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Coxsackievirus B5 (CVB5) is a major pathogen responsible for hand-foot-mouth disease, herpangina, and even severe death. The mechanisms underlying CVB5-induced diseases are not fully elucidated, and no specific antiviral treatments are currently available. Circular RNAs (circRNAs), a closed-loop molecular structure, have been reported to be involved in virus infectious diseases. However, their roles and mechanisms in CVB5 infection remain largely unknown. In this study, we identify that CircPTPN11 is significantly upregulated following CVB5 infection in RD cells. Characteristic analysis reveals that the expression of CircPTPN11 is both time- and dose-dependent upon CVB5 infection and is specific to intestinal tissue. Moreover, CircPTPN11 inhibits CVB5 replication by activating IRF3 in the type-I interferon (IFN-I) pathway. Further underneath mechanism shows that CircPTPN11 indirectly regulates CVB5 replication by sponging miR-152-3p, and miR-152-3p influences CVB5 replication by interacting with the gene coding for signal regulatory protein alpha (SIRPA). In conclusion, this study suggests that CircPTPN11 targets SIRPA by sponging miR-152-3p, thereby inhibiting the replication and proliferation of CVB5. These findings provide a molecular target for the diagnosis and treatment of CVB5 infection., Competing Interests: Declaration of competing interest All authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

Evidence is emerging on the roles of long noncoding RNAs (lncRNAs) as regulatory factors in a variety of viral infection processes, but the mechanisms underlying their functions in coxsackievirus group B type3 (CVB3)-induced acute viral myocarditis have not been explicitly delineated. We previously demonstrated that CVB3 infection decreases miRNA-21 expression; however, lncRNAs that regulate the miRNA-21-dependent CVB3 disease process have yet to be identified. To evaluate lncRNAs upstream of miRNA-21, differentially expressed lncRNAs in CVB3-infected mouse hearts were identified by microarray analysis and lncRNA/miRNA-21 interactions were predicted bioinformatically. MEG3 was identified as a candidate miRNA-21-interacting lncRNA upregulated in CVB3-infected mouse hearts. MEG3 expression was verified to be upregulated in HeLa cells 48 h post CVB3 infection and to act as a competitive endogenous RNA of miRNA-21. MEG3 knockdown resulted in the upregulation of miRNA-21, which inhibited CVB3 replication by attenuating P38-MAPK signaling in vitro and in vivo. Knockdown of MEG3 expression before CVB3 infection inhibited viral replication in mouse hearts and alleviated cardiac injury, which improved survival. Furthermore, the knockdown of CREB5, which was predicted bioinformatically to function upstream of MEG3, was demonstrated to decrease MEG3 expression and CVB3 viral replication. This study identifies the function of the lncRNA MEG3/miRNA-21/P38 MAPK axis in the process of CVB3 replication, for which CREB5 could serve as an upstream modulator., Competing Interests: Declaration of Competing Interest All authors declare that they have no competing interests., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Coxsackievirus B3 (CVB3) is a positive single-strand RNA virus causing myocarditis, pancreatitis and meningitis. During CVB3 infection, various host cellular components, including proteins and non-coding RNAs, interact with the virus and affect viral infection. Poly(rC) binding protein 1 (PCBP1) is a multifunctional RNA binding protein regulating transcription, translation and mRNA stability of a variety of genes. In this study, we observed a significant reduction of PCBP1 protein during CVB3 infection. By bioinformatic prediction and luciferase-assay verification, we confirmed that the expression of PCBP1 was directly inhibited by miR-21, a microRNA upregulated during CVB3 infection. Furthermore, we found that overexpression of PCBP1 promoted CVB3 infection and knocking down of PCBP1 inhibited it. In the subsequent mechanism study, our results revealed that PCBP1 blocked the translation of p62/SQSTM1 (sequestosome 1), an autophagy-receptor protein suppressing CVB3 replication, by interacting with the cis-element in the 5' untranslational region (5' UTR) of p62/SQSTM1. In summary, our studies have identified PCBP1 as a beneficial factor for CVB3 infection. These findings may deepen the understanding of host-virus interactions and provide a potential target for intervention of CVB3 infection., (Copyright © 2022. Published by Elsevier B.V.)

An understanding of how viruses interact with their receptors is vital as this step is a major determinant of host susceptibility and disease. The enterovirus coxsackievirus A9 (CVA9) is an important pathogen responsible for respiratory infections, myocarditis, infections of the central nervous system, chronic dilated cardiomyopathy and possibly type I diabetes. CVA9 harbours an integrin- recognition motif, RGD (Arg-Gly-Asp), in the capsid protein VP1 and this motif is believed to be primarily responsible for binding to integrins αvβ6 and/or αvβ3 during cell entry. Despite the consistent conservation of RGD-flanking amino acids in multiple RGD-containing picornaviruses, the significance of these amino acids to cell tropism has not been thoroughly investigated. In this study we used 10 CVA9 mutants and a panel of cells to analyse cell tropism. We showed that CVA9 infection proceeds by either an RGD- dependent or an apparently RGD- independent pathway. Differences in the amino acid found at the +1 position of the RGD motif affect the cell tropism of CVA9 when an RGD- dependent pathway is used. Naturally occurring CVA9 isolates have either the sequence RGDM and RGDL and we found that the corresponding viruses in our panel infected cells most efficiently. There was also a strong selection pressure for RGDL in adaptation experiments. However, there was also an unexpected selection of an RGDL variant in an apparently RGD- independent cell line. There was also no simple relationship between infection of cells and expression of integrins αvβ3 and αvβ6. The results obtained have greatly improved our understanding of how CVA9 infects cells. This will be useful in the design of antivirus drugs and also gives a framework for the modification of CVA9 or other RGD containing picornaviruses for specific targeting of cancer cells for oncolytic therapy., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The cis-acting replication element (CRE) in the 2C protein coding region [CRE(2C)] of enteroviruses (EV) facilitates the addition of two uridine residues (uridylylation) onto the virus-encoded protein VPg in order for it to serve as the RNA replication primer. We demonstrated that coxsackievirus B3 (CVB3) is replication competent in the absence of a native (uridylylating) CRE(2C) and also demonstrated that lack of a functional CRE(2C) led to generation of 5' terminal genomic deletions in the CVB3 CRE-knock-out (CVB3-CKO) population. We asked whether reversion of the mutated CRE(2C) occurred, thus permitting sustained replication, and when were 5' terminal deletions generated during replication. Virions were isolated from HeLa cells previously electroporated with infectious CVB3-CKO T7 transcribed RNA or from hearts and spleens of mice after transfection with CVB3-CKO RNA. Viral RNA was isolated in order to amplify the CRE(2C) coding region and the genomic 5' terminal sequences. Sequence analysis revealed reversion of the CVB3-CKO sequence to wildtype occurs by 8 days post-electroporation of HeLa cells and by 20days post-transfection in mice. However, 5' terminal deletions evolve prior to these times. Reversion of the CRE(2C) mutations to wildtype despite loss of the genomic 5' termini is consistent with the hypothesis that an intact CRE(2C) is inherently vital to EV replication even when it is not enabling efficient positive strand initiation., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

To determine whether 2A protease of the enterovirus genus with type I internal ribosome entry site (IRES) effect on the viral replication of type II IRES, coxsackievirus B3(CVB3)-encoded protease 2A and encephalomyocarditis virus (EMCV) IRES (Type II)-dependent or cap-dependent report gene were transiently co-expressed in eukaryotic cells. We found that CVB3 2A protease not only inhibited translation of cap-dependent reporter genes through the cleavage of eIF4GI, but also conferred high EMCV IRES-dependent translation ability and promoted EMCV replication. Moreover, deletions of short motif (aa13-18 RVVNRH, aa65-70 KNKHYP, or aa88-93 PRRYQSH) resembling the nuclear localization signals (NLS) or COOH-terminal acidic amino acid motif (aa133-147 DIRDLLWLEDDAMEQ) of CVB3 2A protease decreased both its EMCV IRES-dependent translation efficiency and destroy its cleavage on eukaryotic initiation factor 4G (eIF4G) I. Our results may provide better understanding into more effective interventions and treatments for co-infection of viral diseases., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Echovirus 25 (E-25) is a member of the enterovirus family and a common pathogen that induces hand, foot, and mouth disease (HFMD), meningitis, skin rash, and respiratory illnesses. In this study, we constructed and characterized an infectious full-length E-25 cDNA clone derived from the XM0297 strain, which was the first subgenotype D6 strain isolated in Xiamen, China. The 5'-Untranslated Regions (5'-UTR), P3 (3A-3B, 3D) and P3 (3C) regions of this E-25 (XM0297) strain were highly similar to EV-B77, E-16 and E-13, respectively. Our data demonstrate that the rescued E-25 viruses exhibited similar growth kinetics to the prototype virus strain XM0297. We observed the rescued viral particles using transmission electron microscope (TEM) and found them to possess an icosahedral structure, with a diameter of approximately 30 nm. The cross neutralization test demonstrated that the E-25 (XM0297) strain immune serum could not neutralize EV-A71, CV-A16 or CV-B3; likewise, the EV-A71 and CV-A16 immune serum could not neutralize E-25 (XM0297). The availability of this infectious clone will greatly enhance future virological investigations and possible vaccine development against E-25., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Non-polio enteroviruses (NPEVs) have often been identified in association with acute flaccid paralysis (AFP) in most polio surveillance studies worldwide. In a polio endemic country like Nigeria, there is need for distinction of AFP due to poliovirus and those potentially due to NPEVs. This study was undertaken to characterize the enterovirus (EV) types circulating in both children with and without AFP in Nigeria. Of fecal sample from 966 children with AFP, 96 (10%) were positive for NPEVs in RD cells, while 42 (5.5%) of 756 samples from non-AFP children was positive. Genotyping of all NPEV isolates was done by partial VP1 gene sequencing and phylogenetic analysis. EV-B was the predominant species detected (84%) and infection was common in children with AFP with CVB3, E6, and E11 constituting the predominant types detected. The CVB3 isolates cluster with Chinese CVB3 isolate recently detected in a newborn with AFP. There was also a remarkable clustering of isolates such as E6, E12, E13, E24, E30 and E33 to types previous detected in West Africa suggesting a probable circulation of these lineages in the region. Taken together, this study reveals a diverse species EV-B types in AFP cases and highlights the fact that other neurotropic EVs circulate in asymptomatic persons. Improved continuous surveillance of NPEV is warranted as in the likely attainment of polio eradication, other neurotropic EVs may emerge causing similar paralytic diseases., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In the replication process of RNA(+) viruses both the positive-strand template and the newly synthesized negative strand appear in a double-stranded form, RF. It has been shown for poliovirus that prior to the initiation of positive-strand synthesis, the 5'-terminus of the positive strand must adopt a cloverleaf structure. When that happens, the 3'-terminal region of the negative strand is released from the RF form and is able to form into its own defined structure. In order to determine the secondary structure of this region, a comprehensive approach consisting of experimental mapping methods, phylogenetic analysis and computer predictions was applied. Here we propose the first structural model of the 3'-terminal region of the coxsackievirus B3 (CV-B3) negative strand, approximately 450 nucleotides in length. The region folds into three highly defined structural domains, I'-III'. The most 3'-terminal part of this region is domain I', which folds into a cloverleaf structure similar to that found in the viral RNA strand of positive-polarity. Remarkably, this motif is conserved among all analyzed viral isolates of CV-B3 despite the observed sequence diversity. Several other conserved structural motifs within the 3'-terminal region of the viral negative strand were also identified. The structure of this region may be crucial for the replication complex assembly., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Calpain is a family of neutral cysteine proteinase involved in many physiological and pathological processes including virus replication, autophagy and apoptosis. Previous study has indicated the involvement of calpain in pathogenesis of coxsackievirus B3 (CVB3)-induced myocarditis. Besides, many studies demonstrated that host cell autophagy and apoptosis mechanisms participate in virus life cycle. However, role of calpain in CVB3 replication via autophagy/apoptosis mechanisms has not been reported, which was discussed here in H9c2 cardiomyocytes. The data demonstrated that calpain was activated following CVB3 infection. Calpain inhibition decreased autophagy, indicating role of calpain in enhancing autophagy during CVB3 infection. Both calpain activity and autophagy were involved in facilitating CVB3 replication demonstrated by virus titer and CVB3 capsid protein VP1 expression alterations resulting from calpain inhibitor ALLN and autophagy inhibitor 3MA intervention. We also found that both calpain activity and autophagy suppressed caspase3 activity and host cell apoptosis 5-10h post-infection (p.i.). In summary, the present study shows that CVB3 infection of H9c2 cells hinders caspase3 activity provocation and cell apoptosis at least in the early phase of infection (5-10h p.i.) via calpain-induced autophagy enhancement, which might be a mechanism facilitating CVB3 replication in host cells., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Decay accelerating factor (DAF, CD55) is used by DAF-dependent (Daf+) variants of echovirus 11 (EV11) as a primary cellular receptor. The interaction of EV11 with DAF is completely reversible, therefore DAF-dependent variants require an unidentified coreceptor to initiate uncoating. Daf- variants of EV11, which do not interact with DAF, use an alternative primary cellular receptor. The aim of this study was to test the hypothesis whether the coreceptor, which is necessary for the uncoating of DAF-dependent variants, may act as an alternative primary receptor for the Daf- variants of EV11. By using the model of the two closely related daf+ and daf- clones of EV11 in rhabdomyosarcoma (RD) cell line, it was shown that a single amino acid substitution in the capsid protein VP2 could control the expression of the DAF-dependent phenotype. Anti-DAF monoclonal antibody has blocked the infection of RD cells by the DAF-dependent daf+ clone, but not by the daf- clone of EV11. Since the structural proteins of the two clones differed only in the receptor binding site for DAF, the unidentified non-DAF primary receptor for the daf- clone might have the same conformation as the uncoating coreceptor required for the daf+ clone. Despite the difference in primary receptors, both daf+ and daf- clones were equally inhibited by a monoclonal antibody to beta2-microglobulin. The monoclonal antibody B9.12.1 to class I human leukocyte antigen molecules showed no inhibitory effect in regards to either clone. The hypothesis of convergent intracellular traffic of Daf+ and Daf- variants of EV11 is discussed., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Coxsackievirus B3 (CVB3) is the most important causal agent of viral heart muscle disease, but no specific antiviral drug is currently available. Small interfering RNA (siRNA) has been used as an antiviral therapeutic strategy via posttranscriptional gene silencing. In this study, eleven siRNAs were designed to target seven distinct regions of the CVB3 genome including VP1, VP2, VP3, 2A, 2C, 3C, and 3D. All of the siRNAs were individually transfected into HeLa cells, which were subsequently infected with CVB3. The impacts of RNA interference (RNAi) on viral replication were evaluated using five measures: cytopathic effect (CPE), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 50% tissue culture infectious dose (TCID(50)), real-time RT-PCR, and Western blot. Five of the eleven siRNAs were highly efficient at inhibiting viral replication. This was especially true for siRNA-5, which targeted the ATPase 2C. However, antiviral activity varied significantly among siRNA-9, -10, and -11 even though that they all targeted the 3D region. Our results revealed several effective targets for CVB3 silencing, and provided evidence that sequences except CRE within the 2C region may also be potential targets for CVB3-specific siRNAs design. These data supported a potential role of RNA interference in future antiviral intervention therapies., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In this study we present the comparative sequence analysis of the parental haemagglutinating (daf+) and mutant non-haemagglutinating (daf-) clones of echovirus 11 (EV11) isolated from the prototype strain Gregory. The sequence comparison revealed only a single amino acid substitution in the capsid protein VP2 of each mutant clone. These substitutions were located in the area of viral receptor-binding site for DAF. Since daf- mutants of EV11 did not interact with DAF, they used an alternative receptor for the cell entry. To elucidate the nature of the alternative receptor we used subvariant clones of EV11 adapted to human rhabdomyosarcoma (RD), human carcinoma (HEp-2) and African Green monkey kidney (BGM) cell lines. The usage of the subvariant clones with altered host range and the cell cultures of human and simian origin allowed us to map the amino acid substitutions associated with the adaptation of EV11 to the alternative cellular receptors. These amino acid substitutions were located on the surface of the virion in the canyon area. Hence the virus canyon may serve as the receptor-binding site for the alternative (in respect to DAF) cellular receptor(s).

Echovirus 11 (E-11) has been one of the most frequently discovered human enterovirus (HEV) in Finland during the past few years. We have studied molecular epidemiological patterns of E-11 from 1993 to 2007 exploiting the 257-nucleotide region in the 5'-part of the VP1 used for genetic typing of HEV. Designated genogroup D strains had a striking prevalence among the Finnish strains, a finding in accordance with the recent data from other geographical regions. The subgroup D4, harboring the oldest strains, had become extinct in the beginning of the millennium and D5 strains had taken over. Similarly, a new subgroup of D5 had started to diverge from the main D5 in 2006. However, in addition to endemic D strains, few single strains clustered also to genogroups A and C suggesting importation from more distant locations. The relatively large amino acid sequence variability between and within the genogroups favored the idea of antigenic differences. Neutralization assays confirmed that antigenic differences existed, although all studied E-11 strains were neutralized with antisera against the prototype strain Gregory. Five of the six studied strains belonging to genogroup D were, unexpectedly, also neutralized with antisera against coxsackievirus A9 Griggs.

Two mutants of coxsackievirus B1 (CVB1), CVB1c and CVB1e, with mutations in the stem-loop H of 5'-UTR were generated by site-directed mutagenesis. The A at nt579 of CVB1c was substituted by G. The U at nt573 and A at nt579 of CVB1e were substituted by A and G. The virulences of these mutants had been assessed by means of cytopathic effect (CPE), plaque formation, one-step growth curve, and 50% lethal dose (LD50) assays. The pathogenesis of these mutants was evaluated by attacking suckling Balb/c mice. Plaque assay and one-step growth curve showed that the replication of CVB1c and CVB1e on HeLa cells was significantly faster than that of their prototype CVB1n. Data of CPE assay, LD50, and pathological examination showed that CVB1c and CVB1e were more virulent than CVB1n. These data showed that mutation at nt579 (A-->G) alone and mutations at nt579 (A-->G) and nt573 (U-->A) together within 5'-UTR caused significant augment of the virulence and pathogenesis of coxsackievirus B1, and suggested that nt573 and nt579 might be molecular determinants for the virulence of coxsackievirus B1.

Molecular methods have enabled the rapid identification of new enterovirus (EV) serotypes that are untypeable using existing neutralizing antisera. As a result, sequencing of the VP1 capsid gene has been developed as a surrogate for antigenic typing to distinguish enterovirus types. In this study, 17 enterovirus isolates from four countries were identified as members of 13 new types within the species Human Enterovirus B (HEV-B) by complete genome sequencing. Members of each of these new types are at least 75% identical to one another (91% amino acid identity) in VP1, but members of different types differ from one another and from other enteroviruses by at least 27% in nucleotide sequence (26% amino acid sequence difference). The complete P1 (capsid) sequences of the new types are at least 17% different from those of all other enterovirus serotypes (14.5% amino acid sequence difference), but they are highly conserved within a type (<8% amino acid sequence difference). For both VP1 and P1, the 17 isolates are monophyletic by type with respect to all other EV serotypes. The P2 and P3 sequences are closely related to those of other HEV-B viruses (>93% amino acid identity), confirming that the 17 new strains belong to HEV-B. We propose that these 17 isolates be classified as members of 13 new human enterovirus types, enteroviruses 79-88, 97, and 100-101.

Chemically synthesized small interfering RNA (siRNA) has been used as an anti-coxsackievirus B3 (CVB3) agent. Herein, we investigated whether vector-derived short hairpin RNAs (shRNA) targeting CVB3 can exert antiviral activities, prior to their further application to viral vector system for efficient in vivo administration. Employing transient transfection assays to in vivo mouse models as well as to in vitro Cos-7 cell cultures, we directly demonstrated the potential antiviral activity of shRNAs following challenges with infectious CVB3. Of the six shRNAs that we designed, three prevented cell death from CVB3 infection by suppressing viral replication and viral production in Cos-7 cells. These were shRNA 2, which targeted the capsid protein VP1, and shRNAs 4 and 5, which targeted two different regions of the RNA-dependent RNA polymerase 3D. Furthermore, shRNAs 2 and 5 also exerted strong antiviral effects in viral replication in vivo, accompanied by attenuated pancreatic tissue damage. Through this direct evaluation system we addressed the development and application of vector-derived shRNAs as an anti-CVB3 agent, revealing new target sequences.

MAP kinase signaling has been implicated in coxsackievirus B3 (CVB3) pathogenesis and as necessary in the virus lifecycle. We studied the correlation with extracellular signal-regulated kinase 1/2 (ERK1/2) signaling and virus replication in the presence of coxsackievirus and adenovirus receptor (CAR). In CHO cells that do not expressed CAR, specific ERK1/2 phosphorylation (pERK1/2) was not detected, and progeny virus was not produced after infection. By contrast, in HeLa and CHO-CAR cells, which expressed CAR, the specific early and late pERK1/2 at 0.5 and 8 h were induced, and progeny viruses were produced progressively through 24 h after infection. However, when CHO-CAR cells were infected with replication-defective CVB3, specific pERK1/2 was not detected. In addition, when late pERK1/2 is inhibited by the MEK1 inhibitor PD98059, at 4 h after infection, virus replication significantly decreased. Therefore, our findings suggest that early pERK1/2 is a response to virus binding to CAR, whereas late pERK1/2 is related to the viral replication.

Translation initiation of the coxsackievirus B3 (CVB3) RNA has been shown to be mediated by a highly ordered structure of the 5'-UTR, which harbors an internal ribosome entry site (IRES). In this study, we have investigated the 48S ribosome assembly site and also characterized the intervening spacer region between the cryptic AUG591 and the initiator AUG742. The ribosomal complex formation was mapped by toe-printing experiment using rabbit reticulocyte lysate, which showed a major toe print at nucleotide U570 corresponding to the 48S ribosome assembly site at the putative Shine-Dalgarno like sequence. Elucidation of the secondary structure of a segment encompassing the ribosome binding site and the downstream spacer region by nuclease probing and chemical modifications demonstrated distinct stem and loop structure. Interestingly, a GAGA loop in the apical region of stem-loop H was found to be phylogenetically conserved as a GNRA-loop among the coxsackie B viruses. Deletion or substitution mutation of this apical GAGA loop drastically reduced binding with human La protein and significantly affected the IRES function. The study revealed important insights into the possible role of the intervening spacer region in cellular protein binding and influencing internal initiation of translation of CVB3 RNA.

The complete nucleotide sequence of an enterovirus 77 isolate is reported. The virus designated FR/CF496-99 (France/Clermont-Ferrand 496-1999) was recovered from the feces of a 4-year-old child hospitalized for Salmonella gastroenteritis. The virus was identified by a molecular typing assay based on the genomic sequence encoding the VP1 capsid protein. The phylogenetic analysis based on the VP1 sequence demonstrated that the enterovirus isolated in the child clustered with viruses included in the human enterovirus B species (HEV-B) and was most closely related to enterovirus 77. A sliding window analysis of the complete genome showed an overall nucleotide similarity >80% between the P3 genomic region of the FR/CF496-99 isolate and that of the echovirus 30 prototype strain. A comparative analysis based on partial 3D(pol) sequences showed that the FR/CF496-99 virus was more closely related to recent enteroviruses from different serotypes and different geographical areas than to the prototype strains collected in the 1950s. This suggests that, in this enterovirus, the 3D(pol) encoding sequence is of recent origin.

Echovirus 11 (E11) is among the most commonly isolated human enteroviruses. To examine the range of genetic variation within the E11 serotype, we determined the complete VP1 sequences for 53 geographically dispersed E11 strains isolated in 16 countries from 1953 to 2001. E11 sequences were monophyletic with respect to all other enterovirus serotypes. The sequences clustered into four monophyletic genogroups, A-D; members of each genogroup differed from one another by <20%. Isolates in different genogroups differed from one another by 19-28%. The E11 prototype strain, USA/CA53-Gregory, was the sole member of genogroup B. All recent US isolates were members of one of two discrete lineages within genogroup D. The well-characterized E11 antigenic variant, USA/CA63-Silva, was also a member of genogroup D. Members of genogroups A and C were antigenically similar to USA/CA53-Gregory, as measured by neutralization with anti-Gregory and anti-Silva antisera. Only USA/CA63-Silva was neutralized more efficiently by the anti-Silva antiserum; other genogroup D viruses were Gregory-like or intermediate in their neutralization phenotype. Recent non-US isolates were distributed in genogroups A, C and D. Sequence similarities among genogroup D isolates from North America, Europe, Asia, Australia and North Africa demonstrate that an E11 strain can spread rapidly over a wide geographic area. The aligned sequences were used to develop an E11-specific RT-PCR assay, using degenerate, inosine-containing primers, to amplify all members of all genogroups.

Translation initiation of the coxsackievirus B3 (CVB3) RNA occurs by internal ribosomal entry. The internal ribosomal entry site (IRES) of the virus has been mapped to the 5' untranslated region (5' UTR) of the genome. As well, the 5' UTR has been suggested to play roles in determining the tissue tropism and infectivity of the virus. In this study, we investigated interactions between HeLa cell protein extracts and radiolabeled RNA of CVB3 5' UTR by competitive UV cross-linking. We have observed a number of proteins that specifically interact with the three sub-cloned regions of the 5' UTR. In particular, the molecular weights of five of these proteins resemble those of the eukaryotic translation initiation factors 4A, 4B and 4G, as well as the La autoantigen and the polypyrimidine tract binding protein. Based on this data, we focused on the interaction of the 5' UTR with the La autoantigen, which was purified by the glutathione-S-transferase affinity method. We have confirmed the highly specific interaction of the La autoantigen with the 5' UTR sequence nt 210-529. The core IRES (nt 530-630) and nt 1-209 also appear to bind to the La protein at moderate and weak affinities, respectively. A functional role of the La autoantigen in translation initiation is suggested.

Echovirus 18 (EV18) is one of the echovirus serotypes associated with human diseases and in particular aseptic meningitis. To facilitate studies of the molecular epidemiology of EV18 and the evolution of enteroviruses in general, the complete nucleotide (nt) sequence was determined for the echovirus 18 prototype strain (Metcalf, EV18M). Excluding the poly A sequence, the genome consists of 7410 nt divided into a 740 nt 5' untranslated region (5' UTR), a 6567 nt long open reading frame coding for a 2189 amino acid (aa) polyprotein and a 103 nt 3' UTR. Molecular analysis of the EV18M genome showed a typical enterovirus-like organization. Phylogenetic analysis of the structural and non-structural genes revealed a pattern of different relationships to other echo- and coxsackieviruses. Similarity analysis demonstrated that the Hill strain of echovirus 9 is most likely the result of a previous recombination event between ancestors of the echovirus 9 strain Barty (5' half of the genome) and EV18M (3' half). Using a maximum likelihood approach, the recombination point was mapped to the 2C gene.

We report on clinical samples Stuttgart/97, Berlin/99 and Jasi/99 associated with aseptic meningitis. All three samples contained echovirus 4 (E4) but Stuttgart/97 was simultaneous infected with echovirus 30 (E30). The genetic relationship of the E4 strains was assessed using RT-PCR and direct sequencing of amplicons derived from the genomic region encoding the capsid protein VP1. The sequences have been compared with each other and with sequences of further E4 strains obtained from GenBank. The analysis confirms that sequences of recent isolates have drifted away from elderly strains over a longer period of time. Several amino acid changes in assumed antigenic sites of the VP1 gene may be sufficient to cause changes in antigenic specificity and therefore they may be a reason for failure of serological typing of some new antigenic E4 variants.

To identify the genetic determinants of virulence for swine vesicular disease virus, a panel of recombinant and site-directed mutant viruses were constructed from cDNA clones of a virulent J1'73 strain and an avirulent H/3'76 strain. Initial studies mapped the genetic determinants of virulence to either or both of the two sites at nucleotide (nt) 2842, encoding VP1-132, and nt 3355, encoding 2A-20. To determine their relative importance with regard to virulence, viruses mutated at either of these two sites from the avirulent to the virulent genotype and vice versa were tested in pigs. Viruses, mutated at nt 2842 to the virulent genotype (vSVLS104MJ1) or mutated at nt 3355 to the virulent genotype (vSVLS201MJ1), slightly recovered virulence but were very weak compared with viruses with site-directed mutations at both sites (vSVLS104/201MJ1). On the other hand, viruses, mutated at nt 2842 to the avirulent genotype (vSVLS104M00) or mutated at nt 3355 to the avirulent genotype (vSVLS201M00), did not have attenuated virulence. Sequence analysis of viruses recovered from inoculated pigs revealed that reversion at nt 3355 to the virulent genotype occurred in pigs which had been inoculated with vSVLS201M00. These results suggested that both amino acids determined the virulent phenotype, but that the 2A-20 site might be the major determinant for virulence.

Echoviruses (EV) 1 and 8 were originally considered to be distinct serotypes, but more recently have been considered strains of the same virus. In experiments with chimeric recombinant fusion proteins, both viruses bound to the I domain of the integrin VLA-2, and both required the same receptor residues for attachment. A full-length, infectious cDNA clone encoding EV1 was obtained; its nucleotide sequence was determined, as were the sequences encoding the EV8 capsid. EV1 and 8 show 94% amino acid identity within the capsid region and are more similar to each other than to any other human picornavirus.

The full length sequence for the human pathogen coxsackievirus B6 (CVB6, Schmitt strain) has been determined. We used long RT-PCR to generate full length DNA amplicon of CVB6, and then directly sequenced the amplicons. One-step cloning of the full length amplicon enabled us to obtain an infectious clone of CVB6. RNA generated from CVB6 amplicon DNA or CVB6 clones, by transcription with T7 RNA polymerase, was demonstrated to be infectious upon transfection into HeLa cells in vitro. The CVB6 genome is characteristic of enteroviruses, with a 5'-non-translated region (743 nucleotides) followed by an open reading frame (encoding a 2184 amino acid polyprotein) and a 3'-non-translated region (100 nucleotides) and polyadenylated tail. The predicted amino acid sequence of CVB6 clustered with the other CVB serotypes and swine vesicular disease virus (SVDV).

The human picornavirus coxsackievirus B2 (CVB2) is often linked to several infections, from mild respiratory diseases to more severe illnesses such as myocarditis. In this study, we report the complete genome sequence of CVB2 prototype strain Ohio-1. The genome sequence was determined from reverse transcribed viral RNA, amplified with long distance PCR and used for non-radioactive sequencing. The full length PCR amplicons were used for in vitro transcription and the obtained cRNA was lipofected onto green monkey kidney cells, in order to confirm that the PCR generated sequence reflects a viable virus RNA. The CVB2 genome sequence shows a typical enterovirus genome organization with a total length of 7411 nucleotides. Phylogenetic analysis, using the CVB2 polyprotein in comparison with other enterovirus polyproteins, clearly shows that CVB2 clusters with the coxsackievirus B-like enteroviruses and is more related to coxsackievirus B4 (CVB4) than any other published CVB serotype. The grouping of CVB2 and CVB4 as one subgroup has earlier been reported in connection with receptor usage and ability to replicate in different cell lines. The exposed viral capsid proteins of CVB2 (VP1-VP3) show high similarity to other CVB proteins, except in regions that are likely to be surface epitopes.

Echovirus 5 (EV5) may be isolated from various neurological and exanthematic diseases. To determine the relationship of EV5 to other enteroviruses and for studies of its interactions with the target cell, the complete nucleotide sequence of EV5 was determined. Three overlapping fragments, collectively representing the complete genome, were amplified with RT-PCR and sequenced. Analysis of the EV5 sequence revealed a typical enterovirus-like organization of the genome. To verify that the cDNA generated sequence was derived from infectious viruses, complete EV5 genomes were amplified in one amplicon by long distance PCR. Transfection of in vitro transcribed RNA from these amplicons into cell cultures resulted in replicating EV5. Comparison of the overall nucleotide and amino acid sequences demonstrates that EV5 can be regarded as a coxsackievirus B-like enterovirus. Variable sequences between EV5 and the well characterized coxsackievirus B3 (CVB3) are for the most part observed for amino acid residues that correspond to exposed sequences in the CVB3 capsid. This observation indicates that the reported EV5 strain recently diverged from group B coxsackieviruses.

To define the relationship between echovirus 23 (E23) and other human enteroviruses, we have determined the complete nucleotide sequence of a strain of E23 isolated from a child with high fever in Connecticut in 1986 and compared the nucleotide and deduced amino acid sequences with those of other enteroviruses representing each of the major enterovirus phylogenetic groups, poliovirus type 1, coxsackievirus A16, coxsackievirus B3, echovirus 22 (E22), and enterovirus 70. The genome of E23 (strain CT86-6760) was 7352 nucleotides in length, exclusive of the poly(A) tail, and the genome organization was typical of the picornaviruses. The nucleotide sequence and deduced amino acid sequences were most related to those of E22, a virus with which E23 shares many biological properties, and was quite divergent from the sequences of other enteroviruses (< 20% average amino sequence identity). These data lend further support to the suggestion that E22 and E23 are distinct from members of the Enterovirus genus and that they should be reclassified in a separate genus within the Picornaviridae.

An infectious cDNA of a highly myocarditic coxsackievirus B3 (CVB3m; Nancy strain) was cloned. Sequence data revealed 43 extra non-viral nucleotides upstream of the initial 5' sequence. However, the authentic 5' end sequence was maintained during replication of viral RNA transfected into HeLa cells, suggesting the RNA synthesizing complex edits the picornaviral 5' terminus sequence. Nucleotide sequences of the 5' nontranslated region and the capsid protein gene sequence of CVB3m were compared with the published sequences of five other CVB3 Nancy strains and two main lineages were found. In comparative assays for cardiovirulence, three of four CVB3 tested were cardiovirulent in adolescent male CD-1 mice. Only one of the three available CVB3 strains was neutralized with several anti-CVB3m monoclonal antibodies, suggesting that mutations in the surface epitopes of the capsid polypeptides contribute to antigenic drift within the serotype, perhaps in part through immunoselective pressures. Thus, phenotypic diversity of CVB3 within the prototype Nancy strain is an example of RNA viruses adapting to changing environments (cells, mice and humans) through mutations and selective pressure.

Fifteen clinical coxsackievirus B3 (CVB3) isolates were assessed for cardiopathologic capabilities in adolescent male CD-1 mice in comparison to two well characterized cardiovirulent CVB3 strains. One isolate was cardiovirulent, one minimally cardiovirulent and the remaining 13 isolates were noncardiovirulent. The two cardiovirulent isolates and one well characterized cardiovirulent strain, established higher viremic titers, in comparison to five noncardiovirulent isolates that were examined. The two cardiovirulent isolates and one well characterized cardiovirulent strain replicated to significantly higher titers than five noncardiovirulent isolates in primary cultures of murine neonatal or adolescent cardiac fibroblasts. Nucleotide sequence analysis of an area defined by nucleotides(N)300-N599 in the 5'-nontranslated region were performed on the two well characterized cardiovirulent CVB3 strains, the two cardiovirulent isolates and 12 noncardiovirulent isolates. The data detected a single discriminatory nucleotide position. An A was present at N565 in three of four cardiovirulent CVB3, whereas a U or C was present in this position in 12 of 12 noncardiovirulent CVB3. In toto, these data are compatible with the hypothesis that the type of the nucleotide at N565, a position within the internal ribosome entry site, is associated with capacity of a CVB3 for replication in vivo and in vitro and this capacity for vigorous replication is associated with cardiovirulence.

The 5'-non-translated region (NTR) of enteroviruses contains secondary structures which do not only serve in the initiation of translation but also in the initiation of plus-strand RNA synthesis by binding of viral and cellular proteins. To investigate a very early step of enteroviral replication by cis- and trans-complementation, 220 nucleotides of the 5'-region of coxsackievirus B3 (CVB3) were exchanged with the corresponding region of poliovirus type 1 (PV1) to yield the chimeric virus CVB3[PV5']. The viability of this chimera demonstrates that the polioviral cloverleaf structure of the 5'-NTR is functional in the replication of a chimeric CVB3 RNA. The HeLa-generated chimera reveals a 4-nucleotide deletion (nt 232-235) within a short direct repeat. Besides clearly reduced growth characteristics in all permissive cell lines, the chimera exhibits a small-plaque phenotype. The host range is changed since the virus grows well in human HeLa cells, but does not replicate in murine YAC-1 and Ltk cells, although these cell lines are permissive for the replication of both parental viruses. Moreover, in simian Vero, COS-1, or FRhK-4 cells the HeLa-generated chimera CVB3[PV5'] exhibits a strict temperature sensitivity at 39 degrees C. After infection of simian cells with high m.o.i. in situ hybridization data reveal that the chimera replicates in single cells at almost normal rates indicating that only a small fraction of HeLa-generated virus is able to multiplicate in simian cell lines. After passaging the virus chimera in Vero cells two further mutations occur at nucleotide positions 185 and 227. Since this genome region is known to interact with viral proteins and several host cell factors during the initiation of replication and translation, interactions of such factors with either viral RNA or viral proteins may be disturbed but still functional at permissive temperatures in HeLa cells and simian cell lines, whereas murine cell lines are not permissive. These experiments suggest that phenomena like host range, tissue tropism and cell-type specificity may be explained as a complex interplay of cellular surface receptors and intracellular host factors. Such intracellular factors could be part of the enteroviral initiation complex during the plus-strand RNA synthesis or during translation initiation and could be expressed in a tissue-, organ- or species-specific way or might be regulated developmentally.

Genomic sequences in VP1/2A and 5'-non-coding region of 10 isolates of Coxsackievirus B5 from three outbreaks were compared with published sequences of another Coxsackievirus B5, swine vesicular disease virus, Coxsackievirus B1, Coxsackievirus B3, and Coxsackievirus B4. Isolates of Coxsackievirus B5 from the same outbreak showed close relations, not exceeding 7.2% in nucleotide differences. Differences were greater between isolates from different outbreaks, varying between 8.4 and 16%. We have also shown that Coxsackie B5 viruses from an outbreak in 1967 are more similar to viruses from an outbreak in 1983 than to the viruses isolated from an intervening outbreak in 1972. The sequence comparison of Coxsackievirus B5 isolates with other Coxsackie B viruses and swine vesicular study, cDNA synthesis, polymerase chain reaction, and sequencing, are suitable for rapid Coxsackie B virus detection and identification of genotypic relations between viruses originating from different outbreaks.

Echoviruses are the largest enterovirus subgroup consisting of 32 serotypes. They are common human pathogens causing, for example, meningitis, encephalitis and exanthema, but in spite of their clinical importance, relatively little is known about their biology. To illuminate the molecular characteristics of echoviruses, we have completed the genomic sequence of serotype 11. The RNA genome is 7438 nucleotides in length and it codes for a 2195 amino acid long polyprotein. When compared to other sequenced enteroviruses, echovirus 11 (EV11) shows remarkable similarity with coxsackie B viruses (CBVs) and coxsackievirus A9 (CAV9). On the basis of amino acid sequence homology in the capsid region, CAV9 is the virus most closely related to EV11. These two viruses have an apparent insertion sequence located at the C-terminus of the VP1 polypeptide. EV11, however, lacks the RGD motif found in the corresponding region of CAV9. The organization of the 5' end noncoding region resembles that of other enteroviruses, but contains a 12 nucleotides long poly-U stretch not seen in any other enterovirus sequenced to date.

Coxsackievirus B3 (CVB3) as a potential RNA virus vector for the presentation of foreign antigenic epitopes was further characterized. Insertion mutagenesis of infectious CVB3 cDNA yielded viable antigen chimeras containing variant BC loops of VP1 of coxsackievirus B4 (CVB4). Analysis of three antigen chimeras allowed the mapping of the N-terminal part of the neutralizing antigenic site 1 (N-Ag1) of CVB4 which is located in the BC loop of the structural protein VP1. A significant neutralization of a viable chimera with the deletion of CVB4-specific amino acid Ser-83 at the amino terminus of the VP1 BC loop was obtained with CVB4 serotype-specific polyclonal antisera. This neutralization was reduced after further deletion of the adjacent Ala-84, suggesting that this amino acid either constitutes the beginning of N-Ag1 of CVB4 or is essential for the conformation of the adjacent epitope. In contrast, exchange of amino acid Ser-86 to alanine, in the middle of the BC loop, led to complete loss of reactivity with CVB4-specific antibodies, demonstrating the importance of this residue for binding of CVB4 neutralizing antisera. Furthermore, we observed that manipulations of the VP1 BC loop resulted in increased thermolability of the viable chimeras in comparison to CVB3, although replication efficiencies were similar.

Coxsackieviruses are divided into A and B subgroups on the basis of their pathogenicity in newborn mice. Although used in the classification of these viruses, our understanding of the details of the infection is incomplete due to the lack of sensitive and specific techniques to localize the viruses in affected tissue. We have used in situ hybridization to detect coxsackievirus genomes in tissues of newborn mice after infection by five serotypes (A2, A9, A21, B3 and B4) through different administration routes. Our results indicate that coxsackie A viruses are able to affect both skeletal and heart muscle while the coxsackievirus B subgroup infects a wide range of tissues. In addition to striated muscle these include central nervous system, liver, exocrine pancreas and brown fat. This model will make it possible to analyze molecular factors determining tissue tropism.

The RD variants of group B coxsackieviruses differ from their parental strains in having the ability to replicate in a human rhabdomyosarcoma cell line, RD. The nucleotide sequence of the P1 region of the RD variant of coxsackievirus B3 strain Nancy (CB3NRD) was determined by sequencing cloned cDNAs, obtained by PCR amplification. A comparison between the established nucleotide sequence and that of the P1 region from the parental virus revealed 12 point mutations which corresponded to six amino acid replacements. To identify if the P1 region is responsible for the phenotype of CB3NRD, a chimeric virus was constructed, using an infectious cDNA clone of CB3. The P1 region of the infectious cDNA was replaced by cDNA fragments from CB3N (parental strain Nancy) or CB3NRD and the resulting recombinants were assayed for their ability to infect and replicate in RD cells. The results showed that the RD phenotype of CB3NRD maps in the P1 region. Furthermore, a chimera which only contained the 5' part of the P1 region derived from CB3NRD and the remaining P1 sequence from CB3N was able to replicate in RD cells, suggesting that the VP2 polypeptide contains at least one determinant for the RD phenotype.

We have previously shown that a major determinant of virulence for coxsackievirus B4 mapped to the 5' end of the viral genome. Comparison of the corresponding cDNA sequences of a virulent and a non-virulent virus has allowed the identification of candidate determinants of virulence in the 5' untranslated region and the capsid proteins VP1, VP2 and VP4. Thirteen nucleotide substitutions were observed in a region spanning 3298 nucleotides. Four mutations were detected in the non-coding region. Of the remaining nine mutations, four were silent while five resulted in amino acid substitutions in VP1, VP2 and VP4. The amino acid substitutions in the virulent virus were analyzed in relation to the three-dimensional structures of the capsid proteins of poliovirus. Two substitutions mapped to the amino termini of VP1 and VP4. Of the two substitutions observed in VP2, one mapped to the large loop that connects beta strand E with the radial helix on the back surface of the eight-stranded antiparallel beta barrel while the other mapped to beta strand G. One amino acid substitution in VP1 mapped to the loop connecting beta strands D and E at a site close to a major determinant of attenuation in poliovirus type 2.

The nucleotide sequence from the 5' terminus to nucleotide 3822 has been determined for the genome of the enterovirus, coxsackievirus B3 (CB3). This region encompasses the 5'-terminal 738 nucleotide non-coding sequence and the region which codes for the four viral capsid proteins and for the initial products of the P2 region. Regions in the 5' non-translated RNA sequence which may be involved with a structural and/or regulatory role are discussed.

Coxsackie B viruses are known etiological agents of pancreatic diseases, including diabetes. The pathogenesis of these infections is influenced by both host and viral factors. In this report, we examined whether the outcome of Coxsackie B4 virus infection is dependent on the genes within the major histocompatibility complex (MHC). We generated a pancreatic variant, CB4-V and established an animal model system of pancreatitis with concurrent hypoglycemia in mice. Infection of various B10 H-2 congenic strains of mice revealed that the development of hypoglycemia with accompanying pancreatitis was independent of the MHC haplotype. However, the severity of the disease as monitored by the extent and duration of hypoglycemia and by mortality rate was found to be associated with the H-2 haplotype, specifically the H-2Kq locus. Pancreatic damage induced by CB4-V appeared to be both immune-mediated and viral-mediated. Histological examination of pancreatic tissue from infected B10 H-2 congenic mice revealed an association between acute destruction of the exocrine pancreas and lymphocytic infiltration. This infiltration may correlate with immune-mediated destruction of the infected pancreatic tissue. Since preferential replication of CB4-V was not observed in the most susceptible B10 mouse strain, direct viral destruction may not be the major mechanism of pancreatic injury.