The genome of turkey herpesvirus

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.     

Homologies between herpesvirus of turkey and Marek's disease virus type-1 DNAs within two co-linearly arranged open reading frames, one encoding glycoprotein A

The genomes of two avian herpesviruses, Marek's disease virus type 1 (MDV1) and herpesvirus of turkey (HVT), share close homology only within certain DNA regions. One such homologous region of HVT DNA was cloned and sequenced. Two open reading frames (ORFs) were found in the long unique region, ORF1 encoding the glycoprotein A (gA), and ORF2 encoding a still unidentified protein. These two HVT-ORFs are located at almost the same positions as the homologous MDV1-ORFs. The nucleotide sequence homologies between HVT and MDV1 were 73% and 68% for ORF1 and ORF2, respectively. Both the 5'- and 3'-noncoding regions, however, are less conserved. The third letter within every codon of ORF1 and ORF2 showed a mismatch of greater than 50% between the two viruses. The amino acid (aa) sequence homologies between the corresponding putative viral proteins are 83% and 80% for ORF1 (gA) and ORF2, respectively. More than 90% homology was observed in the C-terminal region of ORF1 (gA). Furthermore, the deduced aa sequences for both of the ORFs in these two viruses showed considerable homology to two adjoining genes in herpes simplex virus type 1, the glycoprotein C and UL45 genes.     

Complete Genome Sequence of a Recombinant Marek's Disease Virus Field Strain with One Reticuloendotheliosis Virus Long Terminal Repeat Insert

Marek''s disease virus (MDV) Chinese strain GX0101, isolated in 2001 from a vaccinated flock of layer chickens with severe tumors, was the first reported recombinant MDV field strain with one reticuloendotheliosis virus (REV) long terminal repeat (LTR) insert. GX0101 belongs to very virulent MDV (vvMDV) but has higher horizontal transmission ability than the vvMDV strain Md5. The complete genome sequence of GX0101 is 178,101 nucleotides (nt) and contains only one REV-LTR insert at a site 267 nt upstream of the sorf2 gene. Moreover, GX0101 has 5 repeats of a 217-nt fragment in its terminal repeat short (TRS) region and 3 repeats in internal repeat short (IRS) region, compared to the other 10 strains with only 1 or 2 repeats in both TRS and IRS.     

Roles of avian herpesvirus microRNAs in infection, latency, and oncogenesis

MicroRNAs have been reported for the avian herpesviruses Marek's disease virus 1 (MDV1; oncogenic), Marek's disease virus 2 (MDV2; non-oncogenic), herpesvirus of turkeys (HVT), and infectious laryngotracheitis virus (ILTV). No obvious phylogenetic relationships exist among the avian herpesvirus microRNAs, but the general genomic locations of microRNA clusters are conserved, with microRNAs being located in the repeat regions of the genomes. In some cases, microRNAs are antisense to open reading frames. Among MDV1 field isolates with different virulence properties, microRNAs are highly conserved, and variations that have been observed lie in putative promoter regions. One cluster of MDV1 microRNAs lies upstream of the meq gene, and this cluster is more highly expressed in tumors caused by an extremely virulent MDV1 isolate compared to tumors caused by a less virulent isolate. Several of the avian herpesvirus microRNAs are orthologs of microRNAs in other species. For example, mdv1-miR-M4 shares a seed sequence with gga-miR-155 (also shared with Kaposi sarcoma herpesvirus (KSHV) kshv-miR-K12), mdv2-miR-M21 shares a seed with miR-29b, and hvt-miR-H14 shares a seed sequence with miR-221. Functional analyses of avian herpesvirus microRNAs include a variety of in vitro assays to demonstrate potential function as well as the use of mutants that can exploit the ability to assess phenotypes experimentally in the natural host. This article is part of a Special Issue entitled:MicroRNA's in viral gene regulation.     

Avirulent Marek’s Disease Virus Type 1 Strain 814 Vectored Vaccine Expressing Avian Influenza (AI) Virus H5 Haemagglutinin Induced Better Protection Than Turkey Herpesvirus Vectored AI Vaccine

Background

Herpesvirus of turkey (HVT) as a vector to express the haemagglutinin (HA) of avian influenza virus (AIV) H5 was developed and its protection against lethal Marek’s disease virus (MDV) and highly pathogenic AIV (HPAIV) challenges was evaluated previously. It is well-known that avirulemt MDV type 1 vaccines are more effective than HVT in prevention of lethal MDV infection. To further increase protective efficacy against HPAIV and lethal MDV, a recombinant MDV type 1 strain 814 was developed to express HA gene of HPAIV H5N1.

Methodology/Principal Findings

A recombinant MDV-1 strain 814 expressing HA gene of HPAIV H5N1 virus A/goose/Guangdong/3/96 at the US2 site (rMDV-HA) was developed under the control of a human CMV immediate-early promoter. The HA expression in the rMDV-HA was tested by immunofluorescence and Western blot analyses, and in vitro and in vivo growth properties of rMDV-HA were also analyzed. Furthermore, we evaluated and compared the protective immunity of rMDV-HA and previously constructed rHVT-HA against HPAIV and lethal MDV. Vaccination of chickens with rMDV-HA induced 80% protection against HPAIV, which was better than the protection rate by rHVT-HA (66.7%). In the animal study with MDV challenge, chickens immunized with rMDV-HA were completely protected against virulent MDV strain J-1 whereas rHVT-HA only induced 80% protection with the same challenge dose.

Conclusions/Significance

The rMDV-HA vaccine was more effective than rHVT-HA vaccine for protection against lethal MDV and HPAIV challenges. Therefore, avirulent MDV type 1 vaccine is a better vector than HVT for development of a recombinant live virus vaccine against virulent MDV and HPAIV in poultry.     

The herpes simplex virus 1 protein kinase encoded by the US3 gene mediates posttranslational modification of the phosphoprotein encoded by the UL34 gene.

Earlier studies have shown that a herpes simplex virus 1 (HSV-1) open reading frame, US3, encodes a novel protein kinase and have characterized the cognate amino acid sequence which is phosphorylated by this enzyme. This report identifies an apparently essential viral phosphoprotein whose posttranslational processing involves the viral protein kinase. Analyses of viral proteins phosphorylated in the course of productive infection revealed a phosphoprotein whose mobility was viral protein kinase and serotype dependent. Thus, the corresponding HSV-1 and HSV-2 phosphoproteins differ in their electrophoretic mobilities, and the phosphoprotein specified by the HSV-1 mutant deleted in US3 (R7041) differs from that of the corresponding HSV-1 and HSV-2 proteins. Analyses of HSV-1 x HSV-2 recombinants mapped the phosphoprotein between 0.42 and 0.47 map units on the prototype HSV-1 DNA map. Within this region, the UL34 open reading frame was predicted to encode a protein of appropriate molecular weight which would also contain the consensus target site for phosphorylation by the viral protein kinase as previously defined with synthetic peptides. Replacement of the native UL34 gene with a UL34 gene tagged with a 17-amino-acid epitope from the alpha 4 protein identified this gene as encoding the phosphoprotein. Finally, mutagenesis of the predicted phosphorylation site on UL34 in the viral genome, and specifically the substitution of threonine or serine with alanine in the product of the UL34 gene, yielded phosphoproteins whose electrophoretic mobilities could not be differentiated from that of the US3- mutant. We conclude that the posttranslational processing of the UL34 gene product to its wild-type phenotype requires the participation of the viral protein kinase. While the viral protein kinase is not essential for viral replication in cells in culture, the UL34 gene product itself may not be dispensable.     

The three-component helicase/primase complex of herpes simplex virus-1

Herpes simplex virus type 1 (HSV-1) is one of the nine herpesviruses that infect humans. HSV-1 encodes seven proteins to replicate its genome in the hijacked human cell. Among these are the herpes virus DNA helicase and primase that are essential components of its replication machinery. In the HSV-1 replisome, the helicase–primase complex is composed of three components including UL5 (helicase), UL52 (primase) and UL8 (non-catalytic subunit). UL5 and UL52 subunits are functionally interdependent, and the UL8 component is required for the coordination of UL5 and UL52 activities proceeding in opposite directions with respect to the viral replication fork. Anti-viral compounds currently under development target the functions of UL5 and UL52. Here, we review the structural and functional properties of the UL5/UL8/UL52 complex and highlight the gaps in knowledge to be filled to facilitate molecular characterization of the structure and function of the helicase–primase complex for development of alternative anti-viral treatments.     

Protection against Marek''s disease by a fowlpox virus recombinant expressing the glycoprotein B of Marek''s disease virus.

Fowlpox virus (FPV) recombinants expressing the glycoprotein B and the phosphorylated protein (pp38) of the GA strain of Marek's disease virus (MDV) were assayed for their ability to protect chickens against challenge with virulent MDV. The recombinant FPV expressing the glycoprotein B gene elicited neutralizing antibodies against MDV, significantly reduced the level of cell-associated viremia, and, similar to the conventional herpesvirus of turkeys, protected chickens against challenge with the GA strain and the highly virulent RB1B and Md5 strains of MDV. The recombinant FPV expressing the pp38 gene failed to either elicit neutralizing antibodies against MDV or protect the vaccinated chickens against challenge with MDV.     

Horizontal transmission of Marek's disease virus requires US2, the UL13 protein kinase, and gC

Marek's disease virus (MDV) causes a general malaise in chickens that is mostly characterized by the development of lymphoblastoid tumors in multiple organs. The use of bacterial artificial chromosomes (BACs) for cloning and manipulation of the MDV genome has facilitated characterization of specific genes and genomic regions. The development of most MDV BACs, including pRB-1B-5, derived from a very virulent MDV strain, involved replacement of the US2 gene with mini-F vector sequences. However, when reconstituted viruses based on pRB-1B were used in pathogenicity studies, it was discovered that contact chickens housed together with experimentally infected chickens did not contract Marek's disease (MD), indicating a lack of horizontal transmission. Staining of feather follicle epithelial cells in the skins of infected chickens showed that virus was present but was unable to be released and/or infect susceptible chickens. Restoration of US2 and removal of mini-F sequences within viral RB-1B did not alter this characteristic, although in vivo viremia levels were increased significantly. Sequence analyses of pRB-1B revealed that the UL13, UL44, and US6 genes encoding the UL13 serine/threonine protein kinase, glycoprotein C (gC), and gD, respectively, harbored frameshift mutations. These mutations were repaired individually, or in combination, using two-step Red mutagenesis. Reconstituted viruses were tested for replication, MD incidence, and their abilities to horizontally spread to contact chickens. The experiments clearly showed that US2, UL13, and gC in combination are essential for horizontal transmission of MDV and that none of the genes alone is able to restore this phenotype.     

Genomic expansion of Marek''s disease virus DNA is associated with serial in vitro passage.

An EcoRI restriction endonuclease pattern of Md11 virus DNA, a very virulent strain of Marek's disease virus (MDV), was obtained by using total cellular DNA from infected cells. With the EcoRI restriction endonuclease pattern and a published BamHI map of MDV (Fukuchi et al., J. Virol. 51:102-109), we constructed a partial EcoRI map of a series of MDV clones (gift from H. J. Kung). The clones were used to identify a region of the Md11 genome which is altered as the oncogenic virus is passaged in vitro. This region was mapped into a 1.8-kilobase segment in the inverted-repeat sequences flanking the long unique region of the virus genome. The alteration appeared to result from multiple DNA insertions that produced an increase of 0.6 to 5.4 kilobases. Although the expansion of this region did not diminish the ability of MDV to replicate in vitro, it may be associated with the loss of Marek's disease oncogenicity.     

The UL13 and US3 Protein Kinases of Herpes Simplex Virus 1 Cooperate to Promote the Assembly and Release of Mature,Infectious Virions

Herpes simplex virus type 1 (HSV-1) encodes two bona fide serine/threonine protein kinases, the US3 and UL13 gene products. HSV-1 ΔUS3 mutants replicate with wild-type efficiency in cultured cells, and HSV-1 ΔUL13 mutants exhibit <10-fold reduction in infectious viral titers. Given these modest phenotypes, it remains unclear how the US3 and UL13 protein kinases contribute to HSV-1 replication. In the current study, we designed a panel of HSV-1 mutants, in which portions of UL13 and US3 genes were replaced by expression cassettes encoding mCherry protein or green fluorescent protein (GFP), respectively, and analyzed DNA replication, protein expression, and spread of these mutants in several cell types. Loss of US3 function alone had largely negligible effect on viral DNA accumulation, gene expression, virion release, and spread. Loss of UL13 function alone also had no appreciable effects on viral DNA levels. However, loss of UL13 function did result in a measurable decrease in the steady-state levels of two viral glycoproteins (gC and gD), release of total and infectious virions, and viral spread. Disruption of both genes did not affect the accumulation of viral DNA, but resulted in further reduction in gC and gD steady-state levels, and attenuation of viral spread and infectious virion release. These data show that the UL13 kinase plays an important role in the late phase of HSV-1 infection, likely by affecting virion assembly and/or release. Moreover, the data suggest that the combined activities of the US3 and UL13 protein kinases are critical to the efficient assembly and release of infectious virions from HSV-1-infected cells.     

Monoclonal antibodies with specificity for three different serotypes of Marek's disease viruses in chickens

A total of 50 antibody-secreting hybridoma cells against Marek's disease virus (MDV) and turkey herpesvirus (HVT) have been produced. Eleven hybridomas were used for serotyping a panel of 15 pathogenic and nonpathogenic strains of MDV and HVT, representing three serotypes. The antibodies from the culture medium have fluorescence antibody (FA) titers of up to 100 and those from mouse ascitic fluid have titers ranging from 10(4) to 10(6). Monoclonal antibody T81 is type-common, i.e., it reacts at equal titer with all MDV and HVT tested. Of the remaining 10 antibodies, eight react only with pathogenic and attenuated strains of MDV (presumably serotype 1), one reacts only with nonpathogenic MDV (presumably) serotype 2), and one reacts only with strains of HVT (presumably serotype 3). Two hybridomas belong to IgG2a and IgG2b subclasses, respectively, and the remaining nine belong to IgG1 subclass. None of the antibodies specific for MDV strains reacted with homologous viruses in serum neutralization (SN), agar gel precipitin (AGP), or membrane immunofluorescence tests. Antibody L78, which is specific for HVT, was reactive with its homologous virus in the SN test; antibody from the culture medium showed an SN titer of 10 and that from mouse ascites a titer of 10,000. None of the antibodies specific for MDV or HVT reacted with other avian or mammalian herpesviruses, avian leukosis viruses (ALV), reticuloendotheliosis viruses (REV), or Marek's disease tumor-associated surface antigen (MATSA) expressed in a lymphoblastoid cell line, MDCC-MSB-1.     

A mutant herpes simplex virus type 1 unable to express glycoprotein L cannot enter cells, and its particles lack glycoprotein H.

Herpes simplex virus type 1 (HSV-1) glycoprotein H (gH) is essential for virus entry into cells and forms a hetero-oligomer with a newly described viral glycoprotein, gL. Normal folding, posttranslational processing, and intracellular transport of both gH and gL depend upon the coexpression of gH and gL in cells infected with vaccinia virus vectors (L. Hutchinson, H. Browne, V. Wargent, N. Davis-Poynter, S. Primorac, K. Goldsmith, A. C. Minson, and D. C. Johnson, J. Virol. 66:2240-2250, 1992). Homologs of gH and gL have been found in herpesviruses of all subgroups, and thus it appears likely that the gH-gL complex serves a highly conserved function during herpesvirus penetration into cells. To examine the role of gL in the infectious cycle of HSV-1, a mutant HSV-1 unable to express gL was constructed by inserting a lacZ gene cassette into the coding sequences of the UL1 (gL) gene. Because gL was found to be essential for virus replication, cell lines capable of expressing gL were constructed to complement the virus mutant. In the absence of gL, virus particles were produced, and these particles reached the cell surface; however, gL-negative particles purified from infected cells were also deficient in gH. Mutant virions lacking gH and gL were able to adsorb onto cells but were unable to enter cells and initiate an infection. Further, the role of gL in fusion of infected cells was reexamined. A mutation in HSV-1 (804) which produces the syncytial phenotype had previously been mapped to a region of the HSV-1 genome which includes the UL1 gene and no other open reading frame. However, in contrast to this previous report, we found that the syncytial mutation in 804 affects the UL53 gene, which encodes gK, a gene commonly mutated in syncytial viruses.     

Functional anatomy of herpes simplex virus 1 overlapping genes encoding infected-cell protein 22 and US1.5 protein

Earlier studies have shown that (i) the coding domain of the alpha22 gene encodes two proteins, the 420-amino-acid infected-cell protein 22 (ICP22) and a protein, US1.5, which is initiated from methionine 147 of ICP22 and which is colinear with the remaining portion of that protein; (ii) posttranslational processing of ICP22 mediated largely by the viral protein kinase UL13 yields several isoforms differing in electrophoretic mobility; and (iii) mutants lacking the carboxyl-terminal half of the ICP22 and therefore DeltaUS1.5 are avirulent and fail to express normal levels of subsets of both alpha (e.g., ICP0) or gamma2 (e.g., US11 and UL38) proteins. We have generated and analyzed two sets of recombinant viruses. The first lacked portions of or all of the sequences expressed solely by ICP22. The second set lacked 10 to 40 3'-terminal codons of ICP22 and US1. 5. The results were as follows. (i) In cells infected with mutants lacking amino-terminal sequences, translation initiation begins at methionine 147. The resulting protein cannot be differentiated in mobility from authentic US1.5, and its posttranslational processing is mediated by the UL13 protein kinase. (ii) Expression of US11 and UL38 genes by mutants carrying only the US1.5 gene is similar to that of wild-type parent virus. (iii) Mutants which express only US1. 5 protein are avirulent in mice. (iv) The coding sequences Met147 to Met171 are essential for posttranslational processing of the US1.5 protein. (v) ICP22 made by mutants lacking 15 or fewer of the 3'-terminal codons are posttranslationally processed whereas those lacking 18 or more codons are not processed. (vi) Wild-type and mutant ICP22 proteins localized in both nucleus and cytoplasm irrespective of posttranslational processing. We conclude that ICP22 encodes two sets of functions, one in the amino terminus unique to ICP22 and one shared by ICP22 and US1.5. These functions are required for viral replication in experimental animals. US1.5 protein must be posttranslationally modified by the UL13 protein kinase to enable expression of a subset of late genes exemplified by UL38 and US11. Posttranslational processing is determined by two sets of sequences, at the amino terminus and at the carboxyl terminus of US1.5, respectively, a finding consistent with the hypothesis that both domains interact with protein partners for specific functions.     

The MHC class I homolog of human cytomegalovirus is resistant to down-regulation mediated by the unique short region protein (US)2, US3, US6, and US11 gene products

Human CMV encodes four unique short region proteins (US), US2, US3, US6, and US11, each independently sufficient for causing the down-regulation of MHC class I molecules on the cell surface. This down-regulation allows infected cells to evade recognition by cytotoxic T cells but leaves them susceptible to NK cells, which lyse cells that lack class I molecules. Another human CMV-encoded protein, unique long region protein 18 (UL18), is an MHC class I homolog that might provide a mechanism for inhibiting the NK cell response. The sequence similarities between MHC class I molecules and UL18 along with the ability of UL18 to form trimeric complexes with beta(2)-microglobulin and peptides led to the hypothesis that if the US and UL18 gene products coexist temporally during infection, the US proteins might down-regulate UL18 molecules, similar to their action on MHC class I molecules. We show here that temporal expression of US and UL18 genes partially overlaps during infection. However, unlike MHC class I molecules, the MHC class I homolog, UL18, is fully resistant to the down-regulation associated with the US2, US3, US6, and US11 gene products. The specific effect of US proteins on MHC class I molecules, but not on UL18, represents another example of how viral proteins have evolved to evade immune surveillance, avoiding fratricide by specifically targeting host proteins.