Search

We have developed a straightforward assay for the rapid typing of enteroviruses using oligonucleotide arrays in microtiter wells. The viral nucleic acids are concomitantly amplified and labeled during reverse transcription-PCR, and unpurified PCR products are used for hybridization. DNA strands are separated by alkaline denaturation, and hybridization is started by neutralization. The microarray hybridization reactions and the subsequent washes are performed in standard 96-well microtiter plates, which makes the method easily adaptable to high-throughput analysis. We describe here the assay principle and its potential in clinical laboratory use by correctly identifying 10 different enterovirus reference strains. Furthermore, we explore the detection of unknown sequence variants using serotype consensus oligonucleotide probes. With just two consensus probes for the coxsackievirus A9 (CVA9) serotype, we detected 23 out of 25 highly diverse CVA9 isolates. Overall, the assay involves several features aiming at ease of performance, robustness, and applicability to large-scale studies.

Calpains are calcium-dependent cysteine proteases that degrade cytoskeletal and cytoplasmic proteins. We have studied the role of calpains in the life cycle of human echovirus 1 (EV1). The calpain inhibitors, including calpeptin, calpain inhibitor 1, and calpain inhibitor 2 as well as calpain 1 and calpain 2 short interfering RNAs, completely blocked EV1 infection in the host cells. The effect of the inhibitors was not specific for EV1, because they also inhibited infection by other picornaviruses, namely, human parechovirus 1 and coxsackievirus B3. The importance of the calpains in EV1 infection also was supported by the fact that EV1 increased calpain activity 3 h postinfection. Confocal microscopy and immunoelectron microscopy showed that the EV1/caveolin-1-positive vesicles also contain calpain 1 and 2. Our results indicate that calpains are not required for virus entry but that they are important at a later stage of infection. Calpain inhibitors blocked the production of EV1 particles after microinjection of EV1 RNA into the cells, and they effectively inhibited the synthesis of viral RNA in the host cells. Thus, both calpain 1 and calpain 2 are essential for the replication of EV1 RNA.

The functional properties of the nonstructural 2A protein are variable among different picornaviruses. The 2A protein of the human parechovirus 1 (HPEV1) has been shown to lack the proteolytic activity found in many other picornaviruses, but no particular function has been identified for HPEV1 2A. To obtain information about the role of HPEV1 2A in the viral life cycle, the protein was expressed in Escherichia coli. A polyclonal antibody was then raised against the protein and employed to investigate its subcellular localization in the infected cells by immunofluorescence microscopy. Typically, a diffuse cytoplasmic staining pattern, concentrated to the perinuclear area, was observed in the infected cells. However, at late stages of infection some infected cells also exhibited diffuse nuclear staining. Viral RNA, visualized by fluorescent in situ hybridization, partly colocalized with 2A in the perinuclear region. Three experimental approaches including Northwestern blot, UV cross-linking, and gel retardation demonstrated that 2A possesses RNA binding activity. Competition experiments with various single-stranded RNA molecules addressed the specificity of 2A binding. These studies revealed that the 2A protein bound RNA corresponding to the 3'-untranslated region (UTR) of the viral genome with highest affinity. At the N- and C-terminal ends of the protein, two regions, necessary for RNA binding, were identified by mutagenesis. In addition, we demonstrated that 2A has affinity to double-stranded RNA containing 3'UTR(+)-3'UTR(-). In conclusion, our experiments showed that HPEV1 2A binds to viral 3'UTR RNA, a feature that could be important for the function of the protein during HPEV1 replication.

The parechoviruses differ in many biological properties from other picornaviruses, and their replication strategy is largely unknown. In order to identify the viral RNA replication complex in human parechovirus type 1 (HPEV-1)-infected cells, we located viral protein and RNA in correlation to virus-induced membrane alterations. Structural changes in the infected cells included a disintegrated Golgi apparatus and disorganized, dilated endoplasmic reticulum (ER) which had lost its ribosomes. Viral plus-strand RNA, located by electron microscopic (EM) in situ hybridization, and the viral protein 2C, located by EM immunocytochemistry were found on clusters of small vesicles. Nascent viral RNA, visualized by 5-bromo-UTP incorporation, localized to compartments which were immunocytochemically found to contain the viral protein 2C and the trans-Golgi marker 1,4-galactosyltransferase. Protein 2C was immunodetected additionally on altered ER membranes which displayed a complex network-like structure devoid of cytoskeletal elements and with no apparent involvement in viral RNA replication. This protein also exhibited membrane binding properties in an in vitro assay. Our data suggest that the HPEV-1 replication complex is built up from vesicles carrying a Golgi marker and forming a structure different from that of replication complexes induced by other picornaviruses.

We used in situ hybridization for the detection of rhinovirus in maxillary sinus biopsy specimens obtained from 14 adult patients with acute sinusitis. In 7 specimens, rhinovirus RNA could be demonstrated in the maxillary sinus epithelium, thereby confirming the etiology of rhinovirus and the clinical suspicion of acute sinusitis.

Although echovirus 22 is presently classified as a member of the enterovirus group in the family of picornaviruses, it has been reported to have exceptional biological properties when compared with other representatives of the group. We have determined the complete nucleotide sequence of the echovirus 22 (Harris strain) genome, which appears to be significantly different from all the other studied picornaviruses. However, the organization of the genome [7339 nucleotides, excluding the poly(A) tract] is similar to that of previously sequenced picornaviruses. This genome includes a 5' untranslated region, relatively well-conserved when compared with aphtho- and cardioviruses, followed by an open reading frame coding for a 2180-amino acid-long polyprotein. The amino termini of capsid polypeptides VP1 and VP3 were determined by direct sequencing, and the other proteolytic cleavage sites in the polyprotein were predicted by comparison with other picornavirus proteins. The amino acid identities of echovirus 22 polypeptides with the corresponding proteins of other picornaviruses are in the 14-35% range, similar to those percentages seen when representatives of the five picornavirus groups (entero-, rhino-, cardio-, aphtho-, and hepatoviruses) are compared. Our results suggest that echovirus 22 belongs to an independent group of picornaviruses.

We have applied a sensitive in situ hybridization method for the detection of coxsackievirus RNA in myocardial tissue. Two radioactive cRNA probes were used: an RNA transcript representing the 5' end of poliovirus 3 which recognizes a highly conserved region among enteroviruses and an RNA transcript of a 1.1 kb fragment from the polymerase gene region of coxsackievirus B3. The reactivity of these probes was tested by dot-blot hybridization against a panel of enteroviruses. Formaldehyde-fixed and paraffin-embedded tissue of experimentally coxsackievirus B3 infected mice was analyzed for the localization of virus RNA. Both the probes gave signals in mouse myocardial cells, disseminated evenly in the heart tissue 7 days postinfection. At this time point, hybridization-positive cells and inflammatory reaction were mainly found in different areas that may be due to the inability of the immune system to recognize the infected cells before cytolysis. The samples were still positive when fixed 52 hours postmortem indicating that virus RNA is in a relatively stable form in the cells.

The presence of mumps virus and enterovirus RNA was studied by in situ hybridization in 15 surgical biopsy specimens from patients with acute pancreatitis. 35S-Labeled cRNA probes, detecting the 5' end of the poliovirus type 3, a 1.1-kb fragment of the polymerase gene region of coxsackievirus B3, and mumps virus mRNA, encoding the nucleocapsid protein, were used. The controls consisted of mumps virus-infected and uninfected cultured cells, normal human and mouse pancreatic tissue, and mouse tissue from experimental coxsackievirus B3-induced pancreatitis. No specific hybridization signal was observed in any of the acute pancreatitis cases. It is concluded that neither mumps virus nor enteroviruses tested were present in pancreatic tissue of advanced human acute pancreatitis.

Aedes aegypti mosquitoes were inoculated intrathoracically with prototype Sindbis virus, held at 26.7 degrees C for from 0-95 h and placed at -70 degrees C. Individual mosquitoes were tested for virus by plaque assay in Vero cells, for viral RNA by nucleic acid hybridization using a cloned cDNA probe, and for viral protein by enzyme-linked immunosorbent assay. Virus was detected by plaque assay as early as 8 h after infection. Sindbis virus RNA was detected by nucleic acid hybridization 18 h after infection and by enzyme-linked immunosorbent assay 10 h after infection. The results of these comparisons suggest that both nucleic acid hybridization and enzyme-linked immunosorbent assay are applicable to direct detection of Sindbis virus in mosquitoes containing virus at levels usually found during arbovirus epidemics.