Identification of gammaherpesvirus infection in free-ranging black bears (Ursus americanus).

Herpesvirus infection was investigated in black bears (Ursus americanus) with neurological signs and brain lesions of nonsuppurative encephalitis of unknown cause. Visible cytopathic effects (CPE) could only be observed on days 3-5 post-infection in HrT-18G cell line inoculated with bear tissue extracts. The observed CPE in HrT-18G cells included syncytia, intranuclear inclusions, and cell detachments seen in herpesvirus infection in vitro. Herpesvirus-like particles were observed in viral culture supernatant under the electron microscope, however, capsids ranging from 60 nm to 100 nm in size were often observed in viral cultures within the first two passages of propagation. Herpesvirus infection in the bear tissues and tissue cultures were detected by PCR using degenerate primers specific to the DNA polymerase gene (DPOL) and glycoprotein B gene (gB). DNA sequencing of the amplicon revealed that the detected herpesvirus has 94-95% identity to Ursid gammaherpesvirus 1 (UrHV-1) DNA sequences of DPOL. Phylogenetic analysis of DPOL sequences indicates that black bear herpesviruses and UrHV-1 are closely related and have small distances to members of Rhadinovirus. Interestingly, black bear herpesvirus infections were also found in bears without neurological signs. The DPOL DNA sequence of black bear herpesviruses detected in neurological bears were similar to the those detected in the non-neurological bears. However, the gB DNA sequence detected from the neurological bear is different from non-neurological bear and has only 64.5%-70% identity to each other. It is possible that at least two different types of gammaherpesviruses are present in the U. americanus population or several gammaherpesviruses exist in ursine species.

Gammaherpesvirus Tegument Protein ORF33 Is Associated With Intranuclear Capsids at an Early Stage of the Tegumentation Process.

UNLABELLED: Herpesvirus nascent capsids, after assembly in the nucleus, must acquire a variety of tegument proteins during maturation. However, little is known about the identity of the tegument proteins that are associated with capsids in the nucleus or the molecular mechanisms involved in the nuclear egress of capsids into the cytoplasm, especially for the two human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), due to a lack of efficient lytic replication systems. Murine gammaherpesvirus 68 (MHV-68) is genetically related to human gammaherpesviruses and serves as an excellent model to study the de novo lytic replication of gammaherpesviruses. We have previously shown that open reading frame 33 (ORF33) of MHV-68 is a tegument protein of mature virions and is essential for virion assembly and egress. However, it remains unclear how ORF33 is incorporated into virions. In this study, we first show that the endogenous ORF33 protein colocalizes with capsid proteins at discrete areas in the nucleus during viral infection. Cosedimentation analysis as well as an immunoprecipitation assay demonstrated that ORF33 is associated with both nuclear and cytoplasmic capsids. An immunogold labeling experiment using an anti-ORF33 monoclonal antibody revealed that ORF33-rich areas in the nucleus are surrounded by immature capsids. Moreover, ORF33 is associated with nucleocapsids prior to primary envelopment as well as with mature virions in the cytoplasm. Finally, we show that ORF33 interacts with two capsid proteins, suggesting that nucleocapsids may interact with ORF33 in a direct manner. In summary, we identified ORF33 to be a tegument protein that is associated with intranuclear capsids prior to primary envelopment, likely through interacting with capsid proteins in a direct manner. IMPORTANCE: Morphogenesis is an essential step in virus propagation that leads to the generation of progeny virions. For herpesviruses, this is a complicated process that starts in the nucleus. Although the process of capsid assembly and genome packaging is relatively well understood, how capsids acquire tegument (the layer between the capsid and the envelope in a herpesvirus virion) and whether the initial tegumentation process takes place in the nucleus remain unclear. We previously showed that ORF33 of MHV-68 is a tegument protein and functions in both the nuclear egress of capsids and final virion maturation in the cytoplasm. In the present study, we show that ORF33 is associated with intranuclear capsids prior to primary envelopment and identify novel interactions between ORF33 and two capsid proteins. Our work provides new insights into the association between tegument proteins and nucleocapsids at an early stage of the virion maturation process for herpesviruses.

Three-dimensional visualization of gammaherpesvirus life cycle in host cells by electron tomography.

Gammaherpesviruses are etiologically associated with human tumors. A three-dimensional (3D) examination of their life cycle in the host is lacking, significantly limiting our understanding of the structural and molecular basis of virus-host interactions. Here, we report the first 3D visualization of key stages of the murine gammaherpesvirus 68 life cycle in NIH 3T3 cells, including viral attachment, entry, assembly, and egress, by dual-axis electron tomography. In particular, we revealed the transient processes of incoming capsids injecting viral DNA through nuclear pore complexes and nascent DNA being packaged into progeny capsids in vivo as a spool coaxial with the putative portal vertex. We discovered that intranuclear invagination of both nuclear membranes is involved in nuclear egress of herpesvirus capsids. Taken together, our results provide the structural basis for a detailed mechanistic description of gammaherpesvirus life cycle and also demonstrate the advantage of electron tomography in dissecting complex cellular processes of viral infection.

Open reading frame 33 of a gammaherpesvirus encodes a tegument protein essential for virion morphogenesis and egress.

Tegument is a unique structure of herpesvirus, which surrounds the capsid and interacts with the envelope. Morphogenesis of gammaherpesvirus is poorly understood due to lack of efficient lytic replication for Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8, which are etiologically associated with several types of human malignancies. Murine gammaherpesvirus 68 (MHV-68) is genetically related to the human gammaherpesviruses and presents an excellent model for studying de novo lytic replication of gammaherpesviruses. MHV-68 open reading frame 33 (ORF33) is conserved among Alpha-, Beta-, and Gammaherpesvirinae subfamilies. However, the specific role of ORF33 in gammaherpesvirus replication has not yet been characterized. We describe here that ORF33 is a true late gene and encodes a tegument protein. By constructing an ORF33-null MHV-68 mutant, we demonstrated that ORF33 is not required for viral DNA replication, early and late gene expression, viral DNA packaging or capsid assembly but is required for virion morphogenesis and egress. Although the ORF33-null virus was deficient in release of infectious virions, partially tegumented capsids produced by the ORF33-null mutant accumulated in the cytoplasm, containing conserved capsid proteins, ORF52 tegument protein, but virtually no ORF45 tegument protein and the 65-kDa glycoprotein B. Finally, we found that the defect of ORF33-null MHV-68 could be rescued by providing ORF33 in trans or in an ORF33-null revertant virus. Taken together, our results indicate that ORF33 is a tegument protein required for viral lytic replication and functions in virion morphogenesis and egress.

Isolation of equine herpesvirus-5 from blood mononuclear cells of a gelding.

Horses are commonly infected by herpesviruses, but isolation of equine herpesvirus-5 (EHV-5) has only infrequently been reported. We describe the isolation and characterization of a strain of EHV-5 from the blood mononuclear cells of a healthy adult horse in California. The virus was initially identified by EHV-5 specific polymerase chain reaction (PCR), and it caused lytic infection of cultured rabbit kidney cells only after repeated serial passage. Virions with characteristic herpesvirus morphology were readily demonstrated in cell culture lysate by transmission electron microscopy. A portion of the glycoprotein B gene of this strain of EHV-5 had 99% identity to the published EHV-5 sequence, and it was clearly distinguishable from other EHV (1-4) by virus-specific PCR assays. Prevalence of EHV-5 infection in a group of young racehorses was estimated at 64% using the EHV-5 specific PCR on nasopharyngeal secretions.