Morphogenesis of pestiviruses: new insights from ultrastructural studies of strain Giraffe-1.

Knowledge on the morphogenesis of pestiviruses is limited due to low virus production in infected cells. In order to localize virion morphogenesis and replication sites of pestiviruses and to examine intracellular virion transport, a cell culture model was established to facilitate ultrastructural studies. Based on results of virus growth kinetic analysis and quantification of viral RNA, pestivirus strain Giraffe-1 turned out to be a suitable candidate for studies on virion generation and export from culture cells. Using conventional transmission electron microscopy and single-tilt electron tomography, we found virions located predominately in the lumen of the endoplasmic reticulum (ER) in infected cells and were able to depict the budding process of virions at ER membranes. Colocalization of the viral core protein and the envelope glycoprotein E2 with the ER marker protein disulfide isomerase (PDI) was demonstrated by immunogold labeling of cryosections. Moreover, pestivirions could be shown in transport vesicles and the Golgi complex and during exocytosis. Interestingly, viral capsid protein and double-stranded RNA (dsRNA) were detected in multivesicular bodies (MVBs), which implies that the endosomal compartment plays a role in pestiviral replication. Significant cellular membrane alterations such as those described for members of the Flavivirus and Hepacivirus genera were not found. Based on the gained morphological data, we present a consistent model of pestivirus morphogenesis.

Use of an immunoperoxidase stain for the demonstration of bovine viral diarrhea virus by light and electron microscopies.

Bovine viral diarrhea (BVD) virus-specific antiserum eluted from an immunoabsorbent column was conjugated with horseradish peroxidase. The immunoperoxidase (IP) conjugate was used to stain BVD virus-inoculated and control tissue cultures processed for light and electron microscopies. Infected cells (in the inoculated cultures) viewed by light microscopy had diffuse staining of the cytoplasm of nonvacuolated cells and a more predominant staining associated with vacuoles as vacuolation occurred. The peroxidase label as demonstrated by electron microscopy was associated primarily with nonenveloped and membrane-associated viral particles. Viral particles 20 to greater than 100 nm (diam) were observed. Some of the particles which were greater than 30 mm in diameter contained multiple nuclear cores. The most intensely stained viral particles were outside of the cells.

Morphology of bovine viral diarrhea virus.

The morphology of bovine viral diarrhea virus (BVDV) was studied by electron microscopy. The NADL strain of BVDV was plaque purified 3 times, concentrated by polyethylene glycol precipitation, and purified by centrifugation to equilibrium in continuous potassium tartrate density-gradients. The virus was examined by negative-stain electron microscopy in the presence or absence of specific antiserum. The density of BVDV was between 1.101 g/cm3 and 1.174 g/cm3, with the peak at maximum infectivity at 1.122 g/cm3. Oval to pleomorphic viral particles, 120 ( +/- 30) nm in diameter, were enriched in the peak of maximum infectivity. The detailed structure of virions was revealed: a 5- to 7-microns thick unit membrane-like envelope layer with numerous projecting knobs, 4 to 5 nm in diameter, surrounding an interior core-like structure. Viral particles measuring 120 ( +/- 30) nm were found in large aggregates in the presence of specific antiserum.

Isolation of bovine viral diarrhea virus-like pestiviruses from roe deer (Capreolus capreolus).

Cytopathogenic pestiviruses were isolated from two seronegative free-ranging roe deer (Capreolus capreolus) from northern Germany (Schleswig-Holstein): an adult female and a young buck collected on 6 December 1990 and 26 July 1991, respectively. The two isolates were identified by polymerase chain reaction as pestiviruses. However, they were negative when primers specific for bovine virus diarrhea virus or classical swine fever virus were used, indicating that the two isolates might belong to a separate group of pestiviruses of wild ruminants different from BVDV.

Virus-like particles in bovine turbinate cells infected with bovine virus diarrhoea/mucosal disease virus.

Thin sections of bovine turbinate cells grown in culture and infected with the NADL strain of bovine virus diarrhoea/mucosal disease virus have been examined by electron microscopy. Infected cells exhibited ultrastructural modification of the rough endoplasmic reticulum and associated with this small numbers of virus-like particles were observed. Advanced stages of cytopathic effect were illustrated by cells showing a marked increase in electron-density. These features were not observed in uninfected control cultures.

The ultrastructure of cell cultures infected with border disease and bovine virus diarrhoea viruses.

The morphology of border disease virus and bovine virus diarrhoea virus in infected bovine embryonic testis cells was examined by electron microscopy. Particles which appeared to be mature virions of both viruses were similar, being roughly circular and approximately 46 nm in diameter with a 20 to 25 nm core. Virus replication took place totally within the cytoplasm in association with structures formed from modified endoplasmic reticulum.

Ruminant pestiviruses.

The ruminant pestiviruses, bovine virus diarrhoea virus (BVDV) and border disease virus (BDV) are highly successful and important pathogens which infect ruminant species worldwide. Although the serological relationships among ruminant pestiviruses require further clarification, there is growing evidence for two antigenic groups, one of which predominates in cattle and one in sheep. The success of pestiviruses stems from the ability of the non-cytopathic (NCP) biotype of the virus to cross the placenta and establish a persistent infection (PI) in the developing foetus. This biotype should be regarded as the 'normal' biotype with the cytopathic (CP) biotype being an abnormal virus that is usually isolated only from PI animals dying from mucosal disease. Recent molecular evidence points to CP viruses arising from their NCP counterparts by recombination events that include the insertion of host RNA and/or the duplication of viral RNA sequences. However, the biological mechanism through which CP viruses kill cells remains unknown. Virtually all CP and NCP viruses cause only mild, transient clinical symptoms in healthy adult animals and stimulate a protective immune response. Despite the urgent requirement for a safe, effective vaccine, there is still no commercial vaccine that has been shown to immunize dams so that foetal infection is prevented. In the absence of an effective vaccine, reliable diagnostic techniques are essential to implement effective control measures. There is now a range of monoclonal antibody-based enzyme-linked immunosorbent assays for identifying PI or convalescent animals. These tests are specific, rapid, sensitive and reliable but may themselves become redundant as they are superceded by ever-increasing molecular biology-based techniques.