Enterovirus Isolates Research Articles

Development of a PCR-based method for the detection of enteroviruses and hepatitis A virus in molluscan shellfish and its application to polluted field samples

The use of the polymerise chain reaction (PCR) for detection of low levels of enteric viruses in bivalve shellfish is hindered by the presence of potent amplification inhibitors. A procedure previously developed for removing the majority of these amplification inhibitors is applied to the detection of enteroviruses and hepatitis A virus in naturally polluted field samples. Quantification of PCR inhibition showed that PCR sample tolerance ranged from 2 to 4.7g shellfish for highly polluted samples. These results indicate the need for adequate controls for PCR inhibition, particularly for negative samples. Reverse transcription (RT)-PCR results were compared with conventional enterovirus isolation for a range of naturally contaminated shellfish. All enterovirus isolation positive samples were also positive by enterovirus RT-PCR. At one field site shellfish were positive by enterovirus RT-PCR but negative for virus isolation. All shellfish tested were negative for hepatitis A by RT-PCR. The procedure for removal of PCR amplification inhibitors should be equally applicable to the detection of Norwalk and related Small Round Structured Viruses (SRSVS) in shellfish.

Identification of enteroviruses by indirect immunofluorescence using monoclonal antibodies

Background: Serum neutralization (Nt) is used most often to type enterovirus isolates, but it is labor-intensive, expensive, and supplies of reference antisera for Nt are limited. Alternative methods of enterovirus typing are needed. Objectives: To investigate the use of indirect immunofluorescence (IFA) with commercially available monoclonal antibodies (MAbs) as an alternative to Nt for the identification of enteroviruses. Study design: Two MAb blends (one for coxsackie B viruses and one for echoviruses 4, 6, 9, 11, 30, and 34) and a coxsackie A9 MAb were used to screen 465 clinical isolates over a period of two years. Virus isolates which tested positive with one of the blends were typed with the individual MAbs of the respective blend. Individual MAbs for polioviruses 1, 2, and 3 acquired late in the study were used to screen 45 viral isolates. Results: The antibodies identified 251 465 (54%) of the total number of isolates tested. IFA results for 451 of 465 viral isolates were in agreement with conventional identification methods. The sensitivity of the IFA screen using the MAb blends and coxsackie A9 MAb was 93% and the specificity was 99%. Thirteen discrepant isolates were negative by IFA, with twelve positive by Nt for echovirus 30 and one isolate positive by Nt for coxsackie A9. The remaining discrepant isolate was positive by IFA for both coxsackie A9 and coxsackie B5, but positive by Nt for coxsackie A9 only. Conclusions: IFA is highly specific for the identification of enteroviruses, but may not be sensitive enough to identify all strains within an enterovirus type. Procedures which utilize an IFA screen and confirm final results by Nt decrease turnaround time and reduce the number of cell culture tubes required for the identification of each enterovirus isolate.

Molecular epidemiology of enterovirus outbreaks in Canada during 1991–1992: Identification of echovirus 30 and coxsackievirus B1 strains by amplicon sequencing

The relatedness of enteroviral isolates associated with two recent outbreaks in Canada was assessed using direct sequencing of amplicons derived from a large portion of the 5' nontranslated region (NTR) of the viral genome. The amplicons of 60 echovirus 30 isolates originating from seven different provinces in 1991 were found to share 99% or greater sequence identity. Recent coxsackievirus B1 isolates characterised in the same manner were identical to each other. When the 5' NTR sequence of these isolates was compared to prototype strains a difference of 11-15% in nucleotide composition was observed. These results indicate that the variability of nucleotide sequence found in 5' NTRs can be utilized to identify rapidly enteroviral strains associated with particular outbreaks and distinguish them from other strains and serotypes.

Diagnosis of enteroviral meningitis with IgG‐EIA using heat‐treated virions and synthetic peptides as antigens

Two recently developed enzyme immunosorbent assays (EIA) for the detection of significant titre increases in enteroviral IgG-antibodies were evaluated as diagnostic tools in 127 etiologically well-characterized patients with aseptic meningitis. One assay was based on heat-treated virions (H-EIA) and one on synthetic peptides (P-EIA) as antigens. The sensitivities, with virus isolation as reference method, were 0.67 by H-EIA and 0.62 by P-EIA, which were higher than by a routinely used complement fixation test (CFT, 0.51) but somewhat lower than the sensitivities found by two previously presented IgM-assays, mu-capture EIA, and solid-phase reverse immunosorbent test (SPRIST). The specificities of the two IgG-EIA techniques and CFT were apparently high, whereas the two IgM-assays showed positive reactions in some non-enteroviral cases. A relatively rapid increase in enteroviral IgG-antibodies was apparent using H-EIA and P-EIA. The two IgG-EIA tests contributed with considerable additional etiological information since significant IgG-rises were obtained in 13 patients by H-EIA and in 19 by P-EIA, respectively, out of the 56 individuals in whom enterovirus isolation was negative and a non-enteroviral diagnosis was not found. Thus, detection of enteroviral IgG-antibodies by H-EIA and P-EIA seems to be a valuable alternative to CFT for the routine diagnosis of enteroviral meningitis. The IgM-assays, mu-capture EIA, and SPRIST, may allow a relatively rapid report of an enteroviral infection. However, since both the IgM-tests are hampered by incomplete specificities, a confirmation of positive results by an IgG-assay should be carried out.

Failure to demonstrate enterovirus aetiology in Swedish patients with dilated cardiomyopathy

Attempts were made to establish a possible relationship between enterovirus infection and dilated cardiomyopathy (DCM) by use of serology, virus isolation from faecal samples, and detection of enteroviral RNA in endomyocardial biopsies (EMB) by the polymerase chain reaction (PCR). Sera from 63 patients with DCM and matched controls were examined for enterovirus infection by mu-capture IgM and indirect IgG ELISA. Thirty-six patients were further tested for the presence of enterovirus group-specific antigen (VP1) in an immunoassay system. The results were consistently negative. Faecal samples from 35 of these patients were negative by enterovirus isolation both when samples were cultured directly and after acid treatment. EMB from 35 patients were examined for enteroviral RNA by PCR; none of the samples was reactive. In conclusion, the results fail to indicate involvement of enteroviruses in the aetiology of DCM.

Serum IgA, IgG, and IgM responses to different enteroviruses as measured by a coxsackie B5-based indirect ELISA.

An enterovirus-specific indirect ELISA, based on a single local isolate of coxsackie B5 as antigen, was used to study the IgA, IgG, and IgM responses in 19 patients with a recent or current enterovirus infection. Twelve different enterovirus serotypes were isolated from 15 patients. Paired serum samples were available from 10 and a single serum from 5 of these 15 patients. In addition, 4 patients diagnosed by a significant titer rise of complement fixing antibodies to enterovirus were included. A serological diagnosis, defined as an increase in titer of enterovirus IgG and/or presence of enterovirus IgM, were established in all 14 patients with paired sera. Enterovirus IgM was present in either a single serum or in both sera in 13 of them. Out of 5 patients with a single serum sample only, enterovirus IgA or enterovirus IgM was found in 4. Specific IgA was present in either a single serum or in both sera in 14 of the 19 patients. Seven of the 10 enterovirus isolate patients with paired sera had a significant titer rise of complement fixing antibodies; however, all 10 were diagnosed by ELISA. Among 64 healthy controls 2 had enterovirus IgA and none had enterovirus IgM. In conclusion, the use of a single antigen-based ELISA was found to be reliable for the diagnosis of recent and current enterovirus infections.

Enteroviral aseptic meningitis in Japan, 1981-1991. A report of the National Epidemiological Surveillance of Infectious Agents in Japan.

In Japan, aseptic meningitis cases due to enterovirus infections increase every summer in various degrees with an incidence peak usually in July. During the past 11 years from 1981 through 1991, a total of 8,595 enterovirus isolations from aseptic meningitis cases were reported from 54 participating laboratories. Eight enterovirus types caused large epidemics; more than 100 isolations of each type from aseptic meningitis cases were reported for every epidemic year of the respective type. They were coxsackievirus (C) types B3 and B5, echovirus (E) types 4, 6, 7, 9, 18 and 30. Among these, the highest meningitis-associating frequency was reported for E30, representing 82.6% of the total isolations reported for the type during this period, followed by E4, 71.1%. The frequencies of E9, E7, E6 and CB5 were in a range from 54.5% to 44.4%, while that of E18 was 37.7% and that of CB3 21.0%. During the epidemics, enterovirus-associated meningitis was most frequently reported among children of 4-7 years of age. High frequencies were also shown in infants less than 1-year of age in some types. A total of 4,240 enteroviruses were isolated from cerebrospinal fluid of aseptic meningitis cases, representing 49.3% of the cases with enterovirus isolation.

A novel approach to enterovirus typing.

A novel method was developed for typing enteroviruses producing cytopathic effect. Monolayers of primary or secondary rhesus monkey kidney cells were prepared and covered with an agarose overlay. Wells were formed in the agarose, the well bottom being optimally determined to be 3 mm from the monolayer and homotypic enterovirus antiserum, intersecting antiserum pool or antiserum-diluent as control was added. After 2 h at 37 degrees C, the test virus isolate was added to each well. Cultures were incubated at 37 degrees C and were examined daily until cytopathic effect was readily observed in the control well. Monolayers were fixed and stained for macroscopic reading. Enterovirus identity based on the inhibition of cytopathic effect was confirmed with a conventional micro-neutralization method. In all, 51 enterovirus isolates were typed. Included were 21 polioviruses, 9 coxsackieviruses and 21 echoviruses, all of which were correctly identified. This method takes advantage of the ability of enterovirus and antibody to diffuse through agarose. It is simple to perform. It does not require preliminary titration of the test virus isolate and tolerates 1,000 fold fluctuations in virus concentration, thereby offering laboratories a more rapid and efficient means of typing enteroviruses.

Detection of hepatitis A virus and other enteroviruses in water by ssRNA probes

Sensitive and specific methods are needed to detect hepatitis A virus (HAV) and other human enteroviruses in environmental samples such as drinking water and foods. Clones of cDNA encoding the 5'-most 1 kb of the HAV and coxsackievirus B3 (CB3) genomes were subcloned into T7/SP6 RNA transcription vectors. In vitro transcribed RNA from the T7 promoter detected their respective HAV or CB3 genomic RNA. Conversely, SP6 transcripts detected viral negative-stranded RNA but not the genome. When both ssRNA probes were tested at high temperature (65°C), they did not hybridize with intracellular RNAs from 6 primate cell cultures used for isolation of HAV and other enteroviruses. The HAV probe did not hybridize with 13 different enteroviruses but detected as little as 500–1000 infectious units of the 7 strains of HAV tested. Conversely, the CB3 probe showed strong homology with all 13 enteroviruses tested but not HAV. The probes were used to detect HAV and other enteroviruses in water samples after virus amplification in cell culture. HAV was detected in water samples obtained during a waterborne hepatitis outbreak using the ssRNA probe. These samples were negative for HAV by direct solid phase radioimmunoassay and were not positive by immunoassays of inoculated cell cultures until several weeks of propagation. The CB3 ssRNA probe detected enteroviruses in samples of surface water and drinking water that were negative for cytopathic effects in inoculated cell cultures.

Effect of enteroviruses on adherence to and invasion of HEp-2 cells by Campylobacter isolates

Coinfection of HEp-2 epithelial cells with coxsackievirus B3, echovirus 7, poliovirus (LSc type 1), porcine enterovirus, and Campylobacter isolates was performed to determine if a synergistic effect could be obtained. The invasiveness of Campylobacter jejuni ATCC 33560 was significantly increased for HEp-2 cells preinfected with echovirus 7, coxsackievirus B3, and UV-inactivated (noninfectious) coxsackievirus B3 particles. Additionally, the invasiveness of C. jejuni M96, a clinical isolate, was significantly increased for HEp-2 cells preinfected with coxsackievirus B3. Poliovirus and porcine enterovirus had no effect on C. jejuni ATCC 33560 adherence and invasiveness. Furthermore, poliovirus had no effect on the ability of C. jejuni M96 to adhere to and invade HEp-2 cells. Campylobacter hyointestinalis and Campylobacter mucosalis, two noninvasive isolates, did not invade virus-infected HEp-2 cells. The increase in the invasiveness of C. jejuni appeared to be the result of specific interactions between the virus and the HEp-2 cell membrane. The data suggest that the invasiveness of Campylobacter spp. is dependent upon the inherent properties of the organism. Virus-induced cell alterations can potentiate the invasiveness of virulent Campylobacter spp. but are not sufficient to allow internalization of noninvasive bacteria.

A serological comparison of 4 Japanese isolates of porcine enteroviruses with the international reference strains.

The reference strains of four serotypes (J6, J8, J9 and J10) of porcine enterovirus isolated in Japan were compared with the international reference strains of 11 serotypes (W1 to W11) by cross neutralization tests. No cross reactions were observed between the two groups, although there were minor one-way crosses between W10 and J10, W11 and J9, and W4 and J9. This leads to our conclusion that all 4 Japanese serotypes can be newly added to 11 international serotypes. J9 virus produced type 1 of CPE (CPE I), J6 and J8 did CPE II, and J10 did CPE III. J10 grew in Vero, HeLa cells, and the primate cells, but J6, J8 and J9 did not.

Isolation of enterovirus 70 (EV70) from patients with acute haemorrhagic conjunctivitis in two areas of Saudi Arabia

This is the first epidemic of acute haemorrhagic conjunctivitis (AHC) reported from Saudi Arabia in which enterovirus 70 (EV70) was implicated as aetiplogical agent. EV70 was isolated from 5 of 29 conjunctival scrapings from patients with AHC. Two human diploid cell lines, human skin fibroblast (HSF) and human embryonic lung fibroblast (MRC-5), were quite sensitive for the isolation of this virus. The relatively high isolation rate could also be attributed to the timing of collection of specimens and perhaps to the fact that conjunctival scrapings contained more virus particles than did eye swabs.

Enterovirus-specific IgM in the diagnosis of meningitis

Samples of serum from 557 patients with a clinical diagnosis of meningitis or encephalitis and referred to the Epsom Public Health Laboratory during a period of 3 years were tested for enterovirus-specific IgM in a mu capture enzyme-linked immunosorbent assay (ELISA). Enterovirus-specific IgM was detected in 45% samples from all age groups. In the 3-5-year age group, 67% specimens were positive. A notable male predominance (73%) was seen in the age group 0-15 years. As predicted, a seasonal increase in incidence was found in the summer and autumn months. Data from a questionnaire sent to the referring laboratories showed only a 5% enterovirus isolation rate from cerebrospinal fluids when isolation of a virus was attempted. The enterovirus IgM ELISA is a sensitive economical and rapid method for use in the diagnosis of viral meningitis.

Isolation and characterisation of cytopathic avian enteroviruses from broiler chicks

Cytopathic viruses were isolated from the faeces, pancreas and caecal contents of broiler chicks in two cases of a stunting syndrome. Two representative strains, designated AAF7 and M-8, were identified as enteroviruses on the basis of their size (25 nm in diameter), RNA in the viral core, virus growth in the cytoplasm, resistance to chloroform, trypsin and acid, and partial heat-stabilisation to molar magnesium chloride. They formed irregular plaques in chick kidney cell cultures. Numerous granula eosinophilic inclusions were induced in the cytoplasm of chicken kidney cell cultures infected with the viruses. Both isolates reacted partially with the G4260 strain of avian nephritis virus but not with the VR strain of avian encephalomyelitis virus.

Isolation of enterovirus and reovirus from sewage and treated effluents in selected Puerto Rican communities

Sewage treatment plant effluents were surveyed for viral contributions to gastroenteritis outbreaks in Puerto Rico. Of the 15 sewage treatment plants studied, all discharged their effluents upstream from water treatment plant intakes. No base-line data on the degree of viral challenge to these sewage treatment plants or the subsequent reduction of viruses before discharge existed. Enterovirus counts were generally much higher than those found in the continental United States. At four plants, viruses in the incoming sewage exceeded 100,000 PFU/liter, and one of these, a trickling filter plant, was discharging 24,000 PFU/liter to receiving waters. Virus identification showed that more than 80% of the enterovirus isolates were coxsackievirus B5. These overwhelming viral numbers pointed to defects in the sewage treatment processes. Without reasonable barriers to protect receiving waters, several of the downstream communities were using raw waters that posed extraordinary demands on the ability of their water treatment plants to supply virologically safe drinking water.

Comparison of cell cultures for rapid isolation of enteroviruses

Cell culture isolation is still the most reliable method for the detection of enteroviruses from clinical specimens. Rapid diagnosis of enterovirus infection affects patient management. To increase yield and enhance the rapidity of enterovirus isolation in cell cultures, we used Buffalo green monkey kidney (BGM) cells and subpassages of primary human embryonic kidney (HEK) cells in addition to the human diploid fibroblast (MRC-5) cells and primary cynomolgus or rhesus monkey kidney (MK) cells routinely used for enterovirus culturing. Growth characteristics of enteroviruses from 421 specimens were studied. All specimens were cultured in MRC-5, MK, and BGM cells, and 204 of these specimens were also cultured in HEK cells. Forty-two percent of the enteroviruses became positive within 3 days, and 85% did so within 7 days. MRC-5 cells provided the highest yield of enteroviruses overall and were the best cell type for the recovery of poliovirus and echovirus. MK cells provided the second best yield but were more useful than MRC-5 cells for coxsackievirus. BGM cells supported the growth of additional isolates of coxsackievirus and enhanced the speed of isolation. HEK cells supported the growth of additional isolates of both coxsackievirus and echovirus, but subculturing was always required for definite enterovirus cytopathic effects. The recovery rate increased 11% when two additional cell lines were used. The use of two tubes of MK cells significantly increased the yield of all enterovirus types. We conclude that the use of multiple appropriate cell lines increases yield and enhances the rapidity of enterovirus isolation.

A microneutralization test for the identification of enterovirus isolates

Two-hundred and thirty-four enterovirus isolates were identified using a recently modified, microneutralization procedure. Neutralization tests were performed in 96 well plastic ‘V’ plates, using 20 μ1 quantities of antisera and virus, then inoculated onto monolayers of buffalo green monkey kidney cells, growing in 48 well tissue culture plates. Utilization of this microneutralization procedure resulted in considerable savings of time, material, and particularly, neutralizing antisera.

A combination of four cell types for rapid detection of enteroviruses in clinical specimens.

Isolation in cell culture is currently the most sensitive and reliable way to demonstrate enterovirus (EV) in clinical specimens. During July-October 1982 and 1983, we studied the impact of adding two new cell lines, Buffalo green monkey kidney (BGM) and human rhabdomyosarcoma (RD), to the more traditional cell combination used for EV isolation, human embryonic lung (HEL) and primary cynomolgus monkey kidney (CMK) cells; 2,558 specimens were studied: 632 fecal, 677 respiratory, 537 CSF, and 712 blood. An EV was isolated from 417 (16%); of these, 77 (18%) were positive only in BGM or RD; 35% (146/417) of the specimens were positive in BGM, RD, or both, at least one day earlier than in the traditional cells. BGM cells were helpful in isolation of group B coxsackieviruses (CB): 99% of 121 positive specimens were detected in BGM vs 73% in CMK and 23% in HEL; 72% of the CB isolates were detected by day 2 in BGM vs 48% in CMK and 0% in HEL. RD cells were helpful in the isolation of echoviruses: 59% of the 189 positive specimens were detected in RD vs 67% in HEL and 58% in CMK. RD was the only positive cell type in 28/189 (15%) positive specimens; 31% of the echovirus isolates were detected by day 2 in RD, vs 20% in HEL and 19% in CMK. Using the cell types described, we provided the clinician with results in 42% of the EV-positive specimens by day 2 after inoculation and in 61% by day 4.

Temporal and geographic patterns of isolates of nonpolio enterovirus in the United States, 1970-1983.

Surveillance data on nonpolio enterovirus (NPEV) from the Centers for Disease Control (Atlanta, Georgia) for the United States from 1970 to 1983 were analyzed for the temporal and geographic patterns of the most common types of NPEV isolated. The number of isolates varied from year to year, partly because of variation in the number of reporting laboratories and partly because of true variation in the rate of isolation. The most common types isolated also varied from year to year, yet the 15 most common types over the entire 14-year period accounted for 65%-89% of isolates for a given year. The 15 most common types of NPEV had two basic patterns of isolation, one in which a type had periodic epidemic years and the other in which it did not. In 11 of the 14 years there was one or more epidemic types, each accounting for greater than or equal to 20% of all isolates of NPEV that year. The six most common isolates in March, April, and May predicted an average of 59% of the total isolates detected in July-December of that year.

558 COXSACKIE A (CA) ANTIGEN DETECTION BY ENZYME IMMUNOASSAY (EIA) IN SUSPECTED SEPSIS (SS)

Previous studies have documented the important association of non-polio enterovirus (E) isolation with SS in young infants. These studies relied on tissue culture alone to identify E. We utilized a double-antibody, solid-phase EIA for CA, in addition to standard methods, in the evaluation of 45 patients <3 months of age admitted to NCMC for SS from 6/1 to 10/31/83. No patient had an obvious focus of infection, abnormal chest roentgenogram, or history of previous antibiotics. 38 patients had a rectal temperature ≥37.8°C on admission, 4 were febrile by history, and 3 were evaluated for lethargy. Blood, cerebrospinal fluid (CSF) and rectal swabs were collected from all patients and urine from 39 for routine bacterial studies. CSF from 42 and nasopharyngeal, throat and rectal swabs from all patients were plated on rhesus monkey and human embryonic kidney cells for viral isolation. Echovirus (8), coxsackie-virus (10), untyped E (2) and parainfluenza (1) were identified by culture and CA antigen was detected in an additional 5 patients. All of the 26 patients with proven viral infections were suspected of having bacterial infections on clinical grounds and 25 were treated with anitbiotics despite negative bacterial cultures. These data support a major role for E in SS in the summer and fall. Further evaluation of rapid diagnostic tests for viruses and their role in preventing unnecessary antibiotic administration are warranted.