Here we present the first complete genomic sequence of Marek's disease virus serotype 3 (MDV3), also known as turkey herpesvirus (HVT). The 159,160-bp genome encodes an estimated 99 putative proteins and resembles alphaherpesviruses in genomic organization and gene content. HVT is very similar to MDV1 and MDV2 within the unique long (UL) and unique short (US) genomic regions, where homologous genes share a high degree of colinearity and their proteins share a high level of amino acid identity. Within the UL region, HVT contains 57 genes with homologues found in herpes simplex virus type 1 (HSV-1), six genes with homologues found only in MDV, and two genes (HVT068 and HVT070 genes) which are unique to HVT. The HVT US region is 2.2 kb shorter than that of MDV1 (Md5 strain) due to the absence of an MDV093 (SORF4) homologue and to differences at the UL/short repeat (RS) boundary. HVT lacks a homologue of MDV087, a protein encoded at the UL/RS boundary of MDV1 (Md5), and it contains two homologues of MDV096 (glycoprotein E) in the RS. HVT RS are 1,039 bp longer than those in MDV1, and with the exception of an ICP4 gene homologue, the gene content is different from that of MDV1. Six unique genes, including a homologue of the antiapoptotic gene Bcl-2, are found in the RS. This is the first reported Bcl-2 homologue in an alphaherpesvirus. HVT long repeats (RL) are 7,407 bp shorter than those in MDV1 and do not contain homologues of MDV1 genes with functions involving virulence, oncogenicity, and immune evasion. HVT lacks homologues of MDV1 oncoprotein MEQ, CxC chemokine, oncogenicity-associated phosphoprotein pp24, and conserved domains of phosphoprotein pp38. These significant genomic differences in and adjacent to RS and RL regions likely account for the differences in host range, virulence, and oncogenicity between nonpathogenic HVT and highly pathogenic MDV1. (+info)
Viral cyclin-cyclin-dependent kinase 6 complexes initiate nuclear DNA replication.
(50/1077)
The cyclins encoded by Kaposi sarcoma-associated herpesvirus and herpesvirus saimiri are homologs of human D-type cyclins. However, when complexed to cdk6, they have several activities that distinguish them from D-type cyclin-cdk6 complexes, including resistance to cyclin-dependent kinase inhibitors and an enhanced substrate range. We find that viral cyclins interact with and phosphorylate proteins involved in replication initiation. Using mammalian in vitro replication systems, we show that viral cyclin-cdk6 complexes can directly trigger the initiation of DNA synthesis in isolated late-G(1)-phase nuclei. Viral cyclin-cdk6 complexes share this capacity with cyclin A-cdk2, demonstrating that in addition to functioning as G(1)-phase cyclin-cdk complexes, they function as S-phase cyclin-cdk complexes. (+info)
Quantitative fluorogenic PCR assay for measuring ovine herpesvirus 2 replication in sheep.
(51/1077)
A fluorogenic PCR specific for ovine herpesvirus 2 (OvHV-2) DNA was developed and compared to a previously established conventional seminested PCR. Testing of a total of 152 blood samples from both positive and negative animals revealed that the results of both assays corresponded to each other in 100% of the cases. A second fluorogenic PCR for genomic sheep DNA was required to normalize the quantity of viral DNA in the sample. Separate standard curves had to be constructed for each PCR. The analytical sensitivity of the new PCRs ranged between at least 10 copies and sometimes even 1 copy of target DNA per reaction mixture. In dilution series of the target DNAs, linear decreases of the signals were observed over 7 orders of magnitude. Thus, it was possible to calculate the amounts of viral DNA in relation to the amounts of cellular DNA by normalizing the absolute quantity of OvHV-2 DNA with the amount of genomic sheep DNA. By this technique, it was possible for the first time to quantitatively characterize the course of OvHV-2 replication in naturally infected sheep. (+info)
Monitoring four herpesviruses in unrelated cord blood transplantation.
(52/1077)
Cord blood transplantation, which has lower risk of graft-versus-host disease than bone marrow transplantation, might have higher risk of infections. A system to quantify four herpesviruses, CMV, human herpesvirus 6 (HHV6), EBV, varicella-zoster virus using the real-time PCR assay was established and applied for prospective viral load monitoring in three recipients undergoing cord blood transplantation. CMV and HHV6 were detected in peripheral blood from all three recipients, while EBV was detected in two. Varicella-zoster virus was not detected at all. At the peak of HHV6 or CMV, each patient showed virus-related symptoms. During the pre-transplant period, CMV DNA was detected in two recipients who later developed CMV-related diseases. These observations indicate that our system is not only useful for managing herpesviruses infections in transplant recipients, but also a powerful method for clarifying the relationships between the viral load and clinical symptoms. (+info)
Genomewide function conservation and phylogeny in the Herpesviridae.
(53/1077)
The Herpesviridae are a large group of well-characterized double-stranded DNA viruses for which many complete genome sequences have been determined. We have extracted protein sequences from all predicted open reading frames of 19 herpesvirus genomes. Sequence comparison and protein sequence clustering methods have been used to construct herpesvirus protein homologous families. This resulted in 1692 proteins being clustered into 243 multiprotein families and 196 singleton proteins. Predicted functions were assigned to each homologous family based on genome annotation and published data and each family classified into seven broad functional groups. Phylogenetic profiles were constructed for each herpesvirus from the homologous protein families and used to determine conserved functions and genomewide phylogenetic trees. These trees agreed with molecular-sequence-derived trees and allowed greater insight into the phylogeny of ungulate and murine gammaherpesviruses. (+info)
Detection and heterogeneity of herpesviruses causing Pacheco's disease in parrots.
(54/1077)
Pacheco's disease (PD) is a common, often fatal, disease of parrots. We cloned a virus isolate from a parrot that had characteristic lesions of PD. Three viral clones were partially sequenced, demonstrating that this virus was an alphaherpesvirus most closely related to the gallid herpesvirus 1. Five primer sets were developed from these sequences. The primer sets were used with PCR to screen tissues or tissue culture media suspected to contain viruses from 54 outbreaks of PD. The primer sets amplified DNA from all but one sample. Ten amplification patterns were detected, indicating that PD is caused by a genetically heterogeneous population of viruses. A single genetic variant (psittacid herpesvirus variant 1) amplified with all primer sets and was the most common virus variant (62.7%). A single primer set (23F) amplified DNA from all of the positive samples, suggesting that PCR could be used as a rapid postmortem assay for these viruses. PCR was found to be significantly more sensitive than tissue culture for the detection of psittacid herpesviruses. (+info)
Constitutive activation of Lck and Fyn tyrosine kinases in large granular lymphocytes infected with the gamma-herpesvirus agents of malignant catarrhal fever.
(55/1077)
Large granular lymphocytes (LGL) with a T or natural killer (NK) lymphoblast morphology and indiscriminate (non-major histocompatibility complex-linked) cytotoxicity for a variety of target cells can be derived in culture from the tissues of animals infected with either alcelaphine herpesvirus-1 (AlHV-1) or ovine herpesvirus-2 (OvHV-2). In this study, LGL survival in the absence of exogenous interleukin-2 was inhibited by the protein kinase inhibitor genestein, but not the p70 s6 kinase inhibitor rapamycin. Constitutive activation of the src kinases Lck and Fyn was demonstrated in a bovine LGL line infected with OvHV-2 and in two rabbit LGL lines infected with AlHV-1. The p44 erk1 and p42 erk2 mitogen-activated protein kinases (MAPK) were also constitutively activated in the LGLs but not control T cells. Lck and Fyn kinase activity in the LGLs did not increase after mitogen (concanavalin A or concanavalin A plus phorbol ester) stimulation of the cells, in contrast to control T cells. Control T cells, but not the LGLs, proliferated after mitogen stimulation. An analysis of tyrosine phosphorylated proteins in the cells indicated that the LGLs exhibited some similarities and differences to activated control T cells. The results demonstrate that the activated phenotype of the LGLs, associated with malignant catarrhal fever virus infection and in the absence of exogenous interleukin-2, involves constitutively activated Lck and Fyn kinases. These are normally crucial for the initial activation of T cells via several cell-surface receptors (e.g. the T-cell receptor and CD2). The inability of the LGLs to proliferate in response to mitogen may be due to an inability of Lck and Fyn to become further activated after mitogen stimulation. (+info)
Genetic and ultrastructural characterization of a European isolate of the fatal endotheliotropic elephant herpesvirus.
(56/1077)
A male Asian elephant (Elephas maximus) died at the Berlin zoological gardens in August 1998 of systemic infection with the novel endotheliotropic elephant herpesvirus (ElHV-1). This virus causes a fatal haemorrhagic disease in Asian elephants, the so-called endothelial inclusion body disease, as reported from North American zoological gardens. In the present work, ElHV-1 was visualized ultrastructurally in affected organ material. Furthermore, a gene block comprising the complete glycoprotein B (gB) and DNA polymerase (DPOL) genes as well as two partial genes was amplified by PCR-based genome walking and sequenced. The gene content and arrangement were similar to those of members of the Betaherpesvirinae. However, phylogenetic analysis with gB and DPOL consistently revealed a very distant relationship to the betaherpesviruses. Therefore, ElHV-1 may be a member of a new genus or even a new herpesvirus subfamily. The sequence information generated was used to set up a nested-PCR assay for diagnosis of suspected cases of endothelial inclusion body disease. Furthermore, it will aid in the development of antibody-based detection methods and of vaccination strategies against this fatal herpesvirus infection in the endangered Asian elephant. (+info)