Biochemistry and pathogenicity of echovirus 9. II. Mutants of echovirus 9, strain Hill, with altered capsid surface properties

The two echovirus 9 strains Hill and Barty have been shown previously not only to differ in pathogenicity for newborn mice but also in a number of in vitro characteristics which depend on viral capsid structure. Three spontaneously occurring mutants of the mouse-apathogenic echovirus 9 prototype strain Hill being resistant to an inhibitor of plaque formation present in agar were isolated and compared biochemically and biophysically to their parent strain and to the mouse-pathogenic echovirus 9 strain Barty, which is resistant to this inhibitor. The mutants differ from their apathogenic parent strain Hill in most of the same in vitro characteristics as strain Barty, namely adsorption to cells in culture, sedimentation behavior in low salt sucrose gradients, distribution of mutant virus particles in isoelectric focusing, and antigenic determinants inducing neutralizing antibodies. For two of the three mutants, Ag2 and Ag3, evidence was obtained from fingerprinting that they differ from their parent strain Hill in VP1; thus, the observed in vitro properties may be caused by the change in this capsid protein. All mutants, however, were found to be apathogenic for newborn mice and do not replicate in the tissues of these animals. It is concluded that the observed changes in capsid structure do not covary with virulence.     

Molecular cloning and sequence determination of the complete genome of the virulent echovirus 9 strain Barty

As part of a study of the molecular basis of pathogenicity of echovirus 9, the complete nucleotide sequence of the mouse-virulent echovirus 9 strain Barty was determined. Excluding the poly(A) tail, the complete RNA genome is composed of 7451 bases. The postulated open reading frame extends from nucleotide (nt) 741 to 7349 and predicts a polyprotein of 2203 amino acids (aa). As compared with the sequence of the echovirus 9 prototype strain Hill, which is apathogenic for newborn mice, 1492 nt are exchanged, leading to 9% divergence of the deduced amino acid sequence. The foremost difference between both strains is located at the C-terminus of the capsid protein VP1. In the case of strain Barty, an additional 10 aa fragment, including an RGD motif, is inserted.     

Molecular and biological analysis of echovirus 9 strain isolated from a diabetic child

The full-length infectious cDNA clone was constructed and sequenced from the strain DM of echovirus 9, which was recently isolated from a 6-week-old child at the clinical onset of type 1 diabetes. Parallel with the isolate DM, the full-length infectious cDNA clone of the prototype strain echovirus 9 Barty (Barty-INF), was constructed and sequenced. Genetic relationships of the sequenced echo 9 viruses to the other members of the human enterovirus type B species were studied by phylogenetic analyses. Comparison of capsid protein sequences showed that the isolate DM was closely related to both prototype strains: Hill and Barty-INF. The only exception was the inner capsid protein VP4 where serotype specificity was not evident and the isolate DM clustered with the strain Hill and the strain Barty-INF with echovirus 30 Bastianni. Likewise, the nonstructural protein coding region, P2P3, of isolate DM was more similar to strain Hill than to strain Barty-INF. However, like echovirus 9 Barty, the isolate DM contained the RGD-motif in the carboxy terminus of capsid protein VP1. By blocking experiments using an RGD-containing peptide and a polyclonal rabbit antiserum to the alpha(v)beta(3)-integrin, it was shown that this molecule works as a cellular receptor for isolate DM. By using primary human islets, it was shown that the isolate DM is capable of infecting insulin-producing beta-cells like the corresponding prototype strains did. However, only isolate DM was clearly cytolytic for beta-cells. The infectious clones that were made allow further investigations of the molecular features responsible for the diabetogenicity of the isolate DM.     

Biochemistry and pathogenicity of echovirus 9

Different clinical isolates of echovirus 9 are known to vary strikingly with regard to pathogenicity. Prototype strain Hill and strain Barty have previously been shown to differ not only in paralytogenic potency for newborn mice but also in a number of in vitro characteristics related to virus capsid structures. A series of mutants of strain Barty, thermosensitive for replication at 40 °C, was isolated after mutagenization with 5-fluorouracil. For all mutants the virus dose required to paralyse 50% of the infected animals was significantly higher than of the parent strain Barty. This reduced pathogenicity was observed at normal room temperature where the baby mice had a body temperature of 32.5 °C, which is even below the permissive temperature for growth of the mutants. The paralytogenic potencies did not further decrease when the mice where kept at elevated room temperature and had a body temperature of 35.1 °C. Thus, the reduced pathogenicity is apparently not a direct consequence of thermosensitivity of growth. Biochemical and biophysical characterization indicated that at least two of the eight mutants have an alteration in capsid protein.     

Isolation and preliminary characterization of antigenic variant of echovirus type 11

Nosocomial infection with echovirus type 11 resulting in aseptic meningitis occurred among newborn babies in a hospital neonatal room at Fukui city. The virus was identified as a variant of echovirus type 11 by cross-neutralization tests with antisera against the prototype Gregory strain and the current Fukui isolate. Fukui isolates expressed strain specific antigen(s) in addition to type specific common antigen(s), but lacked a certain antigen(s) which was present in the prototype strain. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of virus polypeptides revealed that the capsid proteins VP2 and VP3 of Fukui strain migrated more rapidly than the Gregory strain, while both strains had the same migration pattern of VP1 protein on which the antigenic determinants responsible for virus neutralization were present. The current strain produced large plaques and was more thermoresistant, suggesting some alterations in the structural proteins of the virus.     

Echovirus 6 strains derived from a clinical isolate show differences in haemagglutination ability and cell entry pathway

Two echovirus 6 (EV6) strains were isolated from a clinical sample after successive sub-cultures in PLC (human hepatocellular carcinoma) and HeLa (human cervical adenocarcinoma) cells. The first strain retained its haemagglutinating capacity (HAEV6) while the second became non-haemagglutinating (NHAEV6). Virus binding assay showed that HAEV6 was capable of binding to DAF-expressing cells but not NHAEV6 confirming the role of DAF in EV6 haemagglutination. The lack of competition between the two viral strains during coinfections suggested that each strain used a different cell entry pathway. We provide evidence showing that HAEV6 used preferentially the lipid raft-dependent caveolae pathway, whereas NHAEV6 followed the clathrin-mediated pathway. Comparison of the sequences of HAEV6 and NHAEV6 revealed five amino acid changes in the VP1, VP2 and VP3 capsid proteins distributed in domains which are known to be highly immunogenic or suggested to be involved in receptor binding, virion stability and pathogenicity.     

Heterogeneity of capsid proteins of echovirus type 25 wild-type strain and prototype strain, studied by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting were used to compare the capsid proteins of 19 antigenic variants of echovirus type 25 wild-type strains isolated in France between 1976 and 1987 with those of the prototype JV-4 reference strain isolated in 1957. Immunoblots were developed by using polyclonal sera from rabbits and mice immunized with the reference strain. Immunoblotting patterns revealed reactivity only against viral protein VP1 for sera from both animals. Comparative immunoblotting patterns showed differences in the electrophoretic mobilities of viral protein VP1, especially for the Montpellier 76.1262 wild-type strain. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of [35S]methioinine-labeled viral polypeptides revealed that the two variant strains, Montpellier 76.1262 and Thionville 86.222, exhibited significant and reproducible shifts in the relative mobilities of VP1 and VP3 and, to a lesser extent, in those of VP0 and VP2. The relative mobility of VP4 seemed very similar for the JV-4 reference strain and the two variants. Interestingly, the structural differences in VP1 and VP3 of Montpellier 76.1262 were not correlated with the pattern of neutralization by monoclonal antibodies, unlike in our previous study, in which this strain differed from the prototype strain in only two epitopes. We concluded that, in addition to the heterogeneity of their biological and antigenic properties that we observed previously, echovirus type 25 wild-type strains may exhibit differences in their structural proteins.     

Induction of neutralizing antibodies and immunity in vaccinated guinea pigs by cyanogen bromide-peptides of VP3 of foot-and-mouth disease virus.

The specificity of guinea pig antisera against large cyanogen bromide-cleaved peptides of the virus capsid protein VP3 of foot-and-mouth disease virus type O1, strain Kaufbeuren has been characterized by double immunodiffusion, virus neutralization and protection tests. Antibodies to purified 146S particles and the cleavage peptides of VP3 showed an incomplete cross-section against VP3 peptide antigen when reacted in immunodiffusion tests, indicating that new antigenic determinants are exhibited by the peptides which are not recognized by the antiserum against the native virus proteins. The immune response against the reduced, unfolded chain constituents of VP3 was lower in comparison to that of native virus particles but still some immunological determinants remained actively capable of inducing virus-neutralizing antibodies in immunized guinea pigs.     

Molecular analysis of the echovirus 18 prototype: evidence of interserotypic recombination with echovirus 9

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.     

Human parechoviruses--biology and clinical significance

A new genus of the family Picornaviridae, Parechovirus, has recently been recognised on the basis of distinctive biological and molecular properties. In particular: parechoviruses exhibit characteristic effects on the host cell; cleavage of the capsid protein VP0, required for maturation of the virus particle in most other picornaviruses, does not occur; there is a unique extension, which is highly basic in character, to the N-terminus of the capsid protein VP3; and the 2A protein, in common with those of only two other known picornaviruses, is a homologue of a family of cellular proteins involved in the control of cell proliferation. The type member of the Parechovirus genus is a frequent human pathogen, formerly known as echovirus 22, which has been renamed human parechovirus 1. The genus also includes the closely related virus, human parechovirus 2 (formerly echovirus 23). Human parechoviruses generally cause mild, gastrointestinal or respiratory illness, but more serious consequences of infection, such as myocarditis and encephalitis have been reported. Most infections occur in young children. Ljungan virus, a newly identified virus of rodents, shares a number of molecular features with the human parechoviruses, raising important questions about the evolution of parechoviruses and their introduction into the human population.     

Characterization of polyclonal antibodies against the capsid proteins of Ljungan virus

Ljungan virus (LV) is a suspected human pathogen isolated from voles in Sweden and North America. To enable virus detection and studies of localization and activity of virion proteins, polyclonal antibodies were produced against bacterially expressed capsid proteins of the LV strain, 87-012G. Specific detection of proteins corresponding to viral antigens in lysates of LV infected cells was demonstrated by immunoblotting using each one of the generated polyclonal antibodies. In addition, native viral antigens present in cell culture infected with LV strains 87-012G or 145SLG were detected in ELISA and by immunofluorescence using the antibodies against the VP0 and VP1 proteins. The anti-VP3 antibody did not react with native proteins of the LV virion, suggesting that the VP3 is less potent in evoking humoral response and may have a less exposed orientation in the virus capsid. No activity of the antibodies was observed against the closely related human parechovirus type 1. The polyclonal antibody against the VP1 protein was further used for detection of LV infected myocytes in a mouse model of LV-induced myocarditis. Thus, polyclonal antibodies against recombinant viral capsid proteins enabled detection of natural LV virions by several different immunological methods.     

Fractionation of biologically active and inactive populations of human rhinovirus type 2.

Infectious virions of human rhinovirus type 2 (HRV-2) migrate during electrophoresis to pH 6.4 in a sucrose-stabilized pH gradient. As already reported, acidification of HRV-2 produces two kinds of noninfectious components both of which have lost the smallest virion polypeptide, VP4: One has lost RNA in addition to VP4 and sediments at about 80 S, while the other retains RNA and sediments at 135 S. The 80 S particles migrate to pH 4.5 upon electrophoresis while the 135 S particles migrate to pH 4.2. Natural top component of HRV-2 is also separable into two subpopulations that are isoelectric at pH 6.3 and pH 4.5. Only the particles isoelectric at pH 6.3 react with virion (D) specific serum and attach to HeLa cells. The polypeptide compositions of both the attaching and nonattaching fractions of natural top component are the same, and therefore the differences in activity and isoelectric point may reflect the arrangement of the polypeptides in the capsids. After a preparation of purified infectious virus is focused to its isoelectric point some particles are found at pH 4.5. These particles are relatively noninfectious, yet contain RNA and a full complement of polypeptides, and they sediment at 140 S. They are unable to attach to host cells. These results argue against a direct role of VP4 or RNA in the attachment process and support the view that protein conformation changes occur during the inactivation of HRV-2 virions.     

Comparative sensitivities of solid-phase immune electron microscopy and enzyme-linked immunosorbent assay for serotyping of human rotavirus strains with neutralizing monoclonal antibodies.

Suspensions of 24 rotavirus strains, 6 for each known human rotavirus serotype, were serially diluted and titrated by (i) enzyme-linked immunosorbent assay (ELISA) for rotavirus detection, using monoclonal antibodies (MAbs) specific for group-specific sites of the VP6 inner capsid protein; (ii) ELISA for subgrouping, using MAbs reactive with subgroup-specific determinants of rotavirus VP6; (iii) ELISA for serotyping, using MAbs directed to serotype-specific sites of the VP7 outer capsid glycoprotein; and (iv) solid-phase immune electron microscopy (SPIEM) for serotyping, using VP7-specific MAbs. In addition, in each preparation the proportion of double-shelled rotavirus particles were determined by direct electron microscopy. Results showed that SPIEM was 2- to 16-fold more sensitive than ELISA for serotyping of rotavirus. The titers in VP7-specific tests correlated well with the proportion of double-shelled virus particles in each of the samples. Titers obtained by ELISA for serotyping of suspensions containing 20% or fewer complete particles were up to 4,096-fold lower than those obtained by ELISA for detection. ELISA serotyping titers of samples containing 20 to 80% double-shelled rotavirus particles were up to 128-fold lower than ELISA detection titers, whereas preparations with nearly 100% complete particles had ELISA titers that were less different from each other. ELISA subgrouping titers were four- to eightfold lower than corresponding rotavirus detection titers. It was concluded that, although SPIEM appears to be more sensitive than ELISA, the amount of complete virus particles in the specimens is of critical importance for successful serotyping of human rotavirus strains. Samples rich in single-shelled particles but containing low amounts of VP7 outer capsid glycoprotein might even be strongly reactive in assays for rotavirus detection and subgrouping but virtually unreactive in tests for serotyping.     

Molecular typing of echovirus serotype 4 isolates

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.     

Genetic variability within the VP1 coding region of echovirus type 30 isolates

Summary.  Genetic relationships of the prototype Bastianni strain of 1958 and of 13 echovirus type 30 (ECV30) isolates associated with meningitis cases in Germany during a period from 1966 to 1997 were investigated using direct sequencing of amplicons derived from a part of the capsid protein VP1 gene. Sequences were aligned both with each other and with known sequences of other type 30 echovirus strains. Phylogenetic analysis indicated that isolates investigated in this work fell into at least three genetic clusters apart from the prototype Bastianni strain. This suggests that genetically distinct groups of ECV30 variants have developed over time. Received January 17, 2000 Accepted February 22, 2000     

Interactions between multiple genetic determinants in the 5' UTR and VP1 capsid control pathogenesis of chronic post-viral myopathy caused by coxsackievirus B1

Mice infected with coxsackievirus B1 Tucson (CVB1(T)) develop chronic, post-viral myopathy (PVM) with clinical manifestations of hind limb muscle weakness and myositis. The objective of the current study was to establish the genetic basis of myopathogenicity in CVB1(T). Using a reverse genetics approach, full attenuation of PVM could only be achieved by simultaneously mutating four sites located at C706U in the 5' untranslated region (5' UTR) and at Y87F, V136A, and T276A in the VP1 capsid. Engineering these four myopathic determinants into an amyopathic CVB1(T) variant restored the ability to cause PVM. Moreover, these same four determinants controlled PVM expression in a second strain of mice, indicating that the underlying mechanism is operational in mice of different genetic backgrounds. Modeling studies predict that C706U alters both local and long range pairing in the 5' UTR, and that VP1 determinants are located on the capsid surface. However, these differences did not affect viral titers, temperature stability, pH stability, or the antibody response to virus. These studies demonstrate that PVM develops from a complex interplay between viral determinants in the 5' UTR and VP1 capsid and have uncovered intriguing similarities between genetic determinants that cause PVM and those involved in pathogenesis of other enteroviruses.     

Molecular typing and epidemiology of enteroviruses identified from an outbreak of aseptic meningitis in Belgium during the summer of 2000

Non-polio enteroviruses are the most common cause of aseptic meningitis worldwide. From May to September 2000, a major outbreak of aseptic meningitis occurred in Belgium. Cerebrospinal fluid samples (CSF) of 122 patients were found to contain enterovirus RNA using diagnostic RT-PCR that targeted a 231-bp gene fragment in the 5' noncoding region. In addition, a molecular typing method was developed based on RT-nested PCR and sequencing directly from CSF(a) 358-bp fragment in the aminoterminal part of the VP1 capsid protein. To identify the enterovirus type, nucleotide sequences of the VP1 amplicons were compared to all the enterovirus VP1 sequences available in GenBank. Echovirus 30 (31.2%), echovirus 13 (23.8%), and echovirus 6 (20.5%) were identified most frequently during the epidemic. Coxsackievirus B5 was present in 15.6% of the samples, and could be subdivided in two distinct epidemic clusters, coxsackievirus B5a (10.7%) and B5b (4.9%). Other enteroviruses encountered were echovirus 16 (5.7%), echovirus 18 (1.6%), coxsackievirus B4 (0.8%) and echovirus 7 (0.8%). The high prevalence of echovirus 13, considered previously a rare serotype, indicates it is an emerging epidemic type. To verify the typing results and to explore further the intratypical genetic variation, phylogenetic analysis was carried out. Geographical clustering of most of the strains within each type and subtype could be observed. The RT-nested PCR strategy, carried out directly on clinical samples, is a simple and rapid method for adequate molecular typing of the Group B enteroviruses causing aseptic meningitis.     

Analysis of SAT1 type foot-and-mouth disease virus capsid proteins: influence of receptor usage on the properties of virus particles

The three SAT serotype viruses, endemic in Africa, are well known for their difficulty to adapt to cell culture. The viral mechanism involved in foot-and-mouth disease virus (FMDV) tissue tropism and cell-entry is not well understood. A recombinant, small plaque-forming virus (vSAT1tc), derived from a tissue culture-adapted SAT1 virus (SAR/9/81tc), revealed four amino acid substitutions (VP3 Asp192→Tyr; VP3 Ser217→Ile; VP1 Ala69→Gly and VP1 Asn110→Lys) in the capsid, compared to the SAR/9/81wt isolate collected from infected impala epithelium. One substitution added a positively charged lysine residue to the short βF-βG loop of VP1. Furthermore, vSAT1tc displayed a high affinity for CHO-K1 cells possibly via interaction with negatively charged sulphated polysaccharides while SAT1 impala strain relied strongly on α(V)β6 integrin receptors for cell entry. The cell culture adaptation and small plaque phenotype of vSAT1tc was accompanied by differences in particle aggregation and significant differences in acid stability. Based on limited cross neutralization data, the antigenic features seem to be unchanged. Thus, acquisition of positively charged residues in the virion may be beneficial for adaptation of SAT type field strains to cell culture.     

Virion polypeptides and structure of SV 40

Summary Five virion polypeptides, VPI to VPV, have been identified in purified SV40 virus by polyacrylamide gel electrophoresis of which two, VPI and VPII, have been located in the virus capsid. The number of molecular subunits in VPI and VP II calculated from experimental data suggest that they represent the hexons and pentons, respectively, of a 72 unit icosahedral capsid structure.     

The morphogenesis of Theiler’s murine encephalomyelitis virus is strain specific

Summary.  During a single cycle infection with the neurovirulent GDVII- and demyelinating DA-strain of Theiler’s murine encephalomyelitis virus (TMEV) in L-929 cells, different subviral particles were found for both strains. Early in the assembly process, the DA-strain generated 14 S pentamers composed of the viral proteins VP0, VP1 and VP3, while in GDVII-infected cells, particles with the same protein composition but with a sedimentation coefficient of 20 S were found. These newly discovered 20 S particles are probably virion assembly precursors considering their capsid protein composition and their early time of appearance in infected cells. Near the end of the assembly process, VP0, VP1 and VP3 containing 80 S empty capsids became apparent in GDVII-infected cells, while these particles could not be found in DA-infected cells. The significance of these empty capsids will be discussed. After virion assembly, 14 S particles were observed for both strains. These 14 S particles resulted from the degradation of the 160 S virions as indicated by their protein composition (VP1, VP2, VP3) and time of appearance. Our results demonstrate that the assembly of the GDVII-strain differs from that of the DA-strain. In addition, the strain-specific assembly of TMEV implies that not all picornaviruses assemble as proposed by the poliovirus morphogenesis model and thus rendering its general validity questionable. Received October 14, 2002; accepted January 3, 2003 Published online March 21, 2003