[Modern methods of virus diagnosis]. / Moderne Methoden der Virusdiagnostik.

In recent years, a series of new approaches for the laboratory diagnosis of viral infections have been developed. In the present review, we describe the different tests available, such as virus isolation and identification in cell culture, antigen and genome detection, and determination of antibodies by serological methods. In order to chose the appropriate test for a given case, advantages and disadvantages of the laboratory procedures as well as their suitability for specimens taken from different organs have to be taken into account. Such considerations are particularly important for a correct interpretation of the results.

Cellular COPII proteins are involved in production of the vesicles that form the poliovirus replication complex.

Poliovirus (PV) replicates its genome in association with membranous vesicles in the cytoplasm of infected cells. To elucidate the origin and mode of formation of PV vesicles, immunofluorescence labeling with antibodies against the viral vesicle marker proteins 2B and 2BC, as well as cellular markers of the endoplasmic reticulum (ER), anterograde transport vesicles, and the Golgi complex, was performed in BT7-H cells. Optical sections obtained by confocal laser scanning microscopy were subjected to a deconvolution process to enhance resolution and signal-to-noise ratio and to allow for a three-dimensional representation of labeled membrane structures. The mode of formation of the PV vesicles was, on morphological grounds, similar to the formation of anterograde membrane traffic vesicles in uninfected cells. ER-resident membrane markers were excluded from both types of vesicles, and the COPII components Sec13 and Sec31 were both found to be colocalized on the vesicular surface, indicating the presence of a functional COPII coat. PV vesicle formation during early time points of infection did not involve the Golgi complex. The expression of PV protein 2BC or the entire P2 and P3 genomic region led to the production of vesicles carrying a COPII coat and showing the same mode of formation as vesicles produced after PV infection. These results indicate that PV vesicles are formed at the ER by the cellular COPII budding mechanism and thus are homologous to the vesicles of the anterograde membrane transport pathway.

Requirements for assembly of poliovirus replication complexes and negative-strand RNA synthesis.

HeLa cells were transfected with several plasmids that encoded all poliovirus (PV) nonstructural proteins. Viral RNAs were transcribed by T7 RNA polymerase expressed from recombinant vaccinia virus. All plasmids produced similar amounts of viral proteins that were processed identically; however, RNAs were designed either to serve as templates for replication or to contain mutations predicted to prevent RNA replication. The mutations included substitution of the entire PV 5' noncoding region (NCR) with the encephalomyocarditis virus (EMCV) internal ribosomal entry site, thereby deleting the 5'-terminal cloverleaf-like structure, or insertion of three nucleotides in the 3Dpol coding sequence. Production of viral proteins was sufficient to induce the characteristic reorganization of intracellular membranes into heterogeneous-sized vesicles, independent of RNA replication. The vesicles were stably associated with viral RNA only when RNA replication could occur. Nonreplicating RNAs localized to distinct, nonoverlapping regions in the cell, excluded from the viral protein-membrane complexes. The absence of accumulation of positive-strand RNA from both mutated RNAs in transfected cells was documented. In addition, no minus-strand RNA was produced from the EMCV chimeric template RNA in vitro. These data show that the 5'-terminal sequences of PV RNA are essential for initiation of minus-strand RNA synthesis at its 3' end.

Formation of the poliovirus replication complex requires coupled viral translation, vesicle production, and viral RNA synthesis.

Poliovirus (PV) infection induces the rearrangement of intracellular membranes into characteristic vesicles which assemble into an RNA replication complex. To investigate this transformation, endoplasmic reticulum (ER) membranes in HeLa cells were modified by the expression of different cellular or viral membrane-binding proteins. The membrane-binding proteins induced two types of membrane alterations, i.e., extended membrane sheets and vesicles similar to those found during a PV infection. Cells expressing membrane-binding proteins were superinfected with PV and then analyzed for virus replication, location of membranes, viral protein, and RNA by immunofluorescence and fluorescent in situ hybridization. Cultures expressing cellular or viral membrane-binding proteins, but not those expressing soluble proteins, showed a markedly reduced ability to support PV replication as a consequence of the modification of ER membranes. The altered membranes, regardless of their morphology, were not used for the formation of viral replication complexes during a subsequent PV infection. Specifically, membrane sheets were not substrates for PV-induced vesicle formation, and, surprisingly, vesicles induced by and carrying one or all of the PV replication proteins did not contribute to replication complexes formed by the superinfecting PV. The formation of replication complexes required active viral RNA replication. The extensive alterations induced by membrane-binding proteins in the ER resulted in reduced viral protein synthesis, thus affecting the number of cells supporting PV multiplication. Our data suggest that a functional replication complex is formed in cis, in a coupled process involving viral translation, membrane modification and vesicle budding, and viral RNA synthesis.

Competing death programs in poliovirus-infected cells: commitment switch in the middle of the infectious cycle.

Productive poliovirus infection of HeLa cells leads to the canonical cytopathic effect (CPE), whereas certain types of abortive infection result in apoptosis. To define the time course of commitment to the different types of poliovirus-induced death, inhibitors of viral replication (guanidine HCl) or translation (cycloheximide) were added at different times postinfection (p.i.). Early in the infection (during the first approximately 2 h p.i.), predominantly proapoptotic viral function was expressed, rendering the cells committed to apoptosis, which developed several hours after viral expression was arrested. In the middle of infection, concomitantly with the onset of fast generation of viral progeny, the implementation of the viral apoptotic program was abruptly interrupted. In particular, activation of an Asp-Glu-Val-Asp (DEVD)-specific caspase(s) occurring in the apoptosis-committed cells was prevented by the ongoing productive infection. Simultaneously, the cells retaining normal or nearly normal morphology became committed to CPE, which eventually developed regardless of whether or not further viral expression was allowed to proceed. The implementation of the poliovirus-induced apoptotic program was suppressed in HeLa cells overexpressing the Bcl-2 protein, indicating that the fate of poliovirus-infected cells depends on the balance of host and viral pro- and antiapoptotic factors.

A cytopathic and a cell culture adapted hepatitis A virus strain differ in cell killing but not in intracellular membrane rearrangements.

Aside from a common gene organization shared with other picornaviruses, hepatitis A virus (HAV) is characterized by its slow-growth phenotype, the inability to shut off host macromolecular synthesis, and, in general, lack of cytopathic (cp) effects in permissive cell cultures. Nevertheless, several cp HAV strains have been isolated during the past decade. In FRhK-4 cells infected with HM175/24a, a fast-growing cp strain, increasing amounts of viral RNA, detected by fluorescence in situ hybridization, indicated viral RNA replication. An ultrastructural analysis of the infected cells revealed a tubular-vesicular network in close proximity to the rough endoplasmic reticulum. Infection of the same cell type with a cell culture adapted (cc) strain, HM175/P35, divulged membrane alterations indistinguishable from the network induced by the cp strain. The overall appearance of the tubular-vesicular network resembles membrane alterations induced by other picornaviruses. However, the shape of the vesicle-like structures is rather oblong and tubular and, thus, seems to be specific for HAV. By electron microscopic immunocytochemistry (IEM), proteins 2B and 2C were found exclusively on the membranes of the network. Proteins expressed from the open reading frame of the cc HAV variant or 2B proteins originating from HM175 cp, cc, or the wt strain expressed in the absence of other HAV proteins induced membrane alterations resembling those seen in HAV-infected cells. The induction of similar structures suggests that protein 2B is involved in the rearrangement of cellular membranes. In all cases, IEM demonstrated that the 2B protein was closely associated with altered membranes. The extent of membrane changes did not seem to increase for both the cp strain and the cc strain during the infectious cycle. Late in the infection and shortly before the culture died off, a large number of cells infected with HM175/24a showed typical signs of apoptosis, whereas the cc strain did not induce cell killing in the same type of cells. Therefore, we conclude that cell death in HM175/24a-infected cells is induced by apoptosis rather than by cytopathology.

Fluorochrome-labeled RNA as a sensitive, strand-specific probe for direct fluorescence in situ hybridization.

Detection of target RNA by in situ hybridization (ISH) in the classic and confocal fluorescence microscope was performed using strand-specific single-stranded RNA probes labeled directly with the fluorochromes fluorescein isothiocyanate or Texas red. The probes, produced by in vitro transcription from PCR-generated templates with T7 RNA polymerase and fluorochromized UTP, gave ISH signals directly visible by fluorescence microscopy without the use of any immunological detection step. In avoiding antibodies, it was possible to strongly increase the sensitivity of the ISH since antibodies may contain RNase which can reduce hybridization signals considerably, even beyond the detection limit. Fluorescent RNA probes thus allowed for the detection of low numbers of target molecules per cell, such as minus strand intermediates in picornavirus RNA replication. Using appropriate denaturing conditions, the targets could be visualized in a double-stranded configuration as well as in the presence of a 100-fold excess of complementary RNA. Furthermore, double ISH for the simultaneous detection of two different RNA species, such as plus and minus strand RNA of poliovirus, or of different regions of the viral genomic RNA was possible with appropriate fluorescent strand-specific probes labeled with different fluorochromes. Combination of ISH and immunofluorescence was found feasible if RNA was present in relatively large amounts. In addition to the investigation of virus replication, possible applications of fluorochromized RNA probes might include antisense RNA detection as well as plant virus resistance and gene silencing.

Characterisation and mitochondrial localisation of AUH, an AU-specific RNA-binding enoyl-CoA hydratase.

AU-rich elements function as instability elements which direct rapid mRNA degradation. AUH protein exhibits an AU-specific RNA-binding property and an intrinsic enoyl-CoA hydratase activity and may therefore function to link mRNA decay to metabolic processes (. Proc. Natl. Acad. Sci. USA 92, 2051-2055). The sequence encoding the murine protein, muAUH, was established by cloning, and the corresponding polypeptide predicted to have a molecular mass of 37kDa. As shown for the human protein, muAUH is expressed in a 32kDa form and there is 94% homology between the two species. Recombinant muAUH was shown to be an RNA-binding enoyl-CoA hydratase. All murine cells studied contained a single AUH transcript of approx. 1.7kb and an investigation of tissue-specific expression revealed highest levels in kidney, skeletal muscle, heart, liver and spleen. It was further determined, using immunoelectron microscopy, that AUH is located in the mitochondria of mouse cells.

Intracellular localization of poliovirus plus- and minus-strand RNA visualized by strand-specific fluorescent In situ hybridization.

The time courses of poliovirus plus- and minus-strand RNA synthesis in infected HEp-2 cells were monitored separately, using a quantitative RNase assay. In parallel, viral RNA and proteins were located in situ by confocal microscopy within cells fixed by a protocol determined to retain their native size and shape. Plus- and minus-strand RNAs were visualized by fluorescent in situ hybridization (FISH) with strand-specific riboprobes. The probes were labelled with different fluorochromes to allow for the simultaneous detection of plus- and minus-strand RNA. The FISH experiments showed minus-strand RNA to be present in distinct, regularly sized, round structures throughout the viral replication cycle. Plus-strand RNA was found in the same structures and also in smaller clusters of vesicles. Association of viral RNA with membranes was demonstrated by combining FISH with immunofluorescence (IF) detection of the viral 2B- and 2C-containing P2 proteins, which are known to be markers for virus-induced membranes. At early times postinfection, the virus-induced membranous structures were distributed through most of the cytoplasm, whereas around peak RNA synthesis, both RNA-associated membranous structures migrated to the center of the cell. During this process, the plus- and minus-strand-containing larger structures stayed as recognizable entities, whereas the plus-strand-containing granules coalesced into a juxtanuclear area of membranous vesicles. An involvement of Golgi-derived membranes in the formation of virus-induced vesicles and RNA synthesis early in infection was investigated by IF with 2C- and Golgi-specific antibodies.

Two types of death of poliovirus-infected cells: caspase involvement in the apoptosis but not cytopathic effect.

The death of poliovirus-infected cells may occur in two forms: canonical cytopathic effect (CPE) (on productive infections) or apoptosis (when the viral reproduction is hindered by certain drugs or some other restrictive conditions). Morphological manifestations of the CPE and apoptosis, being distinct, share some traits (e.g., chromatin condensation and nuclear deformation). It was shown here that a permeable caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone (zVAD.fmk), prevented the development of the poliovirus-induced apoptosis on abortive infection. The apoptotic pathway could be dissected by an inhibitor of chymotrypsin-like serine proteases, N-tosyl-l-phenylalanine chloromethyl ketone (TPCK), which prevented the cleavage of DNA to oligonucleosome-sized pieces and nuclear fragmentation but did not suppress cellular shrinkage, cytoplasmic blebbing, and partial chromatin condensation. These results demonstrate that caspase activation is involved in the execution phase of the viral apoptosis and suggest that a nuclear subset of the apoptotic program is under a separate control, involving a TPCK-sensitive event. Neither zVAD.fmk nor TPCK, at the concentrations affecting the apoptotic response, exerted appreciable influence on the virus growth or cellular pathological changes on productive infection, indicating that the pathways leading to the poliovirus-evoked CPE and apoptosis are different.

Surveillance of cytomegalovirus after solid-organ transplantation: comparison of pp65 antigenemia assay with a quantitative DNA hybridization assay.

In a multicenter study, 113 blood samples from 19 organ transplant patients were analyzed for cytomegalovirus by the pp65 antigenemia assay and a quantitative DNA hybridization assay. Overall, there was 84% agreement among the results obtained by the two tests. Fifteen of 16 episodes of active infection were detected by both assays. One episode was missed by the pp65 assay, and one patient showed significant DNA-emia but only low-level antigenemia.

Induction of intracellular membrane rearrangements by HAV proteins 2C and 2BC.

Hepatitis A virus (HAV) is distinguished from other picornaviruses by its slow and relatively poor, noncytopathic growth in cultures of mammalian cells. The 2C and 2BC proteins of HAV have been implicated in the determination of virus growth in cultured cells. The homologous proteins from other picornaviruses, such as poliovirus, have been demonstrated to exhibit multiple activities, such as RNA binding, nucleotide binding and NTPase, and membrane binding and reorganization. At least some of these activities are required for viral RNA replication. We report here that HAV 2C and 2BC proteins, like their poliovirus counterparts, can induce rearrangement of intracellular membranes and directly or indirectly interact with membranes. Therefore, the inefficient replication properties of HAV are not consequences of the inherent ability of 2C (2BC) to interact with membranes. The effect of 2C (2BC) protein sequences derived from a cell culture-adapted (cc) strain of HAV was compared with that of corresponding protein sequences from either a wild-type (wt) strain of HAV or a faster replicating cytopathic (cp) strain. The analysis demonstrated that mutations acquired in wt virus during adaptation to cell culture do not change dramatically either the ability of these proteins to associate with membranes and induce membrane alterations or the specific architecture of the induced membrane structures. On the other hand, 2C, but not 2BC, protein from the cp strain of HAV induced different membrane structures.

Poliovirus 2C protein determinants of membrane binding and rearrangements in mammalian cells.

Poliovirus protein 2C is a 329-amino acid-protein that is essential for viral RNA synthesis and may perform multiple functions. In infected cells, it is associated with virus-specific membrane vesicles. Recombinant 2C protein expressed in transfected cells has been shown to associate with and induce rearrangement of the intracellular membrane network. This study was designed to map the determinants of membrane binding and rearrangement in the 2C protein. Computer-assisted analysis of the protein sequence led to a prediction that the protein folds into a structure composed of three domains. Expression plasmids that encode each or combinations of these predicted domains were used to examine the abilities of the partial protein sequences to associate with intracellular membranes and to induce rearrangement of these membranes in HeLa cells. Biochemical fractionation procedures suggested that the N-terminal region of the protein was required for membrane association. Electron microscopic and immunoelectron microscopic observation showed that both the N- and C-terminal regions, but not the central portion, of 2C protein interact with intracellular membranes and induce major changes in their morphology. The central portion, when fused to the N-terminal region, altered the specific membrane architecture induced by the N-terminal region, giving rise to vesicles resembling those observed during poliovirus infection.

Reversible dissociation of the poliovirus replication complex: functions and interactions of its components in viral RNA synthesis.

Membrane-bound replication complexes containing transcriptionally active replicative intermediates (RI) can be isolated from poliovirus-infected HEp-2 cells and consist of rosette-like structures of virus-induced vesicles surrounding the replicating viral RNA. At low ionic strength and low temperature, the rosettes reversibly dissociate into individual tubulated vesicles. As determined by immunoelectron microscopy and immunoprecipitation, the vesicles carry a set of viral structural and nonstructural proteins as well as RI RNA. At 30 degrees C, the vesicles reassociate into rosettes synthesizing plus-strand RNA in the RI. The in vitro transcriptional activities of rosettes and vesicles kept separated by high dilution were assessed by an RNase protection assay. The synthesis of the first 178 nucleotides at the 5' end of the plus strand was considered to reflect initiation, and the detection of a 530-nucleotide fragment in the P2 genomic region was considered to reflect elongation. It could be shown that the initiation and elongation of plus strands on individual vesicles are comparable to those in rosettes, with initiation proceeding in de novo-assembled initiation complexes. By use of detergent treatment it was found that initiation, but not elongation, is dependent on vesicular membranes.

Reverse transcription multiplex PCR for differentiation between polio- and enteroviruses from clinical and environmental samples.

For the rapid detection of polioviruses and their differentiation from nonpoliovirus enteroviruses, we developed a protocol in which clinical or environmental specimens are first inoculated onto cell cultures in tubes. After overnight incubation, the cultures are subjected to reverse transcription multiplex PCR with a primer pair which detects all enteroviruses (T. Hyypiä, P. Auvinen, and M. Maaronen, J. Gen. Virol. 70:3261-3268 1989) and two newly designed primer pairs specific for all 36 poliovirus strains tested. The PCR products can unequivocally be identified by their lengths in agarose gels, whereas the genetic heterogeneity of the poliovirus strains precludes identification by back-hybridization with internal probes. The proposed protocol is highly insensitive to the inhibitory effects of substances in the sample (stool, sewage). It allows for the detection of polioviruses and for polioviruses to be distinguished from nonpoliovirus enteroviruses within 24 h, and it allows for the concomitant isolation of a viable strain suitable for further typing.

Immunocytochemistry and in situ hybridization in the electron microscope: combined application in the study of virus-infected cells.

The present review evaluates methods for electron microscopic immunocytochemistry and in situ hybridization, using post-embedding techniques and colloidal gold as a label. Special emphasis is given to double labeling immunocytochemistry and double in situ hybridization and to their combined application on the same specimen. Brief guidelines are presented for fixation, embedding media, the use of polyclonal and monoclonal antibodies and nucleic acid probes. Conditions for labeling and binding of antibody and nucleic acid probes to the target and protocols for direct and indirect immunodetection are discussed. Combinations of direct and indirect immunodetections in multiple labeling experiments are summarized.

Poliovirus subviral particles associated with progeny RNA in the replication complex.

The poliovirus replication complex (RC), the site of genomic 36S RNA synthesis, was previously shown to contain subviral particles of 5S protomer and 14S pentamer antigenicity. The present investigation demonstrates that 5S/14S antigenic subviral particles can be cross-linked to viral RNA by UV irradiation of a subcellular fraction containing the poliovirus RC. Each capsid protein of the subviral particles, i.e. VP0, VP1 and VP3, was cross-linked to viral RNA. SDS-PAGE analysis of the cross-linked capsid proteins revealed a bandshift for VP1, whereas VP0 migrated in several bands, which were interpreted to be multimers of VP0 linked by short stretches of RNA. It was found that 36S RNA rather than replicative intermediate RNA was cross-linked to capsid proteins. Our results indicate that encapsidation of poliovirus RNA starts in the RC and is initiated by 14S pentamers.