Creation of a bovine herpes virus 1 (BoHV-1) quantitative particle standard by transmission electron microscopy and comparison with established standards for use in real-time PCR.

Standards are pivotal for pathogen quantification by real-time PCR (qPCR); however, the creation of a complete and universally applicable virus particle standard is challenging. In the present study a procedure based on purification of bovine herpes virus type 1 (BoHV-1) and subsequent quantification by transmission electron microscopy (TEM) is described. Accompanying quantitative quality controls of the TEM preparation procedure using qPCR yielded recovery rates of more than 95% of the BoHV-1 virus particles on the grid used for virus counting, which was attributed to pre-treatment of the grid with 5% bovine albumin. To compare the value of the new virus particle standard for use in qPCR, virus counter based quantification and established pure DNA standards represented by a plasmid and an oligonucleotide were included. It could be shown that the numbers of virus particles, plasmid and oligonucleotide equivalents were within one log10 range determined on the basis of standard curves indicating that different approaches provide comparable quantitative values. However, only virus particles represent a complete, universally applicable quantitative virus standard that meets the high requirements of an RNA and DNA virus gold standard. In contrast, standards based on pure DNA have to be considered as sub-standard due to limited applications.

Phosphorylation of Bovine Herpesvirus 1 VP8 Plays a Role in Viral DNA Encapsidation and Is Essential for Its Cytoplasmic Localization and Optimal Virion Incorporation.

UNLABELLED: VP8 is a major tegument protein of bovine herpesvirus 1 (BoHV-1) and is essential for viral replication in cattle. The protein undergoes phosphorylation after transcription through cellular casein kinase 2 (CK2) and a viral kinase, US3. In this study, a virus containing a mutated VP8 protein that is not phosphorylated by CK2 and US3 (BoHV-1-YmVP8) was constructed by homologous recombination in mammalian cells. When BoHV-1-YmVP8-infected cells were observed by transmission electron microscopy, blocking phosphorylation of VP8 was found to impair viral DNA encapsidation, resulting in release of incomplete viral particles to the extracellular environment. Consequently, less infectious virus was produced by the mutant virus than by wild-type (WT) virus. A comparison of mutant and WT VP8 by confocal microscopy revealed that mutant VP8 is nuclear throughout infection while WT VP8 is nuclear early during infection and is associated with the Golgi apparatus at later stages. This, together with the observation that mutant VP8 is present in virions, albeit in smaller amounts, suggests that the incorporation of VP8 may occur at two stages. The first takes place without the need for phosphorylation and before or during nuclear egress of capsids, whereas the second occurs in the Golgi apparatus and requires phosphorylation of VP8. The results indicate that phosphorylated VP8 plays a role in viral DNA encapsidation and in the secondary virion incorporation of VP8. To perform these functions, the cellular localization of VP8 is adjusted based on the phosphorylation status. IMPORTANCE: In this study, phosphorylation of VP8 was shown to have a function in BoHV-1 replication. A virus containing a mutated VP8 protein that is not phosphorylated by CK2 and US3 (BoHV-1-YmVP8) produced smaller numbers of infectious virions than wild-type (WT) virus. The maturation and egress of WT and mutant BoHV-1 were studied, showing a process similar to that reported for other alphaherpesviruses. Interestingly, lack of phosphorylation of VP8 by CK2 and US3 resulted in reduced incorporation of viral DNA into capsids during mutant BoHV-1 infection, as well as lower numbers of extracellular virions. Furthermore, mutant VP8 remained nuclear throughout infection, in contrast to WT VP8, which is nuclear at early stages and Golgi apparatus associated late during infection. This correlates with smaller amounts of mutant VP8 in virions and suggests for the first time that VP8 may be assembled into the virions at two stages, with the latter dependent on phosphorylation.

Kinetics of BoHV-1 dissemination in an in vitro culture of bovine upper respiratory tract mucosa explants.

Bovine herpesvirus 1 (BoHV-1) is a well-known disease-causing agent in cattle. There is little known detailed information on viral behavior with emphasis on host invasion at primary replication sites such as the mucosa of the upper respiratory tract. Therefore, an in vitro system of bovine upper respiratory tract (bURT) mucosa explants was set up to study BoHV-1 molecular/cellular host-pathogen interactions. We performed a thorough morphometrical analysis (epithelial integrity, basement membrane continuity, and lamina propria integrity) using light microscopy and transmission electron microscopy. We applied a terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) staining as a viability test. Bovine upper respiratory tract mucosa explants were maintained in culture for up to 96 hours without any significant changes in morphometry and viability. Next, bURT mucosa explants were infected with BoHV-1 (Cooper) and collected at 0, 24, 48, and 72 hours postinoculation (p.i.). Using a quantitative analysis system to measure plaque latitude and invasion depth, we assessed dissemination characteristics in relation to elapsed time p.i. and found a plaquewise spread of BoHV-1 across the basement membrane as early as 24h p.i., similar to pseudorabies virus (PRV). Moreover, we observed that BoHV-1 exhibited an increased capacity to invade in proximal tracheal tissues compared to tissues of the deeper part of the nasal septum and ventral conchae. Revealing a more distinct invasion of BoHV-1 in proximal trachea, we can conclude that, in order to study an important aspect of BoHV-1 pathogenesis, the bovine upper respiratory tract mucosa explant model is the best suited model.

The UL49 gene product of BoHV-1: a major factor in efficient cell-to-cell spread.

The role of the UL49 gene product, VP22, of bovine herpesvirus type 1 (BoHV-1) in virus replication was characterized with respect to a putative functional interaction of VP22 with the viral glycoprotein E (gE) during BoHV-1 cell-to-cell spread. Deletion of the open reading frames of UL49 and/or gE from an infectious BoHV-1 bacterial artificial chromosome clone did not severely impair the production of viral progeny in single-step growth experiments. However, plaque sizes induced by a VP22-negative BoHV-1 were reduced by 52%, whilst for the gE/VP22-negative double-deletion mutant a reduction of 83% could be observed in comparison with parental and revertant viruses, which was consistent with a marked reduction in multi-step growth experiments at early time points. These results suggest that gE and VP22 are important for BoHV-1 cell-to-cell spread, and that both are likely to act independently of each other in a critical pathway for virus cell-to-cell spread.

Role of the UL28 homologue of bovine herpesvirus 1 in viral DNA cleavage and packaging.

In the aim to study the function of the bovine herpesvirus 1 (BoHV-1) UL28 protein during the replicative cycle, we characterized a UL28 deletion mutant of BoHV-1, BoHV-1 Delta UL28. Productive growth of BoHV-1 Delta UL28 was only observed in a specifically engineered complementing cell line expressing the native UL28 protein, demonstrating that UL28 is essential for virus replication. UL28 deficiency did not compromised viral protein synthesis of the late class as shown by the detection of the viral alpha gene trans-inducing factor protein encoded by UL48, a gene of the gamma2 class. Southern blotting analyses of total DNA extracted from BoHV-1 Delta UL28-infected normal cells revealed that viral DNA replication was not compromised but the process of cleavage of the newly synthesized DNA was defective. Transmission electron microscopy of non-complementing BoHV-1 Delta UL28-infected cells revealed an accumulation of capsids devoid of DNA, suggesting that the DNA packaging was impaired. We conclude that the BoHV-1 UL28 protein is essential for viral replication and is necessary for the formation of mature capsid.

The significance of the Golgi complex in envelopment of bovine herpesvirus 1 (BHV-1) as revealed by cryobased electron microscopy.

Nucleocapsids of herpesviruses originate in the nucleus of host cells and bud through the inner nuclear membrane acquiring tegument and envelope. The release of the enveloped virus particle from the perinuclear space is unknown. Cryobased electron microscopic imaging revealed enveloped virus particles within cisterns associated with the perinuclear space, a pre-Golgi compartment connecting Golgi cisterns to the perinuclear space, and enveloped virus particles in Golgi cisterns where they are packaged into transport vacuoles by membrane fission. To our knowledge, our images show for the first time the connectivity from the perinuclear space to Golgi cisterns. The data strongly indicate an intracisternal transport of enveloped virus particles from the budding site to the packaging site. Budding starts by condensation at the inner membrane. Condensation involving the viral envelope and peripheral tegument was persistent in virus particles within perinuclear space and associated cisterns. Virus particles within Golgi cisterns and transport vacuoles originating by Golgi membrane fission, however, lacked condensation. Instead, spikes were clearly evident. The phenomenon of condensation is considered likely to be responsible for preventing fusion of the viral envelope with cisternal membranes and/or for driving virions from the perinuclear space to Golgi cisterns. Glycoprotein K is discussed to likely play a role in the intracisternal transportation of virions. In addition to the pathway including intracisternal transport and packaging, there were clear indications for the well-known pathway involving wrapping of cytoplasmic nucleocapsids by Golgi membranes. The origin of the cytoplasmic nucleocapsids, however, remains obscure. Lack of evidence for release of nucleocapsids at the outer nuclear membrane suggests that the process is very rapid, or that nucleocapsids pass the nucleocytoplasmic barrier via an alternative route.

Novel entry pathway of bovine herpesvirus 1 and 5.

Herpesviruses enter cells by a yet poorly understood mechanism. We visualized the crucial steps of the entry pathway of bovine herpesvirus 1 (BHV-1) and BHV-5 by transmission and scanning electron microscopy, employing cryotechniques that include time monitoring, ultrarapid freezing, and freeze substitution of cultured cells inoculated with virus. A key step in the entry pathway of both BHV-1 and BHV-5 is a unique fusion of the outer phospholipid layer of the viral envelope with the inner layer of the plasma membrane and vice versa resulting in "crossing" of the fused membranes and in partial insertion of the viral envelope into the plasma membrane. The fusion area is proposed to function as an axis for driving the virus particle into an invagination that is concomitantly formed close to the fusion site. The virus particle enters the cytoplasm through the opened tip of the invagination, and the viral envelope defuses from the plasma membrane. There is strong evidence that the intact virus particle is then transported to the nuclear region.

The role of the major tegument protein VP8 of bovine herpesvirus-1 in infection and immunity.

The tegument of bovine herpesvirus-1 (BHV-1) carries an abundant protein of 96 kDa, termed VP8. Immunolabeling using VP8-specific antiserum and colloidal gold-labeled protein A as the electron-dense marker was used to identify VP8 in the virions and virus-infected cells. VP8 was confirmed to be a tegument protein that, like the herpes simplex virus-1 homologue VP13/14, contains O-linked carbohydrates. VP8 was found in the nucleus of virus-infected cells as early as 2 hr postinfection. Since VP8 is a gamma2 protein, this protein cannot be newly synthesized at this time and must be acquired from the inoculum. This supports the hypothesis that early during infection, VP8 has a function in modulation of alpha gene expression. Later during infection, VP8 was observed in the cytoplasm around nucleocapsids and in dense inclusions, which accumulated in the cisternae of the Golgi. In addition, de novo-synthesized VP8 continued to accumulate in the nucleus in dense areas and around nucleocapsids. In calves, VP8 stimulated T cell proliferation and antibody production, both after BHV-1 challenge and after immunization with purified VP8. These results suggest a role for VP8 in the induction of humoral and specifically cell-mediated immunity to BHV-1.

Functional and topographical analyses of epitopes on bovine herpesvirus type 1 glycoprotein IV.

Bovine herpesvirus type 1 (BHV-1) glycoprotein gIV was purified by affinity chromatography. Purified preparations showed two distinct components of 71 K and 140 K following electrophoresis in sodium dodecyl sulphate polyacrylamide gels. The polypeptides were separated, excised from the gel and used to immunize rabbits; the resulting antisera showed a high degree of cross reactivity indicating that these polypeptides represent monomeric and dimeric forms of the same glycoprotein. Purified gIV was also used to develop a gIV-specific panel of monoclonal antibodies. Neutralizing monoclonal antibodies directed against gIV were conjugated to horseradish peroxidase and subjected to competition binding assays by ELISA. Three distinct neutralizing antigenic domains on gIV were identified. Domain 1 comprised two overlapping epitopes, whereas domain 2 was represented by a single monoclonal antibody. The third antigenic domain was made up of a complex of four identical or overlapping epitopes designated 3a, b, c, and d. Evidence is presented suggesting that domain 1 of gIV may be involved in penetration of the virus into the cell.

Neutralizing monoclonal antibodies directed to infectious bovine rhinotracheitis virus.

Infectious bovine rhinotracheitis virus (IBRV) has been shown in this report to have thirty-three polypeptides. Ten of the eleven polypeptides which can be labeled with (3H)-glucosamine are located on the surface of the virus since they can be surface labeled with sodium boro(3H)hydride. In order to define the immunologically important viral proteins, monoclonal antibodies were prepared against the virus and selected for their ability to neutralize infectivity. Four such hybridoma lines were obtained for characterization of the antigens that elicit neutralizing antibodies. The viral polypeptides were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the specificity of each monoclonal antibody was determined by "Western" blot analysis and/or by immunoprecipitation of (35S)-methionine and (3H)-glucosamine labeled infected cell lysates by the monoclonal antibodies. One monoclonal antibody reacted with two glycoproteins, gp135 and gp78a, on the "Western" blot but immunoprecipitated three glycoproteins, gp135, gp78a, and gp54 from labeled infected cell lysates. The other three monoclonal antibodies immunoprecipitated a single glycoprotein, gp78b, from (3H)-glucosamine labeled infected cell lysates but not from (35S)-methionine labeled infected cell lysates.

Identification of envelope and nucleocapsid proteins of infectious bovine rhinotracheitis virus by SDS-polyacrylamide gel electrophoresis.

Infectious bovine rhinotracheitis (IBR) virus was purified by rate zonal and isopycnic centrifugation in potassium tartrate gradients. Viral nucleocapsids were isolated from purified virions by treatment with the nonionic detergent Triton X-100 followed by high speed centrifugation. This treatment was shown to produce a suspension of 74% completely de-enveloped nucleocapsids, 24% incompletely de-enveloped nucleocapsids, and 2% whole virions. The viral nucleocapsids contained DNA and banded at a density of 1.25 g/cm3. Analysis of the viral polypeptides by gradient SDS-polyacrylamide gel electrophoresis revealed that 33 virion proteins, ranging in molecular weight from 13,000 to 275,000 dalton, were present in the complete virus particle. Detergent treatment of the virus quantitatively removed two of the major proteins (vp8, 90,000 dalton, and vp13, 73,000 dalton) and partially removed eleven other proteins. Fifteen viral polypeptides appeared to remain firmly associated with the viral nucleocapsids.

[Infectious bovine rhinotracheitis (VHB-1). II. Physico-chemical properties of strain L-114]. / Rinotraqueítis infecciosa bovina (VHB-1). II: Propiedades físico-químicas de la cepa L-114.

The thermal stability, density gradient and morphological features of a recently isolated L-114 strain of infectious bovine rhinotracheitis virus were determined. Morphology studies showed an enveloped virus with 200 nm of total diameter, a core diameter of 90 nm and an icosaedral-type structure; purified preparations contained both complete and empty viral particles. More than 90% of the infectivity was lost after 15 hours at 37 degrees C; at 56 degrees C, inactivation was much faster, with a 3 log titer reduction, in 24 minutes. Density gradient studies in cesium chloride, carried out with virus concentrated on sucrose gradient, gave an estimated density of 1.25 g/ml for the purified virus, which fits with light herpesviruses.

Diagnosis of rotavirus, adenovirus, and herpesvirus infections by immune electron microscopy using a serum-in-agar diffusion method.

The sensitivity of immune electron microscopy (IEM) for the detection and identification of bovine rotavirus, infectious bovine rhinotracheitis virus (IBR), and canine adenovirus has been studied by using the serum-in-agar (SIA) method in which a specific antiserum has been incorporated in agar.

Chromic-acid formaldehyde fixation of nucleic acids of bacteriophage phi6 and infectious bovine rhinotracheitis virus.

Bacteriophage phi6 nucleic acid was present as a torus after chromic acid-formaldehyde-OSO4 fixation and acetone and propylene oxide dehydration. A herpes virus, infectious bovine rhinotracheitis virus, had its DNA mostly as a torus, collapsed in the centre, or as a network, after glutaraldehyde-OSO4 fixation, but in an uncollapsed torus or network formation after chromic acid-formaldehyde-OSO4. This fixative stabilized nucleic acids, allowing acetone dehydration and plastic embedding without collapse of nucleic acid to the centre of the virion.

Purification and buoyant density of infectious bovine rhinotracheitis virus.

A purification scheme for infectious bovine rhinotracheitis virus utilizing rate-zonal centrifugation in a 10-40% potassium tartrate gradient was described. The density of IBRV in the potassium tartrate gradient was found to be 1.22 g/cm3. Electron microscopic examination of purified virus preparations revealed homogeneous populations of enveloped virions with minute projections on the envelope surface.

Envelopment and the envelopes of infectious bovine rhinotracheitis virus in ultrathin sections.

An electron microsopic study of cell cultures and bovine foetal tracheal organ cultures infected with infectious bovine rhinotracheitis (IBR) virus showed the following. a) The difference in the site at which the outer envelope is acquired in the infected cell is responsible for some morphological differences between the virions. Where envelopment of the capsids occurs by budding into cytoplasmic tubules, dense material adjacent to the tubules is often incorporated between the capsid and the outer envelope, giving a pleomorphic appearance to the latter. No dense material is seen in the same position if envelopment occurs in the nucleus. b) Dense material adjacent to the cytoplasmic tubules is not only incorporated in the virions, but may also bud into the cytoplasmic tubules without nucleocapsids, thus giving rise to dense bodies. c) Dense material in the virions is closely adjacent to the inner side of the outer envelope, but is separated from the capsid by a lucent zone. A similar zone can be seen around some nucleocapsids in the nucleus. d) The outer envelope of numerous extracellular virions and of those located in the cytoplasmic tubules shows the unit membrane structure with projections. The outer envelope of intranuclear particles and of those located in the perinuclear cisterna had the appearance of a dense membrane.