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Enterovirus 71 isolated from cases of epidemic poliomyelitis-like disease in Bulgaria.

Virological and serological studies of an epidemic disease in Bulgaria, 1975, were carried out. Epidemiologically, clinically and pathomorphologically, the disease simulated almost all known forms of poliomyelitis, acute stem encephalitis, encephalomyocarditis and aseptic meningitis. The studies completely rules out the participation of polioviruses and provided comprehensive evidence for the etiological role of a peculiar enterovirus subsequently identified as enterovirus (EV) type 71 known in the literature since 1974. Altogether, in 1975 and 1976 from 65 cases of poliomyelitis-like disease (PLD) 92 strains of EV71 were isolated, including 37 strains from the brain and medulla, 1 from the cerebrospinal fluid, 10 from mesenterial lymph nodes and tonsils and 44 from faeces. In addition, in 282 convalescent cases of the disease, diagnostic seroconversion or high titers of antibody to this virus were demonstrated. The most successful virus isolation was achieved by inoculation of green monkey kidney cell cultures and newborn white mice. Bulgarian strains of enterovirus 71 regularly caused paralysis in monkeys and morphological poliomyelitis-like lesions in their CNS, and paralysis and myositis with Zenker necrosis in newborn white mice, cotton rats, Syrian hamsters, and 3-week-old cotton rats. The diseased rodents had much more virus in their mucles than in brains.

Adult

Non-polio enterovirus disease in the United States, 1971--1975.

Reports to the Center for Disease Control on isolation of non-polio enteroviruses for the years 1971--1975 were analysed. During the 5 year period, enterovirus isolations were reported from 7 075 individuals. 90% of these occurred in the 7 month interval of May--November. Enteroviruses were isolated more frequently from males than females for all age groups in all 5 years. The incidence of reported isolations decreased with increasing age, and an inverse relationship between severity of disease and age was suggested. Clinical diagnoses associated with enteroviral isolations included aseptic meningitis, encephalitis, upper respiratory tract disease, non-specific febrile illness, gastroenteritis, pneumonia and lower respiratory tract disease, exanthem, and enanthem.

Age Factors

[Detection of small amounts of viruses (enteroviruses) in drinking water (author's transl)].

A simple detection method of enteroviruses and adenoviruses in drinking water has been performed, using adsorption-elution on to cellulose nitrate membranes. Average of enteroviruses recovering is varying from 23 to 80% when 30 liters of water containing 60 PFU are monitored. Only 10% of adenoviruses are recovered in the same conditions. This method allowed us to detect 12 PFU of enteroviruses in 120 liters of water. Recovering of 1 PFU in 10 liters when examining more than 100 liters is reproducible.

Adenoviridae

[Isolation of type 71 enterovirus from patients with a poliomyelitis-like disease during an outbreak in Bulgaria].

Virological examinations of 72 specimens obtained during an outbreak of a poliomyelitis-like disease in Bulgaria, 1975, yielded 23 strains of enterovirus type 71 (E71) and 5 strains of enteroviruses of other types. The viruses were isolated in green monkey kidney cell cultures, in newborn cotton rats, newborn white mice and in monkeys. Most of E71 strains (16 out of 23) were isolated from the autopsy materials (brains and spinal cords) from 9 children dying of poliomyelitis, meningoencephalitis, and encephalitis. The remaining E71 strains and all the strains of other enterovirus serotypes, with the exception of ECHO7, were isolated from stool specimens of children with aseptic meningitis and their contacts. The antigenic similarity of the group of Bulgarian strains and their relationship with the prototype BrCr strain of E71 was established. The conclusion is that E71 was the causative agent of an outbreak of a poliomyelitis-like disease in Bulgaria.

Animals

Global Evolution and Transmission Dynamics of Enterovirus D68.

Enterovirus D68 (EV-D68), a serotype of the enterovirus species D, has garnered significant attention due to outbreaks reported in 2014, 2016, and 2018. In this study, 36 Chinese EV-D68 strains were isolated, sequenced, and combined with all EV-D68 VP1 sequences from GenBank to form a data set of 1679 sequences. This data set served as the basis for phylogenetic, evolutionary dynamics, phylogeographic, and key amino acid site mutation analyses of EV-D68. Based on the VP1 region, EV-D68 is classified into four genotypes (A-D), and seven subgenotypes (B1-B3, D1-D4), with B3 and D3 being the predominant subgenotypes. Bayesian skyline plots indicated that genotypes B and D experienced multiple population expansions, aligning with reported EV-D68 outbreaks. Phylogeographic analyses of the B3 subgenotypes revealed sequences from Europe and North America clustering into a single evolutionary branch, suggesting significant transmission between these regions. Additionally, mutation analysis identified VP1-98 as a high-frequency mutation site, differing significantly between the previously prevalent A and C genotypes and the currently prevalent B and D genotypes. However, the functional implications of this mutation require further investigation. This study provides a solid theoretical basis for epidemiological research, disease surveillance, and prevention efforts related to EV-D68.

Enterovirus Infections

Long-term clinical and genomic surveillance of rare respiratory enterovirus C types in France, 2013-2025.

INTRODUCTION: Rare enterovirus types assigned to species C (EV-C) display respiratory tropism and may be associated with neurological involvement, which display similarities to EV-D68 disease. PATIENTS AND METHODS: We conducted continuous enterovirus/rhinovirus (EV/RV) surveillance between 2013 and 2025, including systematic EV/RV screening of all respiratory samples and reflex VP4/VP2 sequencing (a total of 5855 samples). When an EV-C strain was detected, the VP1-coding and complete genome sequence was sequenced to investigate phylogenetic relationships and to identify any recombinant forms. RESULTS: Over the 13-year period, 30 EV-C infections were identified from respiratory samples, with 23 cases (77%) detected between 2013 and 2018. EV-C105 was the most frequent type (n = 11), followed by EV-C104 (n = 8), EV-C109 (n = 6), and EV-C117 (n = 5). We also document the first detection of EV-C117 in France and only the second case reported in Europe since its initial description in 2011 in Lithuania. Clinical data were available for 25 patients, of whom 19 (76%) were children. Respiratory symptoms predominated (17/25, 68%), and 7 patients presented with lower respiratory tract infections. Hospitalization was required in 18 cases (72%), including three with ICU admissions (12%). Nearly half of the patients (12/25, 48%) had at least one risk factor for severe respiratory disease. Across all respiratory EV-C types, recovered sequences clustered with contemporary global strains. CONCLUSIONS: This long-term surveillance highlights the sustained circulation of multiple respiratory EV-C types in France and underscores the need for continued clinical and genomic monitoring to assess the evolution and pathogenic potential of these enteroviruses.

Complete genome characterization

Non-polio enterovirus activity in Wisconsin based on a 20-year experience in a diagnostic virology laboratory.

Laboratory test results, demographic information, and clinical data on 1364 patients who had specimens submitted to the Wisconsin State Laboratory of Hygiene and from whom non-polio enteroviruses were isolated during 1957--1976 were examined. Echoviruses (echo) and Coxsackievirus group B (CB) and group A (CA) were isolated from 719, 389, and 256 of the patients, respectively. Thirty-five different serotypes were identified in the 20-year period. The six most frequently occurring serotypes (echo 9, CB 5, echo 18, CB 2, CB 4, and echo 6) were associated with 57 per cent of all illnesses. The first three serotypes occurred in an epidemic-like manner whereas the next three appeared to be endemic. Eighty-eight per cent of all isolations were from patients whose illnesses began in July through October. The percentage of CA isolates obtained from adults was much smaller than the corresponding percentages seen with CB or echo. A majority of the non-polio enteroviruses were obtained from males. Central nervous system and gastrointestinal tract involvement was greatest with echo and CB 5 tract and lowest with CA and CB 1--4 illnesses. The highest frequency of respiratory tract involvement was with CA and echo 9 illnesses and rash was most frequent with CA illnesses. The highest frequency of hospitalization was with CB 5 patients.

Adolescent

Genomic surveillance of enterovirus D68 circulating in 2025 reveals the emergence of a novel A2/B3 recombinant lineage.

Enterovirus D68 (EV-D68) has re-emerged over the past decade as a significant respiratory pathogen associated with severe respiratory disease and acute flaccid myelitis. Its circulation has typically followed a biennial pattern, with predominance in late summer and early fall, a pattern that was temporarily disrupted during the COVID-19 pandemic. Surveillance in 2025 revealed off-season circulation of EV-D68. This study describes the genomic characteristics of the 2025 EV-D68 viruses and the clinical features of affected patients. Between May and December 2025, remnant respiratory specimens positive for rhinovirus/enterovirus were screened for EV-D68 and subjected to whole-genome sequencing. Phylogenetic analyses were performed using maximum-likelihood methods. Recombination was assessed using subgenomic phylogenies, SimPlot similarity and BootScan analyses, and read-level inspection. Among 1,321 patients tested, 147 (11.1%) were EV-D68-positive, and 119 (81.0%) yielded complete genomes. EV-D68 positivity increased in July 2025, peaked in August (~21%), and remained elevated through September and October, exceeding levels observed in 2024. Patients had a median age of 36 years, with infections disproportionately affecting older adults. Phylogenetic analysis demonstrated exclusive circulation of subclade A2. Five genomes formed a distinct recombinant lineage (A2-Re). Subgenomic phylogenies showed clustering with A2 viruses in the P1 region and with B3 viruses in the P2-P3 regions. SimPlot and BootScan analyses identified a recombination breakpoint near the 2A/2B junction (~nt 3,700). The recombinant lineage was associated with temporally clustered cases in September-October. These findings demonstrate recombination between distinct EV-D68 subclades and underscore the importance of whole-genome surveillance for accurate viral characterization. Continued genomic monitoring is essential for detecting emerging variants with potential implications for transmissibility, pathogenicity, and public health preparedness.IMPORTANCEThis study highlights an increased off-season circulation of Enterovirus D68 (EV-D68) and a higher burden of disease in adults in 2025. The identification of a novel A2-B3 recombinant lineage provides evidence of ongoing viral evolution through recombination, a mechanism that may alter transmissibility, virulence, or immune responses. Detection of this lineage in temporally clustered cases suggests local transmission and underscores the potential for rapid spread of newly emerged variants. These findings emphasize the limitations of partial genomic approaches and the critical role of whole-genome sequencing in accurately characterizing circulating strains and identifying recombination events. Enhanced genomic surveillance is essential to detect emerging variants in real time, inform diagnostic assay performance, and support public health responses. Continued monitoring of EV-D68 evolution will be important for anticipating changes in disease burden, guiding clinical awareness, and strengthening preparedness for future outbreaks.

Humans

Isolation of a new porcine enterovirus in the United States.

A cytopathogenic virus isolated from an Iowa farm pig with diarrhea was identified as a member of Group 5 of the porcine enteroviruses. Clinical signs observed in other pigs in the herd were diarrhea, debilitation and gauntness. Morbidity and mortality rates were approximately 50% and 3% respectively.A Group 5 porcine enterovirus had not been previously isolated in North America according to the Western Hemisphere Committee on Animal Virus Characterization.

Animals

Development of quantitative methods for the detection of enteroviruses in sewage sludges during activation and following land disposal.

The development and evaluation of methods for the quantitative recovery of enteroviruses from sewage sludge are reported. Activated sewage sludge solids were collected by centrifugation, and elution of the solid-associated virus was accomplished by mechanical agitation in glycine buffer at pH 11.0. Eluted viruses were concentrated either onto an aluminum hydroxide floc or by association with a floc which formed de novo upon adjustment of the glycine eluate to pH 3.5. Viruses which remained in the liquid phase after lowering the pH of glycine eluate were concentrated by adsorption to and elution from membrane filters. The method of choice included high pH glycine elution and subsequent low pH concentration; it yielded an efficiency of recovery from activated sludge of 80% for poliovirus type 1, 68% for echovirus type 7, and 75% for coxsackievirus B3. This method was used to study the survival of naturally occurring virus in sludge at a sewage treatment plant and after subsequent land disposal of the solids after aerobic digestion. Reduction of enterovirus titers per gram (dry weight) of solids were modest during sludge activation but increased to a rate of 2 log 10/week after land disposal.

Buffers

Adsorption of enteroviruses to soil cores and their subsequent elution by artificial rainwater.

The adsorption and elution of a variety of human enteroviruses in a highly permeable, sandy soil was studied by using cores (43 by 125 mm) collected from an operating recharge basin on Long Island. Viruses studied included field and reference strains of polioviruses types 1 and 3 and reference strains of coxsackie virus B3 and echovirus types 1 and 6. Viruses suspended in treated sewage effluent were allowed to percolate through soil cores, and the filtrate was assayed for unadsorbed viruses. To determine the likelihood of desorption and mobilization, soil-bound viruses were subjected to a rinse with either treated sewage effluent or simulated rainwater which reflected the anion, cation, and pH characteristics of a typical northeastern United States rainfall. The results demonstrated that all polioviruses tested, including both reference and field strains, adsorbed extremely well to cores. Adsorption was somewhat reduced when clean, unconditioned soils were used. Soil-bound poliovirus strain LSc was not significantly mobilized by flooding columns with either a sewage effluent or rainwater rinse. One virus was mobilized by both types of rinses. The amount of viruses mobilized by rainwater rinses ranged from 24 to 66%. Variable adsorption-elution results were observed with other enteroviruses. Two guanidine-resistant mutants of poliovirus LSc demonstrated a soil adsorption-elution profile different from that of the parent strain. The data support the conclusion that soil adsorption-elution behavior is strain dependent and that poliovirus, particularly strain LSc, represents an inappropriate model.

Adsorption

Development of a simple method for concentrating enteroviruses from oysters.

The development of a simple method for concentrating enteroviruses from oysters is described. In this method viruses in homogenized oyster tissues are efficiently absorbed to oyster solids at pH 5.5 and low salt concentration. After low-speed centrifugation, the supernatant is discarded and viruses are eluted from the sedimented oyster solids by resuspending them in pH 3.5 glycine-buffered saline. The solids are then removed by low-speed centrifugation, and the virus-containing supernatant is filtered through a 0.2-micronm porosity filter to remove bacteria and other small particulates without removing viruses. The virus-containing filtrate is then concentrated to a volume of a few milliliters by ultrafiltration, and the concentrate obtained is inoculated directly into cell cultures for virus assay. When tested with pools of oysters experimentally contaminated with small amounts of different enteroviruses, virus recovery efficiency averaged 63%.

Adsorption

A comparative study of agar overlay and standard tissue culture methods for isolation of enteroviruses.

A study was made of the isolation of enteroviruses from 62 cerebrospinal fluids and 141 autopsy specimens with standard fluid monolayer tissue culture methods and tissue cultures under agar overlay with and without added MgCl2 (0.025M). Sixteen virus isolations were made only by the overlay method, two only by the standard technique, and six by both procedures. Additional studies were carried out on 975 diagnostic specimens of various types with standard tissue culture methods and with primary rhesus kidney cells under agar overlay containing 0.025M MgCl2. Seventeen virus isolations were made only by the overlay procedure, 20 only by the standard tissue culture method, and 75 by both techniques. Since there were instances in which either the agar overlay or the standard method alone succeeded in isolating a virus, use of both types of procedures would be desirable to isolate as many enteroviruses as possible from clinical specimens.

Agar

Serological comparison of French WD-42 enterovirus isolate with bovine winter dysentery in New York State.

Forty-eight acute and convalescent paired serums from 10 dairy cattle herds naturally infected with winter dysentery and 10 paired serums from laboratory infected heifers were tested for antibody to bovine enterovirus WD-42 which reportedly reproduced the disease syndrome in France. Only 3 naturally infected and 1 experimentally infected animals had 2-fold antibody responses. No clinical symptoms were observed in 2 steers inoculated with WD-42. Each had a 4-fold or greater antibody response to the virus. Results indicate that the winter dysentery syndrome observed in New York is produced by an agent other than WD-42 virus and that it is produced by an agent other than an enterovirus.

Animals

Investigation of physicochemical properties of Bulgarian strain 258 of enterovirus type 71. Brief report.

In the present report some general physicochemical properties and RNA characteristics of strain 258 of enterovirus (EV) 71, isolated in 1975 from cases of poliomyelitis-like disease in Bulgaria (1), are described. It is concluded, that the features of the virus are consistent with the previous identification, based on biological studies, of this virus strain as a member of enterovirus subgroup of picornaviridae family.

Animals

Characterization of an enterovirus associated with acute infectious lymphocytosis.

An enterovirus (EVU-16) previously isolated from children with acute infectious lymphocytosis has been further characterized. The EVU-16 virus sediments as a 135S particle in sucrose gradients, has a density of 1-335 g/ml in CsCl, contains 4 polypeptides and has a single stranded RNA genome sedimenting at 35S. These structural features as well as the presence of a virus-related particle, the procapsid, are similar to those of other enteroviruses. However, the largest polypeptide of EVU-16 is 49000 daltons, which is considerably larger than the corresponding polypeptide from poliovirus; the sizes of the other three viral polypeptides were similar in both viruses. Attempts to induce lymphocytosis by the inoculation of EVU-16 into various animals, including immunologically aberrant 'nude' mice, were unsuccessful.

Cell Line

Replication of enterovirus 70 in non-primate cell cultures.

Replication of the strain J 670/71 of enterovirus 70 (EV70) in non-primate cell cultures at 33 degrees C was studied using strain L (mouse), BHK21 (hamster), RK13 (rabbit), RK17 (rabbit), PK15 (porcine), IB-RS-2 (porcine), ESK (porcine), MDBK (bovine), and BK1 (primary bovine) together with that of the LSc, 2ab strain of poliovrus type 1 (PV1) as a control. All the cells tested adsorbed from 54 to 90% of EV70. The replication with complete c.p.e. was evident in RK13, RK17 and BK1 cells; replication without c.p.e. was shown in L, BHK21, IB-RS-2 and ESK; but PK15 and MDBK were non-permissive despite a high virus adsorption rate. On the contrary, none of these non-primate cells allowed the adsorption and growth of PV1. One-step growth of EV70 in RK13 was almost identical with that in the primate cells. Two other strains of EV70 were found having similar host range in vitro. Therefore, it is concluded that EV70 has a wider host range in vitro than ordinary human enteroviruses, and its implication is discussed.

Adsorption