the CD45R/B220+ phenotype of pre-B and B cells. Whereas viral RNA was also detected in certain CD4+ helper T cells and Mac-1+ macrophages, no enteroviral genomes were identified in CD8+ cytotoxic/suppressor T cells. Later in disease, the localization of enteroviral RNA revealed a persistent type of infection of B cells within the germinal centers of secondary follicles. In addition, detection of the replicative viral minus-strand RNA intermediate provided evidence for virus replication in lymphoid cells of the spleen during the course of the disease. These data indicate that immune cells are important targets of CVB3 infection, providing a noncardiac reservoir for viral RNA during acute and persistent myocardial enterovirus infection.     

Enhancement of enterovirus infectivity in vitro by pretreating host cell monolayers with the cationic polymer polyethyleneimine

Laboratory strains of enteroviruses, as well as viruses isolated from raw wastewater, were found to exhibit enhanced infectivity in vitro when BGM cell monolayers were pretreated with the cationic polymer polyethyleneimine (PEI). Viruses were assayed by the cytopathic effect technique and as PFU under methylcellulose and agar overlays with monolayers treated with 0 to 5.0 x 10(-3)% (wt/vol) PEI in phosphate-buffered saline supplemented with 2% fetal bovine serum. Poliovirus type 1 cytopathic effect occurred at an enhanced rate in cells treated with 5.0 x 10(-3)% PEI compared with untreated cells. PEI-treated cells were found to adsorb viruses much more effectively than untreated cells did. When the methylcellulose overlay procedure was used, rates of infectivity were enhanced as follows: poliovirus type 1, 5.5-fold; echovirus type 1, 1.2-fold; echovirus type 5, 5.2-fold; and coxsackievirus type B5, 4.9-fold. Viruses concentrated from raw wastewater showed a 3.8-fold increase in titer when quantitated by the most-probable-number method and a 3.3-fold increase when quantitated as PFU under an agar overlay.     

Enhancement of enterovirus infectivity in vitro by pretreating host cell monolayers with the cationic polymer polyethyleneimine.

Laboratory strains of enteroviruses, as well as viruses isolated from raw wastewater, were found to exhibit enhanced infectivity in vitro when BGM cell monolayers were pretreated with the cationic polymer polyethyleneimine (PEI). Viruses were assayed by the cytopathic effect technique and as PFU under methylcellulose and agar overlays with monolayers treated with 0 to 5.0 x 10(-3)% (wt/vol) PEI in phosphate-buffered saline supplemented with 2% fetal bovine serum. Poliovirus type 1 cytopathic effect occurred at an enhanced rate in cells treated with 5.0 x 10(-3)% PEI compared with untreated cells. PEI-treated cells were found to adsorb viruses much more effectively than untreated cells did. When the methylcellulose overlay procedure was used, rates of infectivity were enhanced as follows: poliovirus type 1, 5.5-fold; echovirus type 1, 1.2-fold; echovirus type 5, 5.2-fold; and coxsackievirus type B5, 4.9-fold. Viruses concentrated from raw wastewater showed a 3.8-fold increase in titer when quantitated by the most-probable-number method and a 3.3-fold increase when quantitated as PFU under an agar overlay.     

The enter of viruses family Picornaviridae in residents macrophages

Viruses enter in cells through clathrin- and dinamin-mediated uptake route-endocytosis, caveolae-mediated local destruction of cell plasma membranes, and macropinocytosis. The non-enveloped viruses to which Picornaviridae famiy is attributed are important human and animal pathogens. The aim of this study was to examine the mechanisms of penetration of viruses of this family (polio-, echo 11-, entero 71- and coxsackie B1-viruses) into resident macrophages. After attachment to the plasma membrane of macrophages the enterovirus 71 and coxsackievirus B1 penetrated into macrophages by invagination of the plasma membrane and formation of intracytoplasmic vesicules - caveoles. The poliovirus entered macrophages both by caveols formation and local destruction of plasma membranes of the host cells. Macropinocytos of polioviruses was observed after 45 min contact. The echovirus 11 entered in host macrophages by local destruction of their plasma membranes during first 15 min. Then the formation of endocytosed vesicles with included viruses was observed. The echovirus 11 went out of endocytosed vesicles by local destruction of membrane vesicles.     

Defectiveness of Interferon Production and of Rubella Virus Interference in a Line of African Green Monkey Kidney Cells (Vero)

Vero cells, a line of African green monkey kidney cells, failed to produce interferon when infected with Newcastle disease, Sendai, Sindbis, and rubella viruses, although the cells were sensitive to interferon. Further, infection of Vero cells with rubella virus did not result in interference with the replication of echovirus 11, Newcastle disease virus, or vesicular stomatitis virus, even in cultures where virtually every cell was infected with rubella virus. Under the same conditions, BSC-1 cells and other cells of primate origin produced interferon and showed rubella virus interference. The results indicate that the presence of rubella virus in the cell does not in itself exclude multiplication of other viruses and that rubella virus interference appears to be linked to the capability of the cell to produce interferon.     

Biological significance of a human enterovirus B-specific RNA element in the 3' nontranslated region

The secondary structures predicted for the enteroviral 3' nontranslated region (3'NTR) all seem to indicate a conformation consisting of two (X and Y) hairpin structures. The higher-order RNA structure of the 3'NTR appears to exist as an intramolecular kissing interaction between the loops of these two hairpin structures. The enterovirus B-like subgroup possesses an additional stem-loop structure, domain Z, which is not present in the poliovirus-like enteroviruses. It has been suggested that the Z domain originated from a burst of short sequence repetitions (E. V. Pilipenko, S. V. Maslova, A. N. Sinyakov, and V. I. Agol, Nucleic Acids Res. 20:1739-1745, 1992). However, no functional features have yet been ascribed to this enterovirus B-like-specific RNA element in the 3'NTR. In this study, we tested the functional characteristics and biological significance of domain Z. A mutant of the cardiovirulent coxsackievirus group B3 strain Nancy which completely lacked the Z domain and which therefore acquired enterovirus C-like secondary structures exhibited a wild-type growth phenotype, as determined by single-cycle growth analysis with BGM cells. This result proves that the Z domain is virtually dispensable for viral growth in tissue cultures. Partial distortion of the Z domain structure resulted in a disabled virus with reduced growth kinetics, probably due to alternative conformations of the overall structure of the domain. Infection of mice showed that the recombinant coxsackievirus group B3 mutant which completely lacked the Z domain was less virulent. Pancreatic tissues from mice infected with wild-type virus and recombinant virus were equally affected. However, the heart tissue from mice infected with the recombinant virus showed only slight signs of myocarditis. These results suggest that the enterovirus B-like-specific Z domain plays a role in coxsackievirus-induced pathogenesis.     

Simultaneous concentration of four enteroviruses from tap, waste, and natural waters.

The efficiency of virus recovery from water was investigated by using a method which enabled the concentration of a mixture of four enteroviruses with determination of their individual recovery efficiencies. The four viruses used (poliovirus 1, coxsackievirus A9, coxsackievirus B1, and echovirus 7) represented each of the four major subgroups of enteroviruses. This method, which was based on selective antibody neutralization, was used to investigate the effects of input water quality on enterovirus concentration by Balston filters (grade C; Balston, Inc., Lexington, Mass.) and organic flocculation. With tap water, the average recovery efficiency of the four viruses was 97%. Concentration from natural waters, including samples from two lakes (Lake Kinneret and the Hula Nature Reserve) and the Mediterranean Sea, resulted in similarly high average recovery efficiencies. Echovirus 7 was recovered with a slightly lower average efficiency from these types of water than were the other viruses. In comparison with other types of water, virus concentration from Jerusalem wastewater generally had a slightly lower efficiency of recovery, ranging from 63 to 75% for each of the viruses, with an overall average of 68%. The ability of each concentration step, membrane filtration or organic flocculation, to recover the viruses from water was assayed. For the filtration step, although there were not large differences in virus recoveries from tap water, echovirus 7 was recovered with the lowest efficiency (72%), and poliovirus 1 was recovered with the highest (87%) efficiency. Overall virus recovery by the filtration step was least efficient for wastewater (73%) and most efficient for seawater (107%).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Simultaneous concentration of four enteroviruses from tap, waste, and natural waters

The efficiency of virus recovery from water was investigated by using a method which enabled the concentration of a mixture of four enteroviruses with determination of their individual recovery efficiencies. The four viruses used (poliovirus 1, coxsackievirus A9, coxsackievirus B1, and echovirus 7) represented each of the four major subgroups of enteroviruses. This method, which was based on selective antibody neutralization, was used to investigate the effects of input water quality on enterovirus concentration by Balston filters (grade C; Balston, Inc., Lexington, Mass.) and organic flocculation. With tap water, the average recovery efficiency of the four viruses was 97%. Concentration from natural waters, including samples from two lakes (Lake Kinneret and the Hula Nature Reserve) and the Mediterranean Sea, resulted in similarly high average recovery efficiencies. Echovirus 7 was recovered with a slightly lower average efficiency from these types of water than were the other viruses. In comparison with other types of water, virus concentration from Jerusalem wastewater generally had a slightly lower efficiency of recovery, ranging from 63 to 75% for each of the viruses, with an overall average of 68%. The ability of each concentration step, membrane filtration or organic flocculation, to recover the viruses from water was assayed. For the filtration step, although there were not large differences in virus recoveries from tap water, echovirus 7 was recovered with the lowest efficiency (72%), and poliovirus 1 was recovered with the highest (87%) efficiency. Overall virus recovery by the filtration step was least efficient for wastewater (73%) and most efficient for seawater (107%).(ABSTRACT TRUNCATED AT 250 WORDS)