Global exploration of the metabolic requirements of gallid alphaherpesvirus 1.

Although therapeutics targeting viral metabolic processes have been considered as promising strategies to treat herpesvirus infection, the metabolic requirements of gallid alphaherpesvirus 1 (ILTV), which is economically important to the poultry industry worldwide, remain largely unknown. Using the ILTV-susceptible but nonpermissive chicken cell line DF-1 and the ILTV-permissive chicken cell line LMH as models, the present study explored the metabolic requirements of ILTV by global transcriptome analysis and metabolome assays of ILTV infected cell lines in combination with a set of functional validations. The extensive metabolic exploration demonstrated that ILTV infection tended to promote a metabolic shift from glycolysis to fatty acid (FA) and nucleotide biosynthesis and utilizes glutamine independently of glutaminolysis, without significant general effect on the TCA cycle. In addition, different metabolic pathways were found to be required for distinct stages of ILTV replication. Glucose and glutamine were required for the transcription of viral immediate early gene ICP4 and subsequent steps of viral replication. However, FA synthesis was essential for assembly but not required for other upstream steps of ILTV replication. Moreover, the metabolic requirements of ILTV infection revealed in chicken cell lines were further validated in chicken primary cells isolated from chicken embryo kidneys and chicken embryo livers. The present study, to the best of our knowledge, provides the first global metabolic profile of animal herpesviruses and illustrates the main characteristics of the metabolic program of ILTV.

Identification and functional analysis of membrane proteins gD, gE, gI, and pUS9 of Infectious laryngotracheitis virus.

Herpesvirus envelope proteins are of particular interest for development of attenuated live, marker, and subunit vaccines, as well as development of diagnostic tools. The unique short genome region of the chicken pathogen infectious laryngotracheitis virus (ILTV, Gallid herpesvirus 1) contains a cluster of six conserved alphaherpesvirus genes encoding membrane proteins, of which up to now only glycoproteins gG and gJ have been analyzed in detail. We have now prepared monospecific rabbit antisera against ILTV gD, gE, and gI, and the ILTV type II membrane protein pUS9, each of which showed specific immunofluorescence reactions, and detected proteins of approximately 65 and 70 kDa (gD), 62 kDa (gI), 75 kDa (gE), or 37 kDa (pUS9) in western blot analyses of infected chicken cells. The proteins gD, gI, and gE, but not pUS9, were identified as abundant virion proteins, and gE and gI were shown to be N-glycosylated. We also isolated gE-, gI-, and pUS9-deleted ILTV recombinants, whereas it was not possible to purify gD-negative ILTV to homogeneity, indicating that gD, like in other alphaherpesviruses, is essential for receptor binding and virus entry. The pUS9-deleted ILTV exhibited almost wild-type-like replication properties in cell culture. The gE- and gI-negative viruses showed significantly reduced plaque sizes, whereas virus titers were barely affected. Since homologous gene-deletion mutants of other alphaherpesviruses are in use as live vaccines, the generated ILTV recombinants might be also suitable for this application.

Development of an enzyme-linked immunosorbent assay to detect chicken serum antibody to glycoprotein G of infectious laryngotracheitis virus.

Infectious laryngotracheitis (ILT) is a significant upper respiratory tract disease of chickens and has a worldwide distribution. Diagnostic enzyme-linked immunosorbent assays (ELISAs) are commonly used in ILT disease control programs. These ELISAs generally detect serum antibody to infectious laryngotracheitis virus (ILTV) and frequently utilize whole virus as the ELISA antigen. This study investigated the use of recombinant glycoprotein G (gG) of ILTV as an alterative to the use of whole virus antigen. Codon-optimized ILTV gG was expressed in Escherichia coli as a fusion protein with a maltose binding protein tag (gG-MBP). Another gG fusion protein with a 6-histidine tag (gG-His) was expressed in a baculovirus expression system. Following purification, the proteins were assessed for their suitability to be used as an antigen in an ELISA to detect ILTV-specific antibodies in sera from commercial and specific-pathogen-free (SPF) birds. The gG-MBP antigen showed some nonspecific reactions with chicken sera, but the gG-HIS antigen was found to be suitable for differentiating between sera collected from ILTV-vaccinated and unvaccinated chickens. The highest levels of agreement between the results from the gG-HIS ELISA and the commercial Trop-ILT ELISA were achieved using a cut-off value for positivity equal to the geometric mean antibody concentration of the sera from the unvaccinated birds plus 1 SD. This produced a very good level of agreement (kappa [kappa] value of 0.821) using sera from commercial birds and a moderate level of agreement (kappa value of 0.506) using sera from SPF birds. Importantly, this ELISA was also tested for its ability to discriminate between sera collected from SPF chickens vaccinated with a gG deletion mutant candidate vaccine strain of ILTV (gG-ve ILTV) and sera collected from SPF chickens vaccinated with other ILTV strains. The results showed that the gG-His ELISA has the potential to serve as a companion diagnostic tool in conjunction with the gG-ve ILTV vaccine in a 'differentiating infected from vaccinated animals' approach to the control of ILT.

Molecular biology of avian infectious laryngotracheitis virus.

Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that causes an economically important chicken disease, which results in delayed growth, reduced egg production, and also frequently in death of the animals. After acute infection of the upper respiratory tract, the virus can establish latency in the central nervous system, and subsequent reactivations can lead to infection of naive chickens. For prevention of ILT, conventionally attenuated live vaccines are available. However, these vaccine strains are genetically not characterized, and reversions to a virulent phenotype occur. Although molecular analyses of ILTV are hampered by the lack of an optimal cell culture system, the complete nucleotide sequence of the ILTV genome has recently been elucidated, and several ILTV recombinants lacking nonessential, but virulence determining genes have been constructed. Animal trials indicated that genetically engineered stable gene deletion mutants are safe alternatives to the current vaccine strains. Furthermore, since live ILTV vaccines are suitable for fast and inexpensive mass administration, they are promising as vectors for immunogenic proteins of other chicken pathogens. Thus, immunization with ILTV recombinants expressing avian influenza virus hemagglutinin was shown to protect chickens against ILT and fowl plague. Using monospecific antisera and monoclonal antibodies several virion proteins of ILTV have been identified and characterized. Since they include immunogenic envelope glycoproteins, these results can contribute to the improvement of virus diagnostics, and to the development of marker vaccines.

Glycoprotein G is a virulence factor in infectious laryngotracheitis virus.

Infectious laryngotracheitis virus (ILTV; Gallid herpesvirus 1) is an alphaherpesvirus that causes acute respiratory disease in chickens. The role of glycoprotein G (gG) in vitro has been investigated in a number of alphaherpesviruses, but the relevance of gG in vivo in the pathogenicity of ILTV or in other alphaherpesviruses is unknown. In this study, gG-deficient mutants of ILTV were generated and inoculated into specific-pathogen-free chickens to assess the role of gG in pathogenicity. In chickens, gG-deficient ILTV reached a similar titre to wild-type (wt) ILTV but was significantly attenuated with respect to induction of clinical signs, effect on weight gain and bird mortality. In addition, an increased tracheal mucosal thickness, reflecting increased inflammatory cell infiltration at the site of infection, was detected in birds inoculated with gG-deficient ILTV compared with birds inoculated with wt ILTV. The reinsertion of gG into gG-deficient ILTV restored the in vivo phenotype of the mutant to that of wt ILTV. Quantitative PCR analysis of the expression of the genes adjacent to gG demonstrated that they were not affected by the deletion of gG and investigations in vitro confirmed that the phenotype of gG-deficient ILTV was consistent with unaltered expression of these adjacent genes. This is the first reported study to demonstrate definitively that gG is a virulence factor in ILTV and that deletion of gG from this alphaherpesvirus genome causes marked attenuation of the virus in its natural host.

The nonessential UL49.5 gene of infectious laryngotracheitis virus encodes an O-glycosylated protein which forms a complex with the non-glycosylated UL10 gene product.

The UL10 and UL49.5 genes of avian infectious laryngotracheitis virus (ILTV) encode putative envelope proteins which are conserved in Alpha, Beta, and Gammaherpesvirinae. Many of the corresponding gene products have been shown to be glycosylated and to form heterodimeric protein complexes with each other. Unlike the homologous gM proteins of other herpesviruses, the UL10 protein of ILTV is not detectably glycosylated [Fuchs, W., Mettenleiter, T.C., 1999. DNA sequence of the UL6 to UL20 genes of infectious laryngotracheitis virus and characterization of the UL10 gene product as a nonglycosylated and nonessential virion protein. J. Gen. Virol. 80, 2173-2182]. Using a monospecific antiserum, we now identified the UL49.5 gene product of ILTV as an O-glycosylated membrane protein (gN). Correct processing of gN was shown to depend on the presence of the UL10 protein. Both gN and UL10 could be co-immunoprecipitated from ILTV-infected cell lysates with antisera against either of the proteins, indicating stable protein-protein interactions. For functional analysis parts of the UL10 and UL49.5 open reading frames were deleted from the ILTV genome, and replaced by a beta-galactosidase expression cassette. The resulting virus mutants were isolated and propagated in non-complementing chicken cells, which demonstrated that the UL10 and UL49.5 genes are not essential for in vitro replication of ILTV.

Infectious laryngotracheitis virus, an alpha herpesvirus that does not interact with cell surface heparan sulfate.

Among the alpha herpesviruses studied to date, the initial stage of wild-type virus attachment involves an interaction between virally encoded structural envelope glycoproteins (predominantly glycoprotein C) and cell surface heparan sulfate proteoglycans. An analysis of the infectious laryngotracheitis virus (ILTV) glycoprotein C and glycoprotein B sequences suggested that these proteins lacked consensus heparin-binding domains. This indicated that ILTV might attach to its host cell in a heparan-independent manner, distinct from other alpha herpesviruses. The infectivity of two ILTV strains, a tissue-culture-adapted vaccine strain and a virulent field challenge strain, were found to be insensitive to the presence of exogenous heparin or chondroitin. Furthermore, infectivity was retained in chicken embryonic liver cells treated with heparinase. However, 4 degrees C attachment studies and penetration studies in the presence of citrate buffer clearly demonstrated that ILTV attaches stably to and effectively penetrates chicken embryonic liver cells. Consequently, ILTV represents an alpha herpesvirus whose initial attachment step does not involve interactions with heparan or chondroitin sulfate containing proteoglycans.