Isolation and partial characterization of a novel virus from different carp species suffering gill necrosis - ultrastructure and morphogenesis.

Two isolates of a novel enveloped RNA virus were obtained from carp and koi carp with gill necrosis. Both isolates behaved identically and could be propagated in different cyprinid cell lines forming large syncytia. The virus was sensitive to lipid solvents and neither exhibited haemadsorption/haemagglutination nor reverse transcriptase activity. Mature virus particles displayed a spherical shape with diameter of 100-350 nm after negative staining and 100-300 nm in ultrathin sections, covered by short projections of 8-10 nm in length. Maturation of virus progeny was shown to occur by budding and envelopment of the filamentous helical nucleocapsids at the cell surface. A detailed comparison of ultrastructure and morphogenesis of the novel virus isolates with selected arena-, ortho- and paramyxoviruses as possible candidates for evaluation of taxonomic classification yielded no consistency in all phenotypic features. Thus, on the basis of ultrastructure the novel virus isolates could not be assigned unequivocally to any established virus family.

Neospora caninum and Waddlia chondrophila strain 2032/99 in a septic stillborn calf.

Characteristics of an intracellularly growing micro-organism isolated from an aborted bovine foetus are described. The organism replicated within cytoplasmic vacuoles, was resistant to penicillin and exhibited structural characteristics compatible with Waddlia chondrophila. An ELISA specific for Chlamydia spp., immunofluorescence tests using antibodies directed against Chlamydia spp. or Simkania negevensis, and PCR using Chlamydia-specific primers showed that the agent was distinct from Chlamydiae or S. negevensis. Determination of 16S and partial 23S ribosomal RNA gene sequences in combination with the PCR results and the morphological, antigenic and developmental characteristics provided evidence that the isolate 2032/99 can be classified as W. chondrophila or a closely related organism.

Pseudorabies virus UL37 gene product is involved in secondary envelopment.

Herpesvirus envelopment is a two-step process which includes acquisition of a primary envelope resulting from budding of intranuclear capsids through the inner nuclear membrane. Fusion with the outer leaflet of the nuclear membrane releases nucleocapsids into the cytoplasm, which then gain their final envelope by budding into trans-Golgi vesicles. It has been shown that the UL34 gene product is required for primary envelopment of the alphaherpesvirus pseudorabies virus (PrV) (B. G. Klupp, H. Granzow, and T. C. Mettenleiter, J. Virol. 74:10063-10073, 2000). For secondary envelopment, several virus-encoded PrV proteins are necessary, including glycoproteins E, I, and M (A. R. Brack, J. M. Dijkstra, H. Granzow, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 73:5364-5372, 1999). We show here that the product of the UL37 gene of PrV, which is a constituent of mature virions, is involved in secondary envelopment. Replication of a UL37 deletion mutant, PrV-DeltaUL37, was impaired in normal cells; this defect could be complemented on cells stably expressing UL37. Ultrastructural analysis demonstrated that intranuclear capsid maturation and budding of capsids into and release from the perinuclear space were unimpaired. However, secondary envelopment was drastically reduced. Instead, apparently DNA-filled capsids accumulated in the cytoplasm in large aggregates similar to those observed in the absence of glycoproteins E/I and M but lacking the surrounding electron-dense tegument material. Although displaying an ordered structure, capsids did not contact each other directly. We postulate that the UL37 protein is necessary for correct addition of other tegument proteins, which are required for secondary envelopment. In the absence of the UL37 protein, capsids interact with each other through unknown components but do not acquire the electron-dense tegument which is normally found around wild-type capsids during and after secondary envelopment. Thus, apposition of the UL37 protein to cytoplasmic capsids may be crucial for the addition of other tegument proteins, which in turn are able to interact with viral glycoproteins to mediate secondary envelopment.

Egress of alphaherpesviruses: comparative ultrastructural study.

Egress of four important alphaherpesviruses, equine herpesvirus 1 (EHV-1), herpes simplex virus type 1 (HSV-1), infectious laryngotracheitis virus (ILTV), and pseudorabies virus (PrV), was investigated by electron microscopy of infected cell lines of different origins. In all virus-cell systems analyzed, similar observations were made concerning the different stages of virion morphogenesis. After intranuclear assembly, nucleocapsids bud at the inner leaflet of the nuclear membrane, resulting in enveloped particles in the perinuclear space that contain a sharply bordered rim of tegument and a smooth envelope surface. Egress from the perinuclear cisterna primarily occurs by fusion of the primary envelope with the outer leaflet of the nuclear membrane, which has been visualized for HSV-1 and EHV-1 for the first time. The resulting intracytoplasmic naked nucleocapsids are enveloped at membranes of the trans-Golgi network (TGN), as shown by immunogold labeling with a TGN-specific antiserum. Virions containing their final envelope differ in morphology from particles within the perinuclear cisterna by visible surface projections and a diffuse tegument. Particularly striking was the addition of a large amount of tegument material to ILTV capsids in the cytoplasm. Extracellular virions were morphologically identical to virions within Golgi-derived vesicles, but distinct from virions in the perinuclear space. Studies with gB- and gH-deleted PrV mutants indicated that these two glycoproteins, which are essential for virus entry and direct cell-to-cell spread, are dispensable for egress. Taken together, our studies indicate that the deenvelopment-reenvelopment process of herpesvirus maturation also occurs in EHV-1, HSV-1, and ILTV and that membrane fusion processes occurring during egress are substantially different from those during entry and direct viral cell-to-cell spread.

Equine herpesvirus 1 (EHV-1) glycoprotein M: effect of deletions of transmembrane domains.

Equine herpesvirus 1 (EHV-1) recombinants that carry either a deletion of glycoprotein M (gM) or express mutant forms of gM were constructed. The recombinants were derived from strain Kentucky A (KyA), which also lacks genes encoding gE and gI. Plaques on RK13 cells induced by the gM-negative KyA were reduced in size by 80%, but plaque sizes were restored to wild-type levels on gM-expressing cells. Electron microscopic studies revealed a massive defect in virus release after the deletion of gM in the gE- and gI-negative KyA, which was caused by a block in secondary envelopment of virions at Golgi vesicles. Recombinant KyA expressing mutant gM with deletions of predicted transmembrane domains was generated and characterized. It was shown that mutant gM was expressed and formed dimeric and oligomeric structures. However, subcellular localization of mutant gM proteins differed from that of wild-type gM. Mutant glycoproteins were not transported to the Golgi network and consequently were not incorporated into the envelope of extracellular virions. Also, a small plaque phenotype of mutant viruses that was indistinguishable from that of the gM-negative KyA was observed. Plaque sizes of mutant viruses were restored to wild-type levels by plating onto RK13 cells constitutively expressing full-length EHV-1 gM, indicating that mutant proteins did not exert a transdominant negative effect on wild-type gM.

Primary envelopment of pseudorabies virus at the nuclear membrane requires the UL34 gene product.

Primary envelopment of several herpesviruses has been shown to occur by budding of intranuclear capsids through the inner nuclear membrane. By subsequent fusion of the primary envelope with the outer nuclear membrane, capsids are released into the cytoplasm and gain their final envelope by budding into vesicles in the trans-Golgi area. We show here that the product of the UL34 gene of pseudorabies virus, an alphaherpesvirus of swine, is localized in transfected and infected cells in the nuclear membrane. It is also detected in the envelope of virions in the perinuclear space but is undetectable in intracytoplasmic and extracellular enveloped virus particles. Conversely, the tegument protein UL49 is present in mature virus particles and absent from perinuclear virions. In the absence of the UL34 protein, acquisition of the primary envelope is blocked and neither virus particles in the perinuclear space nor intracytoplasmic capsids or virions are observed. However, light particles which label with the anti-UL49 serum are formed in the cytoplasm. We conclude that the UL34 protein is required for primary envelopment, that the primary envelope is biochemically different from the final envelope in that it contains the UL34 protein, and that perinuclear virions lack the tegument protein UL49, which is present in mature virions. Thus, we provide additional evidence for a two-step envelopment process in herpesviruses.

Chimeric bovine respiratory syncytial virus with glycoprotein gene substitutions from human respiratory syncytial virus (HRSV): effects on host range and evaluation as a live-attenuated HRSV vaccine.

We recently developed a system for the generation of infectious bovine respiratory syncytial virus (BRSV) from cDNA. Here, we report the recovery of fully viable chimeric recombinant BRSVs (rBRSVs) that carry human respiratory syncytial virus (HRSV) glycoproteins in place of their BRSV counterparts, thus combining the replication machinery of BRSV with the major antigenic determinants of HRSV. A cDNA encoding the BRSV antigenome was modified so that the complete G and F genes, including the gene start and gene end signals, were replaced by their HRSV A2 counterparts. Alternatively, the BRSV F gene alone was replaced by that of HRSV Long. Each antigenomic cDNA directed the successful recovery of recombinant virus, yielding rBRSV/A2 and rBRSV/LongF, respectively. The HRSV G and F proteins or the HRSV F in combination with BRSV G were expressed efficiently in cells infected with the appropriate chimeric virus and were efficiently incorporated into recombinant virions. Whereas BRSV and HRSV grew more efficiently in bovine and human cells, respectively, the chimeric rBRSV/A2 exhibited intermediate growth characteristics in a human cell line and grew better than either parent in a bovine line. The cytopathology induced by the chimera more closely resembled that of BRSV. BRSV was confirmed to be highly restricted for replication in the respiratory tract of chimpanzees, a host that is highly permissive for HRSV. Interestingly, the rBRSV/A2 chimeric virus was somewhat more competent than BRSV for replication in chimpanzees but remained highly restricted compared to HRSV. This showed that the substitution of the G and F glycoproteins alone was not sufficient to induce efficient replication in chimpanzees. Thus, the F and G proteins contribute to the host range restriction of BRSV but are not the major determinants of this phenotype. Although rBRSV/A2 expresses the major neutralization and protective antigens of HRSV, chimpanzees infected with this chimeric virus were not significantly protected against subsequent challenge with wild-type HRSV. This suggests that the growth restriction of rBRSV/A2 was too great to provide adequate antigen expression and that the capacity of this chimeric vaccine candidate for replication in primates will need to be increased by the importation of additional HRSV genes.

Role of the cytoplasmic tail of pseudorabies virus glycoprotein E in virion formation.

Glycoproteins M (gM), E (gE), and I (gI) of pseudorabies virus (PrV) are required for efficient formation of mature virions. The simultaneous absence of gM and the gE/gI complex results in severe deficiencies in virion morphogenesis and cell-to-cell spread, leading to drastically decreased virus titers and a small-plaque phenotype (A. Brack, J. Dijkstra, H. Granzow, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 73:5364-5372, 1999). Serial passaging in noncomplementing cells of a virus mutant unable to express gM, gE, and gI resulted in a reversion of the small-plaque phenotype and restoration of infectious virus formation to the level of a gM(-) mutant. Genetic analyses showed that reversion of the phenotype was accompanied by a genomic rearrangement which led to the fusion of a portion of the gE gene encoding the cytoplasmic domain to the 3' end of the glycoprotein D gene, resulting in expression of a chimeric gD-gE protein. Since this indicated that the intracytoplasmic domain of gE was responsible for the observed phenotypic alterations, the UL10 (gM) gene was deleted in a PrV mutant, PrV-107, which specifically lacked the cytoplasmic tail of gE. Regarding one-step growth, plaque size, and virion formation as observed under the electron microscope, the mutant lacking gM and the gE cytoplasmic tail proved to be very similar to the gE/I/M triple mutant. Thus, our data indicate that it is the cytoplasmic tail of gE which is responsible for the observed phenotypic effects in conjunction with deletion of gM. We hypothesize that the cytoplasmic domain of gE specifically interacts with components of the capsid and/or tegument, leading to efficient secondary envelopment of intracytoplasmic capsids.

Intranuclear inclusions in cells infected with Newcastle disease virus.

Cells infected by Newcastle Disease Virus were observed to contain both intracytoplasmic and intranuclear inclusion bodies. Ultrastructurally, they consisted of twisted strands of about 18-20 nm diameter resembling nucleocapsids. The presence of these inclusions was detected irrespective of host cell or pathogenicity of the virus. In immunofluorescence and immunogold labelling experiments, these structures were tagged by an anti-P protein monoclonal antibody. In summary, we show that intracytoplasmic and intranuclear inclusion bodies, hitherto used as a taxonomic characteristic for the genus Morbillivirus of the Paramyxoviridae, also occur in a member of the genus Rubulavirus.

Pathogenic, antigenic and molecular properties of rabbit haemorrhagic disease virus (RHDV) isolated from vaccinated rabbits: detection and characterization of antigenic variants.

Rabbit haemorrhagic disease virus (RHDV) isolates were obtained from several animals previously vaccinated with an inactivated vaccine. Seven isolates were analyzed by immunological and molecular biological methods and compared to reference strains. Antigenic characterization with monoclonal antibodies as well as haemagglutination assays demonstrated considerable differences between individual isolates. However, sequencing of the capsid protein genes revealed a high degree of homology between five of these isolates and the reference strain FRG. In contrast, two isolates specified remarkably different capsid proteins with a degree of variation not observed so far in RHDV. Amino acid alterations were found clustered between residues 301 and 328 (region C), 344 and 434 (region E) and also in the 3' region of the capsid protein gene. Interestingly, experimental vaccination of rabbits followed by challenge with the heterologous variant strains showed restricted cross-protection against one of the strains. In summary, we found a level of antigenic variation not detected in RHDV so far, and describe two distinct new antigenic variants.

Inhibition of virion maturation by simultaneous deletion of glycoproteins E, I, and M of pseudorabies virus.

Glycoprotein M (gM), the product of the UL10 gene of pseudorabies virus (PrV), is one of the few nonessential glycoproteins conserved throughout the Herpesviridae. In contrast to wild-type PrV strains, the UL10 gene product of the attenuated PrV vaccine strain Bartha (PrV-Ba) is not modified by N-glycans due to a mutation in the DNA sequence encoding the consensus N-glycosylation motif. To assay function of the UL10 protein in PrV-Ba, a UL10-deletion mutant (PrV-Ba-UL10(-)) was isolated. Surprisingly, in contrast to gM-deleted wild-type PrV, PrV-Ba-UL10(-) was severely impaired in plaque formation, inducing only foci of very few infected RK13, Vero, and PSEK cells and tiny plaques on MDBK cells. Since this effect was significantly more dramatic than in wild-type PrV, additional mutations known to be present in PrV-Ba were analyzed for their contribution to this phenotype. trans-complementation of the mutated PrV-Ba UL21 or gC protein by the wild-type version had no influence on the observed phenotype. In contrast, complementation of the gE/gI deletion rescued the phenotype. The synergistic effect of deletions in gE/gI and gM on plaque size was verified by construction of a gE/I/M triple mutant derived from wild-type PrV which exhibited the same phenotype. The dramatic effect of deletion of gM on plaque size in a gE/I- virus background was mainly attributable to a function of gM, and not of the gM/gN complex, as shown by analysis of a gE/I/N triple mutant. Interestingly, despite the strong effect on plaque size, penetration was not significantly impaired. In noncomplementing cells infected with the gE/I/M triple mutant, electron microscopy showed absence of secondary envelopment in the cytoplasm but occurrence of intracytoplasmic accumulations of nucleocapsids in association with electron dense material, presumably tegument proteins. These structures were not observed after infection of cells expressing either gE/I or gM. We suggest that gE/I and gM are required for late stages in virion morphogenesis prior to final envelopment in the cytoplasm.

The subgenus-specific C-terminal region of protein IX is located on the surface of the adenovirus capsid.

We have investigated the antigenicity of the C- and N-terminal halves of pIX of human adenovirus types 2 and 3 (Ad2 and Ad3) as well as their orientations in virions. We found that only the C-terminal halves of Ad2 pIX and Ad3 pIX reacted in a subgenus-specific manner by enzyme-linked immunosorbent assay and immunoblot analysis. Based on immunoelectron microscopy experiments, pIX in viral capsids appears to be positioned such that the C-terminal part of pIX constitutes the surface domain whereas the N terminus of the protein makes up the internal domain in icosahedral Ad capsids.

Arterivirus PRRSV. Experimental studies on the pathogenesis of respiratory disease.

Pigs were infected with the porcine respiratory and reproductive syndrome virus (PRRSV) by the oronasal route. We studied the development of histological lesions, sites of virus infection and of inflammatory infiltrates by quantitative evaluation of reactive cells. The animals developed a multifocal interstitial pneumonia. Clinical signs of pneumonia were observed from day 7 to 21. In the first stage, an acute alveolitis was found, which was characterised by a hyperplasia of type II pneumocytes within the septa and an accumulation of macrophages in the alveolar spaces. Within 2-4 days p.i., virus infected cells were prominent in lymphatic organs, but their number declined rapidly during the following days. In the following period, the number of virus antigen positive cells increased in the lung. An interesting discrepancy existed between the relatively small number of virus specific cells and the degree of intensive pneumonia. As a first step to analyse mechanisms leading to the induction of pneumonia, we studied transcriptional expression of cytokines and other immunomodulatory molecules by semiquantitative RT-PCR.

[Characterization of viral hemorrhagic septicemia (VHS) virus isolates in trout]. / Charakterisierung von Isolaten des Virus der Viralen Hämorrhagischen Septikämie (VHS) der Forellen.

Virus diseases of fish can seriously impair the economy of aquacultur. Control and prevention of fish diseases in the European Union (EU) are focussed on the viral haemorrhagic septicaemia (VHS) and the infectious haematopoeitic necrosis (IHN). The diagnosis of VHS and IHN is performed in the Federal Republic of Germany (FRG) on the basis of the legislation of the EU. Since 1994 we received an increasing number of VHS virus (VHSV) isolates which did not react with a commercially available anti-VHSV monoclonal antibody (MAb) in the indirect immuno fluorescence test. With our own MAb ID8, however, as well as with additional diagnostic methods these virus isolates could be identified. These isolates of rainbow trouts were designated as VHSV type "Wi". Electron microscopically all stages of rhabdovirus maturation could be detected. Morphologically the isolates were undistinguishable from other rhabdoviruses. By immuno electron microscopy using the MAb ID8 rhabdovirus nucleocapsid structures were demonstrated. The virulence of the new VHSV type Wi was not different from that of a VHSV isolate with conventional reaction patterns as well as of a VHSV laboratory strain.

Simple and rapid purification of alphaherpesviruses by chromatography on a cation exchange membrane.

A simple and rapid method is described for the purification of two alphaherpesviruses, pseudorabies virus (PrV) and bovine herpesvirus 1, by chromatography on a cation exchange membrane. Cell culture supernatants were passed over a sulfonic-acid modified filter membrane and virions were eluted with a potassium chloride-containing buffer. Over 85% of the virus was eluted within a single fraction and specific infectivity of the resulting virus preparation was over 10-fold higher than that of sucrose gradient-purified virions. Cation exchange was also used for purification of PrV mutants deleted in several glycoproteins which grow in cell culture to titers 10- to 100-fold lower than those obtained by wildtype PrV. For PrV, the presence of non-essential glycoprotein gC, which mediates interaction of virions with cell surface heparin sulfate during attachment, was crucial for the successful purification by cation exchange.

Pseudorabies virus glycoprotein gK is a virion structural component involved in virus release but is not required for entry.

The pseudorabies virus (PrV) gene homologous to herpes simplex virus type 1 (HSV-1) UL53, which encodes HSV-1 glycoprotein K (gK), has recently been sequenced (J. Baumeister, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 69:5560-5567, 1995). To identify the corresponding protein, a rabbit antiserum was raised against a 40-kDa glutathione S-transferase-gK fusion protein expressed in Escherichia coli. In Western blot analysis, this serum detected a 32-kDa polypeptide in PrV-infected cell lysates as well as a 36-kDa protein in purified virion preparations, demonstrating that PrV gK is a structural component of virions. After treatment of purified virions with endoglycosidase H, a 34-kDa protein was detected, while after incubation with N-glycosidase F, a 32-kDa protein was specifically recognized. This finding indicates that virion gK is modified by N-linked glycans of complex as well as high-mannose type. For functional analysis, the UL53 open reading frame was interrupted after codon 164 by insertion of a gG-lacZ expression cassette into the wild-type PrV genome (PrV-gKbeta) or by insertion of the bovine herpesvirus 1 gB gene into a PrV gB- genome (PrV-gK(gB)). Infectious mutant virus progeny was obtained only on complementing gK-expressing cells, suggesting that gK has an important function in the replication cycle. After infection of Vero cells with either gK mutant, only single infected cells or small foci of infected cells were visible. In addition, virus yield was reduced approximately 30-fold, and penetration kinetics showed a delay in entry which could be compensated for by phenotypic gK complementation. Interestingly, the plating efficiency of PrV-gKbeta was similar to that of wild-type PrV on complementing and noncomplementing cells, pointing to an essential function of gK in virus egress but not entry. Ultrastructurally, virus assembly and morphogenesis of PrV gK mutants in noncomplementing cells were similar to wild-type virus. However, late in infection, numerous nucleocapsids were found directly underneath the plasma membrane in stages typical for the entry process, a phenomenon not observed after wild-type virus infection and also not visible after infection of gK-complementing cells. Thus, we postulate that presence of gK is important to inhibit immediate reinfection.

A second form of infectious bursal disease virus-associated tubule contains VP4.

Preparations of density gradient-purified infectious bursal disease virus (IBDV) were found to contain full and empty icosahedral virions, type I tubules with a diameter of about 60 nm, and type II tubules 24 to 26 nm in diameter. By immunoelectron microscopy we demonstrate that virions and both types of tubular structures specifically react with anti-IBDV serum. In infected cells intracytoplasmic and intranuclear type II tubules reacted exclusively with an anti-VP4 monoclonal antibody, as did type II tubules in virion preparations. The immunofluorescence pattern with the anti-VP4 antibody correlated with electron microscopical findings. Neither purified extracellular nor intracellular virions were labeled with the anti-VP4 MAb. Our data show that the type II tubules contain VP4 and suggest that VP4 is not part of the virus particle.

Functional complementation of UL3.5-negative pseudorabies virus by the bovine herpesvirus 1 UL3.5 homolog.

The UL3.5 gene is positionally conserved but highly variable in size and sequence in different members of the Alphaherpesvirinae and is absent from herpes simplex virus genomes. We have shown previously that the pseudorabies virus (PrV) UL3.5 gene encodes a nonstructural protein which is required for secondary envelopment of intracytoplasmic virus particles in the trans-Golgi region. In the absence of UL3.5 protein, naked nucleocapsids accumulate in the cytoplasm, release of infectious virions is drastically reduced, and plaque formation in cell culture is inhibited (W. Fuchs, B. G. Klupp, H. Granzow, H.-J. Rziha, and T. C. Mettenleiter, J. Virol. 70:3517-3527, 1996). To assay functional complementation by a heterologous herpesviral UL3.5 protein, the UL3.5 gene of bovine herpesvirus 1 (BHV-1) was inserted at two different sites within the genome of UL3.5-negative PrV. In cells infected with the PrV recombinants the BHV-1 UL3.5 gene product was identified as a 17-kDa protein which was identical in size to the UL3.5 protein detected in BHV-1-infected cells. Expression of BHV-1 UL3.5 compensated for the lack of PrV UL3.5, resulting in a ca. 1,000-fold increase in virus titer and restoration of plaque formation in cell culture. Also, the intracellular block in viral egress was resolved by the BHV-1 UL3.5 gene. We conclude that the UL3.5 proteins of PrV and BHV-1 are functionally related and are involved in a common step in the egress of alphaherpesviruses.

The UL20 gene product of pseudorabies virus functions in virus egress.

The UL20 open reading frame is positionally conserved in different alphaherpesvirus genomes and is predicted to encode an integral membrane protein. A previously described UL20- mutant of herpes simplex virus type 1 (HSV-1) exhibited a defect in egress correlating with retention of virions in the perinuclear space (J. D. Baines, P. L. Ward, G. Campadelli-Fiume, and B. Roizman, J. Virol. 65:6414-6424, 1991). To analyze UL20 function in a related but different herpesvirus, we constructed a UL20- pseudorabies virus (PrV) mutant by insertional mutagenesis. Similar to HSV-1, UL20- PrV was found to be severely impaired in both cell-to-cell spread and release from cultured cells. The severity of this defect appeared to be cell type dependent, being more prominent in Vero than in human 143TK- cells. Surprisingly, electron microscopy revealed the retention of enveloped virus particles in cytoplasmic vesicles of Vero cells infected with UL20- PrV. This contrasts with the situation in the UL20- HSV-1 mutant, which accumulated virions in the perinuclear cisterna of Vero cells. Therefore, the UL20 gene products of PrV and HSV-1 appear to be involved in distinct steps of viral egress, acting in different intracellular compartments. This might be caused either by different functions of the UL20 proteins themselves or by generally different egress pathways of PrV and HSV-1 mediated by other viral gene products.

Ultrastructural analysis of the replication cycle of pseudorabies virus in cell culture: a reassessment.

We reinvestigated major steps in the replicative cycle of pseudorabies virus (PrV) by electron microscopy of infected cultured cells. Virions attached to the cell surface were found in two distinct stages, with a distance of 12 to 14 nm or 6 to 8 nm between virion envelope and cell surface, respectively. After fusion of virion envelope and cell membrane, immunogold labeling using a monoclonal antibody against the envelope glycoprotein gE demonstrated a rapid drift of gE from the fusion site, indicating significant lateral movement of viral glycoproteins during or immediately after the fusion event. Naked nucleocapsids in the cytoplasm frequently appeared close to microtubules prior to transport to nuclear pores. At the nuclear pore, nucleocapsids invariably were oriented with one vertex pointing to the central granulum at a distance of about 40 nm and viral DNA appeared to be released via the vertex region into the nucleoplasm. Intranuclear maturation followed the typical herpesvirus nucleocapsid morphogenesis pathway. Regarding egress, our observations indicate that primary envelopment of nucleocapsids occurred at the inner leaflet of the nuclear membrane by budding into the perinuclear cisterna. This nuclear membrane-derived envelope exhibited a smooth surface which contrasts the envelope obtained by putative reenvelopment at tubular vesicles in the Golgi area which is characterized by distinct surface projections. Loss of the primary envelope and release of the nucleocapsid into the cytoplasm appeared to occur by fusion of envelope and outer leaflet of the nuclear membrane. Nucleocapsids were also found engulfed by both lamella of the nuclear membrane. This vesiculation process released nucleocapsids surrounded by two membranes into the cytoplasm. Our data also indicate that fusion between the two membranes then leads to release of naked nucleocapsids in the Golgi area. Egress of virions appeared to occur via transport vesicles containing one or more virus particles by fusion of vesicle and cell membrane. Our data thus support biochemical data and mutant virus studies of (i) two steps of attachment, (ii) the involvement of microtubules in the transport of nucleocapsids to the nuclear pore, and (iii) secondary envelopment in the trans-Golgi area in PrV infection.