Direct next-generation sequencing diagnosis of echovirus 9 meningitis, France.

The prognosis of central nervous system infections caused by enteroviruses partially depends on the viral genotype, which is not provided by current point-of-care diagnostic methods. In this study, next-generation sequencing identified an echovirus 9 directly from the cerebrospinal fluid of a patient presenting with meningitis.

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.

Acute onset of type I diabetes mellitus after severe echovirus 9 infection: putative pathogenic pathways.

Enterovirus infections have been implicated in the development of type I diabetes mellitus. They may cause beta cell destruction either by cytolytic infection in the pancreas or indirectly by contributing to autoimmune reactivity. We sought evidence for these 2 mechanisms in a case of acute-onset diabetes mellitus that occurred during severe echovirus 9 infection. The virus was isolated and administered to cultured human beta cells. No viral proliferation was observed, and no beta cell death was induced, while parallel exposure to Coxsackie B virus serotype 3 resulted in viral proliferation and massive beta cell death. Although the viral protein 2C exhibited a sequence similar to that of the beta cell autoantigen glutamic acid decarboxylase (GAD(65)), no cross-reactive T cell responses were detected. The patient did not develop antibodies to GAD(65) either. Absence of evidence for direct cytolytic action or an indirect effect through molecular mimicry with GAD(65) in the present case raises the possibility of another indirect pathway through which enteroviruses can cause diabetes mellitus.

Determinants of pathogenicity of echovirus 9 in men: significance of a functional RGD-motif.

In this study, we investigated nine independent echovirus 9 isolates obtained from sick children in 1995. It is discovered that these isolates differ in respect to their pathogenicity for newborn mice indicating that the degree of human pathogenicity of an echovirus 9 variant does not necessarily correlate with mouse pathogenicity. Nevertheless, all virus variants are found to code for an RGD-motif within their VP1 protein. Hence, the RGD-motif and its highly conserved flanking regions are the conditio sine qua non, but, as expected, not sufficient for the mouse-pathogenic character.

Cell attachment and mouse virulence of echovirus 9 correlate with an RGD motif in the capsid protein VP1.

The recently analyzed sequences of the nonpathogenic prototype strain Hill and the mouse-virulent strain Barty of the human echovirus 9 differ particularly in an insertion coding for an RGD motif at the C-terminus of the capsid protein VP1 in the genome of strain Barty. To investigate molecular determinants of virulence, we generated a panel of recombinant viruses derived from cDNA clones of strains Hill and Barty. In this communication, we show that the mouse-pathogenic character of strain Barty correlates with a 310-aa segment including the RGD motif. By mutating the RGD to an RGE tripeptide, the infectivity of the resulting echovirus 9 clones for GMK cells is lost. Furthermore, we could show that synthetic peptides containing the RGD sequence influence binding of mouse-virulent echovirus 9 strains to GMK cells, whereas binding of apathogenic strains is not affected. These results suggest that the RGD motif is a significant factor affecting pathogenicity of echovirus 9 strains.

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.

Acid stability of hepatitis A virus.

The acid stability of unpurified and highly purified hepatitis A virus (HAV) was tested and compared with that of poliovirus type 1, coxsackievirus types A9 and B1 and echovirus type 9. Only HAV had a high residual infectivity after 2 h of exposure to pH 1 at room temperature, remaining infectious for up to 5 h. At 38 degrees C, pH 1, HAV remained infectious for 90 min. Highly purified HAV was found to be infectious for 8 h at pH 1 and room temperature. This indicates that the increased stability is not due to protection by cellular material attached to the virus, but is a virus-specific marker. Under the same conditions, at pH 1 and room temperature, unpurified and highly purified HAV antigens were traceable for 5 and 4 h respectively.

Morphological changes and membrane potential of human granulocytes under influence of chemotactic peptide and/or echo-virus, type 9.

The membrane potential, Em, of human granulocytes (PMNs), was recorded using glass microelectrodes. The membrane potential Em exhibited potential fluctuations accompanied by characteristic changes of cell shape. The periodic potential fluctuations (7-s, 70-s, and 260-s periodicities) ascertained by the autocorrelation technique, suggested the existence of an internal clock. The chemoattractant f-Met-Leu-Phe (FMLP) had no influence on the periodicities, whereas the amplitudes of the fluctuations were increased by it. Treatment of PMNs with Echo 9 virus also resulted in hyperpolarization. The 70-s periodicity disappeared under virus treatment indicating a virus-induced change of the internal program and loss of chemotactic activity.

Biochemistry and pathogenicity of echovirus 9. III. Thermosensitive mutants of echovirus 9, strain Barty, with reduced pathogenicity for newborn mice.

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 degrees C, was isolated after mutagenization with 5-fluorouracil. For all mutants the virus dose required to paralyze 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 degrees 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 degrees 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.

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.

Susceptibility of endothelial cells derived from different blood vessels to common viruses.

We examined whether endothelial cells derived from different blood vessels vary in their susceptibility to viral infection. Five common viral pathogens of humans (herpes simplex 1, measles, mumps, echo 9, and coxsackie B4 viruses) were evaluated for growth in endothelial cells derived from bovine fetal pulmonary artery, thoracic aorta, and vena cava. All five viruses replicated in each type of endothelial cell. There were apparent differences in the quantities of measles and mumps viruses produced in pulmonary artery endothelium compared with thoracic aorta and vena cava when endothelial cells were obtained from different animals. However, when pulmonary artery endothelial cells were compared with vena cava cells from the same animal, growth of each virus was similar in the two cell types. Four of the viruses replicated in the various endothelial cells without producing appreciable changes in cell morphology. These results indicate that endothelial cells from different blood vessels are equally susceptible to the human viruses evaluated, and that viral replication can occur without major alterations in cell morphology. Endothelial cells could serve as permissive cells permitting viruses to leave the circulation and initiate infection in adjacent tissues, including subendothelial smooth muscle cells.

Virus infection of endothelial cells.

Endothelial injury is important in the pathogenesis of thrombosis, atherosclerosis, disseminated intravascular coagulation, and vasculitis. The ability of several common human viruses to infect cultures of endothelial cells obtained from human umbilical veins or bovine thoracic aorta was demonstrated. Indicators of infection included cytopathology, viral growth curves, and antigen detection by immunofluorescence. Herpes simplex virus type 1, adenovirus type 7, measles virus, and parainfluenza virus type 3 infected both human venous and bovine aorta endothelium. Mumps virus, poliovirus type 1, and echovirus type 9 grew only in human venous cells; coxsackievirus B4 infected only bovine arterial cultures; and cytomegalovirus, influenza A/Victoria/75 (H3N2) virus, and respiratory syncytial virus failed to grow in either cell culture. During replication some viruses caused acute lytic changes; some produced chronic, less destructive alterations; and other induced no apparent cytopathology. The results suggest that viral replication within endothelium may be important in the pathogenesis of viral disease of initiation of vessel-wall injury.