Infectious Laryngotracheitis Virus Replication Research Articles

P53 maintains gallid alpha herpesvirus 1 replication by direct regulation of nucleotide metabolism and ATP synthesis through its target genes.

P53, a well-known tumor suppressor, has been confirmed to regulate the infection of various viruses, including chicken viruses. Our previous study observed antiviral effect of p53 inhibitor Pifithrin-α (PFT-α) on the infection of avian infectious laryngotracheitis virus (ILTV), one of the major avian viruses economically significant to the poultry industry globally. However, the potential link between this antiviral effect of PFT-α and p53 remains unclear. Using chicken LMH cell line which is permissive for ILTV infection as model, we explore the effects of p53 on ILTV replication and its underlying molecular mechanism based on genome-wide transcriptome analysis of genes with p53 binding sites. The putative p53 target genes were validated by ChIP-qPCR and RT-qPCR. Results demonstrated that, consistent with the effects of PFT-α on ILTV replication we previously reported, knockdown of p53 repressed viral gene transcription and the genome replication of ILTV effectively. The production of infectious virions was also suppressed significantly by p53 knockdown. Further bioinformatic analysis of genes with p53 binding sites revealed extensive repression of these putative p53 target genes enriched in the metabolic processes, especially nucleotide metabolism and ATP synthesis, upon p53 repression by PFT-α in ILTV infected LMH cells. Among these genes, eighteen were involved in nucleotide metabolism and ATP synthesis. Then eight of the 18 genes were selected randomly for validations, all of which were successfully identified as p53 target genes. Our findings shed light on the mechanisms through which p53 controls ILTV infection, meanwhile expand our knowledge of chicken p53 target genes.

PFT-α inhibits gallid alpha herpesvirus 1 replication by repressing host nucleotide metabolism and ATP synthesis

Therapeutics targeting virus–host interactions have been considered promising strategies for treating herpesvirus infection. Our previous study on avian infectious laryngotracheitis virus (ILTV), an avian herpesvirus economically important to the poultry industry worldwide, identified the small molecule Pifithrin-α (PFT-α) as a potential therapeutic agent. However, the underlying mechanisms of its antiviral function remain largely unknown. Using the ILTV-permissive chicken cell line LMH as the model, we found that PFT-α effectively suppressed the transcription and genome replication of ILTV and greatly reduced the level of infectious virions. Genome-wide transcriptome analysis revealed extensive repression of the metabolic processes of infected cells by PFT-α administration. Further metabolome assays of ILTV-infected cells using liquid chromatography coupled with mass spectrometry suggest host nucleotide metabolism and ATP synthesis as the key targets of PFT-α treatment during its repression of ILTV replication, which was experimentally supported by the reduced transcription of many key enzymes essential to nucleotide metabolism and ATP synthesis. The present study provides insights into the mechanisms by which PFT-α inhibits ILTV infection, which may increase the probability of successful clinical application of this molecule.

Fos Facilitates Gallid Alpha-Herpesvirus 1 Infection by Transcriptional Control of Host Metabolic Genes and Viral Immediate Early Gene.

Gallid alpha-herpesvirus 1, also known as avian infectious laryngotracheitis virus (ILTV), continues to cause huge economic losses to the poultry industry worldwide. Similar to that of other herpesvirus-encoded proteins, the expression of viral genes encoded by ILTV is regulated by a cascade, and the underlying regulatory mechanism remains largely unclear. The viral immediate-early (IE) gene ICP4 plays a prominent role in the initiation of the transcription of early and late genes during ILTV replication. In this study, we identified AP-1 as the key regulator of the transcription of ILTV genes by bioinformatics analysis of genome-wide transcriptome data. Subsequent functional studies of the key members of the AP-1 family revealed that Fos, but not Jun, regulates ILTV infection through AP-1 since knockdown of Fos, but not Jun, by gene silencing significantly reduced ICP4 transcription and subsequent viral genome replication and virion production. Using several approaches, we identified ICP4 as a bona fide target gene of Fos that regulated Fos and has Fos response elements within its promoter. Neither the physical binding of Jun to the promoter of ICP4 nor the transcriptional activity of Jun was observed. In addition, knockdown of Fos reduced the transcription of MDH1 and ATP5A1, genes encoding two host rate-limiting enzymes essential for the production of the TCA intermediates OAA and ATP. The biological significance of the transcriptional regulation of MDH1 and ATP5A1 by Fos in ILTV infection was supported by the fact that anaplerosis of OAA and ATP rescued both ICP4 transcription and virion production in infected cells under when Fos was silenced. Our study identified the transcription factor Fos as a key regulator of ILTV infection through its transcription factor function on both the virus and host sides, improving the current understanding of both avian herpesvirus–host interactions and the roles of AP-1 in viral infection.

Simultaneous Deletion of Virulence Factors and Insertion of Antigens into the Infectious Laryngotracheitis Virus Using NHEJ-CRISPR/Cas9 and Cre-Lox System for Construction of a Stable Vaccine Vector.

Infectious laryngotracheitis virus (ILTV) is a promising vaccine vector due to its heterologous gene accommodation capabilities, low pathogenicity, and potential to induce cellular and humoral arms of immunity. Owing to these characteristics, different gene-deletion versions of ILTVs have been successfully deployed as a vector platform for the development of recombinant vaccines against multiple avian viruses using conventional recombination methods, which are tedious, time-demanding, and error-prone. Here, we applied a versatile, and customisable clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 accompanied with Cre–Lox system to simultaneously delete virulence factors and to insert foreign genes in the ILTV genome. Using this pipeline, we successfully deleted thymidine kinase (TK) and unique short 4 (US4) genes and inserted fusion (F) gene of the Newcastle disease virus without adversely affecting ILTV replication and expression of the F protein. Taken together, the proposed approach offers novel tools to attenuate (by deletion of virulence factor) and to generate multivalent (by insertion of heterologous genes) vaccine vectors to protect chickens against pathogens of poultry and public health importance.

Comparision of different cell cultures for replication of infectious laryngotracheitis virus from chickens

The propagation of infectious laryngotracheitis virus (ILTV) has been described using primary cell cultures derived from chicken embryo liver and kidney or embryonated eggs, but these cultures use Specific Pathogen Free (SPF) eggs that are time and cost expensive. Since cell line cultures are easier to maintain in laboratory conditions, the growth of ILTV was evaluated in five different cell cultures: chicken embryo related cells (CER), a cell hybrid derived from chicken embryo fibroblasts cells and BHK-21; Vero, from African green monkey kidney cells; HD11, a chicken macrophage cell line; CEC-32, an avian fibroblast cell line and a primary cell culture of chicken embryo fibroblasts (CEF). Cytophatic effect was observed until 96 hours following inoculation and the detection of the viral DNA was performed by PCR. The HD11 and CEC-32 cell lines did not support the virus growth but CEF and Vero, as already described were permissive cultures for propagation of ILTV. The results also showed that the CER cell line can be used for primary isolation and replication of ILTV.

Genetic Diversity of Infectious Laryngotracheitis Virus during In Vivo Coinfection Parallels Viral Replication and Arises from Recombination Hot Spots within the Genome.

Recombination is a feature of many alphaherpesviruses that infect people and animals. Infectious laryngotracheitis virus (ILTV; Gallid alphaherpesvirus 1) causes respiratory disease in chickens, resulting in significant production losses in poultry industries worldwide. Natural (field) ILTV recombination is widespread, particularly recombination between attenuated ILTV vaccine strains to create virulent viruses. These virulent recombinants have had a major impact on animal health. Recently, the development of a single nucleotide polymorphism (SNP) genotyping assay for ILTV has helped to understand ILTV recombination in laboratory settings. In this study, we applied this SNP genotyping assay to further examine ILTV recombination in the natural host. Following coinoculation of specific-pathogen-free chickens, we examined the resultant progeny for evidence of viral recombination and characterized the diversity of the recombinants over time. The results showed that ILTV replication and recombination are closely related and that the recombinant viral progeny are most diverse 4 days after coinoculation, which is the peak of viral replication. Further, the locations of recombination breakpoints in a selection of the recombinant progeny, and in field isolates of ILTV from different geographical regions, were examined following full-genome sequencing and used to identify recombination hot spots in the ILTV genome.IMPORTANCE Alphaherpesviruses are common causes of disease in people and animals. Recombination enables genome diversification in many different species of alphaherpesviruses, which can lead to the evolution of higher levels of viral virulence. Using the alphaherpesvirus infectious laryngotracheitis virus (ILTV), we performed coinfections in the natural host (chickens) to demonstrate high levels of virus recombination. Higher levels of diversity in the recombinant progeny coincided with the highest levels of virus replication. In the recombinant progeny, and in field isolates, recombination occurred at greater frequency in recombination hot spot regions of the virus genome. Our results suggest that control measures that aim to limit viral replication could offer the potential to limit virus recombination and thus the evolution of virulence. The development and use of vaccines that are focused on limiting virus replication, rather than vaccines that are focused more on limiting clinical disease, may be indicated in order to better control disease.

Impairment of infectious laryngotracheitis virus replication by deletion of the UL[-1] gene.

Infectious laryngotracheitis virus (ILTV) encodes several unique genes, including a pair of unique nuclear proteins UL0 and UL[-1] that are expressed during replication in cell culture. Although the UL0 gene has been shown to be dispensable for replication, the role of UL[-1] has not been elucidated. In this study a deletion mutant of ILTV lacking the UL[-1] gene was constructed using homologous recombination. The coding sequences of the gene were replaced with the gene for enhanced green fluorescent protein and the cytomegalovirus major immediate early promoter element. The progeny virus carrying the reporter gene was readily identified using fluorescent microscopy, but was unable to propagate in the permissive cells in the absence of wild type ILTV. Even after plaque purification and fluorescent associated cell sorting the recombinant virus deficient in UL[-1] gene could not be successfully isolated. Our findings suggest that the UL[-1] gene has an important role in ILTV replication.

Induction of Toll-like receptor 4 signaling in avian macrophages inhibits infectious laryngotracheitis virus replication in a nitric oxide dependent way

LPS is one of the pathogen associated molecular patterns that activates Toll-like receptor 4 (TLR4) signaling pathway eliciting antiviral host responses in mammals although information on such responses in avian species is scarce. Our objectives were to characterize the LPS induced innate responses particularly the expression of LPS receptors (TLR4, CD14) in avian macrophages and observe whether TLR4 mediated induction of NO can elicit antiviral response against infectious laryngotracheitis virus (ILTV) replication. We found that LPS was capable of inducing the expression of TLR4, CD14 and NO production but not the type 1 interferons in an avian macrophage cell line, MQ-NCSU. We also showed that TLR4 mediated NO production can lead to antiviral response against ILTV replication when MQ-NCSU cells were treated with LPS and the resultant supernatant was then transferred to ILTV replicating cells to assess antiviral activity. Antiviral activity of NO was blocked by a selective inhibitor, S-methylisothiourea sulfhate that inhibits inducible NO synthase. This observation confirms that the antiviral activity is positively correlated with NO production. The data show that LPS can be a potential innate immune stimulant that can be used against ILTV infection in chickens that require further evaluation in vivo.

Dynamic distribution and tissue tropism of infectious laryngotracheitis virus in experimentally infected chickens

Infectious laryngotracheitis (ILT), caused by infectious laryngotracheitis virus (ILTV), is an Office International des Epizooties (OIE) notifiable disease. However, we have not clearly understood the dynamic distribution, tissue tropism, pathogenesis, and replication of ILTV in chickens. In this report, we investigated the dynamic distribution and tissue tropism of the virus in internal organs of experimentally infected chickens using quantitative real-time polymerase chain reaction (qPCR) and a histopathological test. The study showed that ILTV could be clearly detected in eight internal organs (throat, trachea, lung, cecum, kidney, pancreas, thymus and esophagus) of infected chickens, whereas the virus was difficult to detect in heart, spleen, proventriculus, liver, brain and bursa. Meanwhile, the thymidine kinase (TK) gene levels in eight internal organs increased from 3 days to 5 days postinfection, and then decreased from 6 days to 8 days postinfection. The log copy number of ILTV progressively increased over 3 days, which corresponds to the clinical score and the result of the histopathological test. The results provide a foundation for further clarification of the pathogenic mechanism of ILTV in internal organs and indicate that throat, lung, trachea, cecum, kidney, pancreas and esophagus may be preferred sites of acute infection, suggesting that the tissue tropism and distribution of ILTV is very broad.

The UL47 gene of avian infectious laryngotracheitis virus is not essential for in vitro replication but is relevant for virulence in chickens

The genome of infectious laryngotracheitis virus (ILTV) exhibits several differences from those of other avian and mammalian alphaherpesviruses. One of them is the translocation of the conserved UL47 gene from the unique long (UL) to the unique short (US) genome region, where UL47 is inserted upstream of the US4 gene homologue. As in other alphaherpesviruses, UL47 encodes a major tegument protein of ILTV particles, whereas the US4 gene product is a non-structural glycoprotein, gG, which is secreted from infected cells. For functional characterization, an ILTV recombinant was isolated in which US4 together with the 3'-terminal part of UL47 was replaced by a reporter gene cassette encoding green fluorescent protein. From this virus, UL47 and US4 single-gene deletion mutants without foreign sequences were derived and virus revertants were also generated. In vitro studies revealed that both genes were non-essential for ILTV replication in cultured cells. Whereas US4-negative ILTV exhibited no detectable growth defects, maximum virus titres of the double deletion mutant and of UL47-negative ILTV were reduced about 10-fold compared with those of wild-type virus and rescued virus. Experimental infection of chickens demonstrated that UL47-negative ILTV was significantly attenuated in vivo and was shed in reduced amounts, whereas wild-type and rescued viruses caused severe disease and high mortality rates. As all immunized animals were protected against subsequent challenge infection with virulent ILTV, the UL47 deletion mutant might be suitable as a live-virus vaccine.

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 β-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.

The non-essential UL50 gene of avian infectious laryngotracheitis virus encodes a functional dUTPase which is not a virulence factor.

The DNA sequence of the infectious laryngotracheitis virus (ILTV) UL50, UL51 and UL52 gene homologues was determined. Although the deduced UL50 protein lacks the first of five conserved domains of the corresponding proteins of mammalian alphaherpesviruses, the ILTV gene product was also shown to possess dUTPase activity. The generation of UL50-negative ILTV mutants was facilitated by recombination plasmids encoding green fluorescent protein (GFP), and expression constructs of predicted transactivator proteins of ILTV (alphaTIF, ICP4) were successfully used to increase the infectivity of viral genomic DNA. A GFP-expressing UL50-deletion mutant of ILTV showed reduced cell-to-cell spread in vitro, and was attenuated in vivo. A similar deletion mutant without the foreign gene, however, propagated like wild-type ILTV in cell culture and was pathogenic in chickens. We conclude that the viral dUTPase is not required for efficient replication of ILTV in the respiratory tract of infected animals. The replication defect of the GFP-expressing ILTV recombinant is most likely caused by toxic effects of the reporter gene product, since spontaneously occurring inactivation mutants exhibited wild-type-like growth.

Replication of Infectious Laryngotracheitis Virus in a Quail Cell Line, QT-35

The relative resistance of the quail fibroblastic cell line QT-35 to infection by infectious laryngotracheitis virus (ILTV) was circumvented by the continual passaging of infected cells in the presence of uninfected monolayers. Such virus-containing cells were used to extend the infection to an otherwise refractory quail liver cell line IQ1A, as well as to a normally permissive chicken liver cell line LMH, with nearly equal efficiency. In contrast, extracellular virus that had been derived from either QT-35 or IQ1A cells could not readily infect either quail cell line, although LMH cells were still susceptible. Therefore, the block to ILTV replication in quail cells is probably at the adsorption/penetration stage.