Notices

A virus isolated from the lung of an aborted Hereford fetus was shown to possess the physical and chemical properties of the herpesvirus group. The virus designated bovine herpesvirus (BH-1247) was isolated in cultures of Madin-Darby bovine kidney (MDBK) and primary bovine embryonic kidney (BEK) cells. Electron microscopic studies revealed typical forms of virus particles of the herpesvirus group. The virus was sensitive to chloroform and virus replication was inhibited by the addition of 5-iododeoxyuridine into the cell culture medium. The characteristic features of the cytopathic changes were syncytial formations with intranuclear inclusion bodies. Discrete plaques were formed in MDBK cell cultures overlaid with agar. Virus growth studies in BEK cells revealed infectious virus to be cell associated and replicated at low titer. By serum neutralization tests the virus was shown to be distinct from bovine herpesviruses; infectious bovine rhinotracheitis, DN-599, Movar 33/63, bovine mammallitis, malignant catarrhal fever and feline viral rhinotracheitis, equine herpesvirus I and pseudorabies. The isolate was nonpathogenic to mice inoculated subcutaneously, intracerebrally and intraperitoneally. Virus replication was not demonstrated when inoculated on the chorioallantoic membrane of embryonated eggs.     

Cloning and sequence of an infectious bovine rhinotracheitis virus (BHV-1) gene homologous to glycoprotein H of herpes simplex virus.

A homologue to the glycoprotein H (gH) gene of herpes simplex virus (HSV) has been identified in the genome of infectious bovine rhinotracheitis virus (IBR, BHV-1). The gene is located immediately downstream from the thymidine kinase gene, and codes for an open reading frame (orf) of 842 amino acids. The orf has the characteristics of a membrane glycoprotein, including an N-terminal hydrophobic region resembling a signal sequence, a C-terminal region which is probably a transmembrane domain, and six potential sites for N-linked glycosylation. This orf shows significant homology to the gH sequences of both HSV and pseudorabies virus (PRV). We conclude that this gene encodes BHV-1 gH.     

Regulation of persistent infection with herpes simplex virus in vitro by hydrocortisone.

About 1% of Raji cells showed sensitivity to herpes simplex virus type 2 (HSV-2) infection when tested by infectious center assays or immunofluorescence tests, and the percentage did not change during cell passage. The addition of hydrocortisone to Raji cells persistently infected with HSV-2 caused a marked increase in virus production and in the number of HSV-producing cells. In the case of HSV-1, it was shown that the addition of hydrocortisone was required to maintain persistent infection. These observations suggest that control of replication of HSV-1 and HSV-2 in these cells is regulated by different mechanisms.     

Bovine Respiratory Syncytial Virus

A large epizootic of an acute respiratory disease of cattle occurred in Japan during the months from October 1968 to May 1969. A virus was recovered in primary cultures of calf kidney and testicle cells from nasal swabs of affected cattle. Neutralization tests revealed the virus to be closely related to the Long strain of human respiratory syncytial virus. The virus induced cytopathic changes including the formation of syncytia and acidophilic-cytoplasmic inclusions in calf kidney and testicle cell cultures. A calf inoculated with the virus by the respiratory route developed an illness resembling the natural disease. Most cattle clinically diagnosed as having the disease showed significant rises of neutralizing antibody titer for the isolated virus, whereas none or only small fractions of those animals showed serological evidence for recent infection with bovine ephemeral fever virus, infectious bovine rhinotracheitis virus, Ibaraki virus, bovine diarrhea virus, bovine adenovirus Type 7 and parainfluenza virus Type 3. Neutralization tests on paired sera revealed a wide dissemination of the isolated virus among cattle in many areas of the country during the epizootic. All these findings leave no doubt that the epizootic was caused by bovine respiratory syncytial virus. This is the first study that ever shows the presence of infection of cattle with this virus in Japan.     

Proteins Specified by bovine herpesvirus 1 (infectious bovine rhinotracheitis virus)

An electrophoretic analysis of radioactively labeled, purified, "empty" and DNA-containing infectious bovine rhinotracheitis virions revealed the presence of 25 to 33 structural (virion) polypeptides. A total of 11 of these polypeptides could be labeled with [3H]glucosamine and were identified as glycoproteins. In addition to the 25 structural polypeptides, infectious bovine rhinotracheitis virus infected cells also contained at least 15 nonstructural (nonvirion) polypeptides that were not present in purified virions. Expression of the viral polypeptides in infected cells was controlled temporally. Thus, most viral polypeptides could be categorized as "alpha" (immediate early), "beta" (early), or "gamma" (late) on the basis of their order of appearance in infected cells and whether their syntheses were dependent upon prior viral protein or DNA synthesis. None of the glycoproteins belongs to the alpha class, although at least one (GVP11) was synthesized in the absence of viral DNA synthesis. Serum from a cow in which infectious bovine rhinotracheitis virus lesions were reactivated by dexamethasone precipitated both structural and nonstructural polypeptides.     

Microtiter Tests for Detecting Antibody in Bovine Serum to Parainfluenza 3 Virus, Infectious Bovine Rhinotracheitis Virus, and Bovine Virus Diarrhea Virus

Microneutralization tests for detection of antibody in bovine serum to three bovine viruses are described. The Madin-Darby bovine kidney cell line was used with parainfluenza 3 virus (PI 3), whereas serially cultivated bovine embryonic kidney cells were used for infectious bovine rhinotracheitis virus and bovine virus diarrhea virus. Comparison of micro-hemagglutination-inhibition (HI) with micro-serum-neutralization (SN) tests for PI 3 showed the SN test to be more sensitive, more specific, and therefore more useful than the HI test for detecting antibody. Although the effect of trypsin-periodate treatment of serum was to reduce the HI titer of numerous sera by a twofold dilution, sufficient evidence could not be found to indicate that nonspecific HI inhibitors to PI 3 are present in bovine sera.     

Neutralizing antibodies specific for glycoprotein H of herpes simplex virus permit viral attachment to cells but prevent penetration.

Monoclonal antibodies specific for gH of herpes simplex virus were shown previously to neutralize viral infectivity. Results presented here demonstrate that these antibodies (at least three of them) block viral penetration without inhibiting adsorption of virus to cells. Penetration of herpes simplex virus is by fusion of the virion envelope with the plasma membrane of a susceptible cell. Electron microscopy of thin sections of cells exposed to virus revealed that neutralized virus bound to the cell surface but did not fuse with the plasma membrane. Quantitation of virus adsorption by measuring the binding of purified radiolabeled virus to cells revealed that the anti-gH antibodies had little or no effect on adsorption. Monitoring cell and viral protein synthesis after exposure of cells to infectious and neutralized virus gave results consistent with the electron microscopic finding that the anti-gH antibodies blocked viral penetration. On the basis of the results presented here and other information published elsewhere, it is suggested that gH is one of three glycoproteins essential for penetration of herpes simplex virus into cells.     

Parasites, diseases, and health status of sympatric populations of sika deer and white-tailed deer in Maryland and Virginia

In July 1981, investigations on parasites, diseases, and herd health status were conducted on sympatric populations of sika deer (Cervus nippon) and white-tailed deer (Odocoileus virginianus) from Blackwater National Wildlife Refuge (Maryland) and Chincoteague National Wildlife Refuge (Virginia) on the Delmarva Peninsula. Five adult deer of each species were collected from each location and subjected to thorough necropsy examinations and laboratory tests. White-tailed deer at both locations harbored protozoan, helminth, and arthropod parasites typically associated with this species throughout the southeastern United States. In contrast, sika deer at both locations harbored only light burdens of ticks, chiggers, and sarcocysts. Serologic tests for antibodies to seven infectious disease agents revealed evidence of exposure to bovine virus diarrhea (BVD) virus, infectious bovine rhinotracheitis virus, and parainfluenza3 virus in white-tailed deer, but only BVD virus in sika deer. At both locations the general health status of sika deer was superior to that of white-tailed deer.     

Identification and characterization of a mouse cell mutant defective in the intracellular transport of glycoproteins

We have isolated a mutant line of mouse L cells, termed gro29, in which the growth of herpes simplex virus (HSV) and vesicular stomatitis virus (VSV) is defective. The block occurs late in the infectious cycle of both viruses. We demonstrate that HSV and VSV enter gro29 cells normally, negotiate the early stages of infection, yet are impaired at a late stage of virus maturation. During VSV infection of the mutant cell line, intracellular transport of its glycoprotein (G protein) is slowed. Pulse-chase experiments showed that oligosaccharide processing is impeded, and immunofluorescence localization revealed an accumulation of G protein in a juxtanuclear region that contains the Golgi complex. We conclude that export of newly made glycoproteins is defective in gro29 cells, and speculate that this defect may reflect a lesion in the glycoprotein transport apparatus.     

Properties of Hamster Embryo Fibroblasts Transformed In Vitro After Exposure to Ultraviolet-Irradiated Herpes Simplex Virus Type 2

An in vitro method which led to the transformation of hamster embryo fibroblasts after exposure to herpes simplex virus type 2 (HSV-2) inactivated with ultraviolet irradiation is described. The transformed cells (333-8-9) produced tumors when inoculated into newborn Syrian hamsters but not when injected into weanling Syrian hamsters of the same LSH inbred strain. However, after one in vivo passage, the 333-8-9 cells became highly oncogenic in weanling hamsters. No infectious virus was recovered from these cells. Herpes simplex virus antigens were detected in the transformed cells by the indirect immunofluorescence technique. Sera from tumor-bearing hamsters contained antibody with highly specific neutralizing activity against HSV-2. These studies indicate the continued involvement of the HSV-2 genome in an oncogenic cell line.     

Monoclonal antibodies suppress replication of herpes simplex virus type 1 in trigeminal ganglia.

The effect of monoclonal antibodies on the growth of herpes simplex virus type 1 in trigeminal ganglia was investigated. Four-week-old mice were infected on an abrased cornea with herpes simplex virus type 1. Forty-eight hours after infection, trigeminal ganglia ipsilateral with infected eyes were removed and placed in culture. Incubation of infected ganglia in the presence of a pool of nonneutralizing monoclonal antibodies specific for glycoproteins of gB and gE suppressed virus growth by greater than 90%. This was comparable to the amount of suppression observed when infected ganglia were incubated in hyperimmune serum. Individual monoclonal antibodies were less efficient, being able to inhibit virus growth by only two- to threefold. The mechanism of suppression was examined. Reduction in virus growth was observed under conditions in which all susceptible ganglion cells were infected in vitro before nonneutralizing monoclonal antibody was added. Similar results were obtained in tests with virus-infected neuroblastoma cells. Furthermore, suppression of infectious progeny was seen in the absence of complement and immunologically reactive cells. Thus, neither virus neutralization nor immunocytolysis could account for the effects of antibody on virus growth. Rather, the data suggest that antibody can bind to herpes simplex virus type 1-infected neuronal cells and suppress intracellular virus replication.     

Alterations in Glycosphingolipid Patterns in a Line of African Green Monkey Kidney Cells Infected with Herpesvirus

The major glycosphingolipids (GSLs) of a line of African green monkey kidney cells (BGM) were characterized as glucosylceramide, lactosylceramide, galactosyl-galactosyl-glucosylceramide, and N-acetylgalactosaminyl-galactosyl-galactosyl-glucosylceramide. Neutral GSLs accounted for approximately 80% of the total GSLs isolated. The predominant gangliosides were N-acetylneuraminyl-galactosyl-glucosylceramide, N-acetylgalactosaminyl-N-acetylneuraminyl-galactosyl- glucosylceramide, and galactosyl-N-acetylgalactosaminyl-N-acetylneuraminyl -galactosyl-glucosylceramide. The incorporation of labeled galactose into GSLs was compared in mock-infected and herpes simplex virus type 1-infected BGM cells. Herpes simplex virus type 1 infection resulted in a three- to four-fold increase in galactose incorporation into glucosylceramide and a decrease in galactose incorporation into galactosyl-galactosyl-glucosylceramide and N-acetyl-galactosaminyl-galactosyl-galactosyl-glucosylceramide. The virus-induced alteration in the GSL labeling pattern occurred early in infection, before the release of infectious virus, and was not prevented by the presence of cytosine arabinoside. Treatment of uninfected BGM cells with cycloheximide resulted in alterations in the GSL pattern which were similar to those observed in herpes simplex virus type 1-infected cells. These observations suggest that an early virus function such as inhibition of host cell protein synthesis is responsible for the observed alterations of GSL metabolism. Experiments with a syncytium-producing strain of herpes simplex virus type 1, herpes simplex virus type 2, and pseudorabies virus indicated that other herpes viruses altered GSL metabolism in a manner similar to herpes simplex virus type 1.     

The effect of substrate on the production of infectious virus by cells in culture

Herpes simplex virus type I (HSV-1), infectious bovine rhinotracheitis virus (IBR) and turkey herpesvirus were examined for growth in cells cultured on three different substrates. The substrates were glass, DEAE-dextran and collagen gel. With two of the viruses, HSV-1 and IBR, there were no apparent differences in production as a function of substrate. In contrast, the amount of the turkey herpesvirus which was recovered varied greatly with the substrate. Titers were highest on glass, followed by DEAE-dextran and then collagen gel. Our previous studies have indicated that the substrate on which anchorage-dependent cells are grown in vitro has an affect on a number of biological and biochemical properties. The present study indicates that the production of commercially important biologicals can be affected by the substrate.     

A neutrophil-derived antiviral protein: induction requirements and biological properties

Polymorphonuclear neutrophilic granulocytes (PMN) have been implicated as playing a role in antiviral defense. In addition to having phagocytic and cytotoxic activities, PMN may produce an antiviral substance with interferon (IFN)-like activity. The product, for which the name polyferon (PF) has been coined, is produced upon direct encounter of PMN with bovine herpesvirus 1 (BHV-1)-infected bovine cells or membranes thereof. Exposure to purified virus only does not induce PF. The intimate interaction between PMN and the membranes was also revealed by electron microscopy studies. Bovine cells infected with herpes simplex virus type 1 could also induce PF production by bovine PMN, whereas cells infected with BHV-2, herpes simplex virus type 2, equine herpesvirus 1, bovine respiratory syncytial virus, bovine viral diarrhea virus, or parainfluenza virus 3 were unable to do so. Results obtained in experiments using transfected cells expressing BHV-1 glycoproteins as well as blocking experiments using BHV-1 glycoprotein-monospecific antibodies suggested that a combination of both viral product(s) and host cell factor(s) unique to bovine cells is required for induction of PF production by PMN. PF, which appeared in detectable amounts 12 to 18 h after exposure of PMN to the appropriate inducer, could not be neutralized by antibodies to bovine IFN-alpha, -beta, and -gamma. PF may nevertheless belong to the IFN family of proteins, as indicated by its ability to induce 2',5'-oligoadenyl synthetase in various cell types that are responsive to bovine IFNs and by its antiviral spectrum. It does, however, differ from the other cytokines in most immunological characteristics tested so far, including major histocompatibility complex class II antigen induction, cell migration, and cytotoxicity.     

Serological evidence of herpesvirus infection in gibbons

Background  

Herpesviruses are not only infectious agents of worldwide distribution in humans, but have also been demonstrated in various non-human primates as well. Seventy-eight gibbons were subjected to serological tests by ELISA for herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), Epstein-Barr virus (EBV) and cytomegalovirus (CMV).     

Prevalence of infectious agents in free-ranging white-tailed deer in northeastern Mexico

The objectives of this study were to determine the prevalence of antibodies against brucellosis, leptospirosis, infectious bovine rhinotracheitis virus, and bovine viral diarrhea virus (BVDV) in white-tailed deer (Odocoileus virginianus) in northeastern Mexico. Deer (n=521) were captured from helicopter using a netgun on 15 ranches covering 62,114 ha in the states of Coahuila, Nuevo Leon, and Tamaulipas during spring 2004. The prevalence of antibodies against Leptospira, infectious bovine rhinotracheitis, BVDV, and brucellosis were 5.6, 41.1, 63.5, and 0%, respectively, indicating that white-tailed deer and cattle may share disease agents when cohabiting in northeastern Mexico.     

Monensin inhibits the processing of herpes simplex virus glycoproteins, their transport to the cell surface, and the egress of virions from infected cells.

HEp-2 cells or Vero cells infected with herpes simplex virus type 1 were exposed to the ionophore monensin, which is thought to block the transit of membrane vesicles from the Golgi apparatus to the cell surface. We found that yields of extracellular virus were reduced to less than 0.5% of control values by 0.2 microM monensin under conditions that permitted accumulation of cell-associated infectious virus at about 20% of control values. Viral protein synthesis was not inhibited by monensin, whereas late stages in the post-translational processing of the viral glycoproteins were blocked. The transport of viral glycoproteins to the cell surface was also blocked by monensin. Although the assembly of nucleocapsids appeared to be somewhat inhibited in monensin-treated cells, electron microscopy revealed that nucleocapsids were enveloped to yield virions, and electrophoretic analyses showed that the isolated virions contained immature forms of the envelope glycoproteins. Most of the virions which were assembled in monensin-treated cells accumulated in large intracytoplasmic vacuoles, whereas most of the virions produced by and associated with untreated cells were found attached to the cell surface. Our results implicate the Golgi apparatus in the egress of herpes simplex virus from infected cells and also suggest that complete processing of the viral envelope glycoproteins is not essential for nucleocapsid envelopment or for virion infectivity.     

传染性牛鼻气管炎病毒和山羊痘病毒从细胞表面释放的研究

利用扫描电镜和细胞表面复型透射电镜技术,研究了传染性牛鼻气管炎病毒和山羊痘病毒分别从牛肾细胞和羊睾丸细胞表面的释放。根据表面形态结果观察,说明病毒是以出芽、细胞裂解、特殊通道和通过绒毛等多种方式释放。本文对病毒的形态、释放数量和分布以及同细胞之间的关系也进行了描述。     

Further evidence for entry of infectious bovine rhinotracheitis virus into bovine kidney cells

The penetration of bovine kidney cells by infectious bovine rhinotracheitis virus, a member of the herpesvirus group, was investigated using the direct immunoferritin labeling technique. Electron microscopic examination of infected cells after 10 min at 37°C revealed fusion between viral envelope and cell membrane; the former reacted with the ferritin particles conjugated with antiviral antibody. However, shortly after penetration of the nucleocapsid, viral-specific antigenic sites on the plasma membrane were not detected by the immunoferritin technique. Antigenically reactive structures in a disorganized array were frequently detected extracellularly, situated above the penetration sites as indicated by the internalized nucleocapsids.