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Infectious laryngotracheitis virus growth, DNA replication, and protein synthesis
Abstract
The polypeptides associated with infection of primary chicken kidney (CK) cells with infectious laryngotracheitis virus (ILTV) were examined by metabolic labelling with [35S]methionine and SDS-PAGE. Polypeptide synthesis was followed over the first 24 h post-infection (p.i.) as this was shown to be the period of viable virus production. A total of 16 ILTV encoded or induced polypeptides were identified using metabolic labelling. The use of inhibitors of protein and DNA synthesis in conjunction with metabolic labelling and viral DNA replication studies enabled a cascade pattern of gene expression, similar to that of herpes simplex virus type 1 (HSV-1), to be established for ILTV. Representatives of alpha, beta, gamma 1 and gamma 2 classes of genes were identified. In contrast to infection with HSV types 1 and 2, which leads to a rapid inhibition of total host cell polypeptide synthesis, ILTV infection of CK cells did not result in a complete inhibition of cellular protein synthesis, with only a small number of host cell polypeptides absent from infected cells.
... Transcription of ILTV DNA proceeds in a highly regulated sequentially ordered cascade-like manner, similar to other alphaherpesviruses (120)(121)(122)(123)(124)(125). In ILTV there are at least 4 types of genes, according to their order of expression during infection and their dependence on prior transcription or DNA replication (124). ...
... Transcription of ILTV DNA proceeds in a highly regulated sequentially ordered cascade-like manner, similar to other alphaherpesviruses (120)(121)(122)(123)(124)(125). In ILTV there are at least 4 types of genes, according to their order of expression during infection and their dependence on prior transcription or DNA replication (124). An early study in 1992 by Prideaux et al. using chicken kidney cells (CK), metabolic inhibitors and an Australian vaccine strain (SA2) was the first to describe a cascade-like pattern of ILTV gene-expression, demonstrating that ILTV has a similar gene expression pattern to that of HSV-1 (124). ...
... In ILTV there are at least 4 types of genes, according to their order of expression during infection and their dependence on prior transcription or DNA replication (124). An early study in 1992 by Prideaux et al. using chicken kidney cells (CK), metabolic inhibitors and an Australian vaccine strain (SA2) was the first to describe a cascade-like pattern of ILTV gene-expression, demonstrating that ILTV has a similar gene expression pattern to that of HSV-1 (124). In this study polypeptides were classified as alpha, beta, gamma 1 or gamma 2 according to the description used for HSV-1 gene-expression (124). ...
Infectious laryngotracheitis virus (ILTV; Gallid alphaherpesvirus-1) is an alphaherpesvirus that causes respiratory disease in chickens, resulting in significant production losses in poultry industries worldwide. Recombination between different ILTV strains has recently been identified. Recombination in alphaherpesviruses was first described more than sixty years ago and since then, different techniques have been used to detect recombination in both natural (field) and experimental settings. In the past, natural recombination events between ILTV strains has resulted in the emergence of virulent recombinant viruses that have caused severe disease outbreaks in Australia.
In this work, a single nucleotide polymorphism (SNP) genotyping assay was developed to study ILTV recombination in vivo. This assay was used to study recombination in a large number of viruses retrieved from chickens co-inoculated with two Australian ILTV field strains. Further application of this SNP genotyping assay helped unveil other aspects of ILTV recombination such as viral diversity over time and dominant recombination patterns in the recombinant progeny. Whole genome sequencing (WGS) of dominant viruses was performed in order to analyse their recombination breakpoint locations. This latter analysis revealed the presence of recombination hot-spots. The location of these hotspots were consistent with those found after the analysis of publicly available whole genome sequences of ILTV from different geographical regions, such as Australia and the United States (US).
Additionally, the recombination output was determined in chickens after vaccination with three commercially available Australian ILTV vaccines (SA2, A20 and Serva), or two vaccines from the US (CEO-Tachivax and HVT-LT). For this later analysis a second SNP genotyping assay was develop to detect recombination between the USA field strains of ILTV used in that study. Results from these analyses indicated that vaccination can limit the number and diversity of recombinant progeny viruses and introduced new research questions about the role of the immune system in limiting recombination.
The studies reported in this thesis have provided new insights into recombination in alphaherpesviruses that will be useful for future studies regarding vaccine development and use in both Australia and elsewhere.
... After attachment, the tegument and nucleocapsid get transported into the cytoplasm and the viral DNA released from the nucleocapsid enter into the nucleus through nuclear pores (Trus et al. 2004;Cardone et al. 2007). The highly regulated transcription and replication of ILTV DNA occur within the nucleus by utilizing the host cell machinery (Prideaux et al. 1992;Guo et al. 1993). Three classes of genes, namely immediate early (α), early (β), and late (γ) are expressed during the viral transcription and translation process (Honess and Roizman 1974). ...
... Three classes of genes, namely immediate early (α), early (β), and late (γ) are expressed during the viral transcription and translation process (Honess and Roizman 1974). The non-structural protein products of α genes play a key role in the expression of β genes between 4 to 16 hrs post-infection (Prideaux et al. 1992). The β gene proteins are critical for viral replication and regulate the production of viral structural proteins encoded by late γ genes. ...
... After attachment, the tegument and nucleocapsid get transported into the cytoplasm and the viral DNA released from the nucleocapsid enter into the nucleus through nuclear pores (Trus et al. 2004;Cardone et al. 2007). The highly regulated transcription and replication of ILTV DNA occur within the nucleus by utilizing the host cell machinery (Prideaux et al. 1992;Guo et al. 1993). Three classes of genes, namely immediate early (a), early (b), and late (c) are expressed during the viral transcription and translation process (Honess and Roizman 1974). ...
... Three classes of genes, namely immediate early (a), early (b), and late (c) are expressed during the viral transcription and translation process (Honess and Roizman 1974). The non-structural protein products of a genes play a key role in the expression of b genes between 4 to 16 hrs post-infection (Prideaux et al. 1992). The b gene proteins are critical for viral replication and regulate the production of viral structural proteins encoded by late c genes. ...
Infectious laryngotracheitis (ILT) is a highly contagious upper respiratory tract disease of chicken caused by a Gallid herpesvirus I (GaHV-1) belonging to the genus Iltovirus, and subfamily Alphaherpesvirinae within Herpesviridae family. The disease is characterized by conjunctivitis, sinusitis, oculo-nasal discharge, respiratory distress, bloody mucus, swollen orbital sinuses, high morbidity, considerable mortality and decreased egg production. It is well established in highly dense poultry producing areas of the world due to characteristic latency and carrier status of the virus. Co-infections with other respiratory pathogens and environmental factors adversely affect the respiratory system and prolong the course of the disease. Latently infected chickens are the primary source of ILT virus (ILTV) outbreaks irrespective of vaccination. Apart from conventional diagnostic methods including isolation and identification of ILTV, serological detection, advanced biotechnological tools such as PCR, quantitative real-time PCR, next generation sequencing, and others are being used in accurate diagnosis and epidemiological studies of ILTV. Vaccination is followed with the use of conventional vaccines including modified live attenuated ILTV vaccines, and advanced recombinant vector vaccines expressing different ILTV glycoproteins, but still these candidates frequently fail to reduce challenge virus shedding. Some herbal components have proved to be beneficial in reducing the severity of the clinical disease. The present review discusses ILT with respect to its current status, virus characteristics, epidemiology, transmission, pathobiology, and advances in diagnosis, vaccination and control strategies to counter this important disease of poultry.
... chment to cell receptors followed by fusion of the envelope with the host cell plasma membrane. The nucleocapsid is released into the cytoplasm and transported to the nuclear membrane; viral DNA is released from the nucleocapsid and migrates into the nucleus through nuclear pores. Transcription and replication of viral DNA occur within the nucleus (Prideaux et. al., 1992;Guo et. al., 1993) Transcription of ILTV DNA occurs in a highly regulated, sequentially ordered cascade similar to that of other alphaherpesviruses (Prideaux et. al., 1992). Approximately 70 virus-coded proteins are produced; several are enzymes and DNA-binding proteins that regulate viral DNA replication, but most are viral structural p ...
... ar membrane; viral DNA is released from the nucleocapsid and migrates into the nucleus through nuclear pores. Transcription and replication of viral DNA occur within the nucleus (Prideaux et. al., 1992;Guo et. al., 1993) Transcription of ILTV DNA occurs in a highly regulated, sequentially ordered cascade similar to that of other alphaherpesviruses (Prideaux et. al., 1992). Approximately 70 virus-coded proteins are produced; several are enzymes and DNA-binding proteins that regulate viral DNA replication, but most are viral structural proteins. Viral DNA replication occurs by a rolling circle mechanism with the formation of concatemers which are cleaved into monomeric units and packaged into preformed nuc ...
Infectious laryngotracheitis (ILT) herpesvirus continues to cause outbreaks of respiratory disease in chickens world-wide. Sporadic cases of ILT occur in all classes of birds, including hobby/show/game chickens, broilers, heavy breeders, and commercial laying hens. These epornitics of ILT tend to occur where there are large populations of naïve, unvaccinated birds, i.e., in concentrated areas of broiler production. ILT virus can be transmitted through (a) chickens with acute upper respiratory tract disease, (b) latently infected "carrier" fowls, and (c) fomites and contaminated persons. Chicken flocks which are endemic infected with ILT virus occur only in some regions of countries or even in particular multiple-age production farms. In these cases modified live vaccines are actually used, even though these biological products, as well as wild ILTV strains, can establish latent infections. In the case of heavy breeders and laying hens, which are typically vaccinated against ILT, sporadic cases are often related to errors in vaccine application and to biosecurity failures.
... In Australia, the disease is controlled by the vaccine strain SA-2, which still retains some pathogenicity in younger birds (Sinkovic and Hunt, 1968;Bagust, 1982). A previous study on the synthesis of ILTV proteins showed that viral proteins were regulated in a cascade fashion (Prideaux et al., 1992), in a similar pattern as shown for herpes simplex virus [HSVI (Honess and Roizman, 1974;Mackem and Roizman, 1981;Fenwick, 1984). Representatives of alpha, beta, gamma 1 and gamma 2 classes of genes were identified. ...
... Representatives of alpha, beta, gamma 1 and gamma 2 classes of genes were identified. Analysis of ILTV immediate early polypeptides using cycloheximide showed at 4 h post-infection (p.i.> the accumulation of a single alpha polypeptide having a molecular weight of 200 kDa (Prideaux et al., 1992). Since DNA replication did not begin until 8 to 12 h p.i., this alpha polypeptide is likely to play a role in regulation of gene expression early in infection. ...
The infectious laryngotracheitis virus (ILTV) gene encoding a homologue to the ICP4 protein of herpes simplex virus (HSV) has been mapped to the inverted repeat region. The complete nucleotide sequence of ILTV ICP4 has been determined. The ILTV ORF encoding ICP4 is 4386 nucleotides long, calculated from the first of four ATG codons, and has an overall G+C content of 59%. The ILTV ICP4 contains two domains of high homology which have been reported in other studies to be conserved in the ICP4 homologues of alphaherpesviruses, and to be functionally important. Several regulatory features were identified including a serine-rich domain in region one. A more extensive serine-rich domain was located in region five which is also found in varicella-zoster virus (VZV) and bovine herpesvirus 1. A 5.4 kb immediate early transcript was identified in infected primary kidney cells.
... In our study, in 2 cases, death occurred within 12 h of admission. In other avian respiratory herpesviruses, the inclusion bodies take longer to develop in cell culture (Reynolds et al. 1968, Prideaux et al. 1992, hence we consider this as evidence that infection occurred in the wild. ...
Rehabilitation is an important strategy for the conservation of the Endangered African penguin Spheniscus demersus, and disease has been raised as a concern in the management of the species, both in the wild and in rehabilitation centres. We report 8 cases of herpes - virus-like respiratory infection in African penguin chicks undergoing rehabilitation between 2010 and 2013 at a facility in Cape Town, South Africa. Infection was confirmed through the identification of viral inclusions in the tracheal epithelium and demonstration of particles consistent with herpesvirus by electron microscopy, whereas virus isolation in eggs, serology and PCR testing failed to detect the virus. Only penguin chicks were affected; they were in poor body condition, and in 2 cases infection occurred prior to admission to the rehabilitation centre. The role played by the herpesvirus-like infection in the overall respiratory disease syndrome is uncertain, due to identification of lesions in only a small proportion of the chicks as well as to the occurrence of other concurrent pathological processes. Further studies are advised to characterise the specific virus involved through the development of sensitive diagnostic methods and to clarify the epidemiology and significance of these infections in wild African penguins.
... Replication of ILTV is similar to other alphaherpesviruses, such as pseudorabies virus and herpes simplex virus (Prideaux et al., 1992). The virus attaches to receptors on the cell surface. ...
... Infectious laryngotracheitis virus (ILTV) is a non-neoplastic avian herpesvirus which causes severe upper respiratory tract infections of chickens, resulting in substantial losses to the poultry industry (Bagust & Johnson, 1995 ; Cover, 1996). The ILTV genome has been mapped and numerous ILTV-encoded polypeptides have been identified on polyacrylamide gels (Johnson et al., 1991 ; Prideaux et al., 1992 ; Wild et al., 1996). ...
Infectious laryngotracheitis virus (ILTV) is an alpha-herpesvirus that causes severe upper respiratory infections in chickens. Although ten putative ILTV glycoprotein genes have been identified by sequence analysis, no ILTV glycoprotein has been extensively characterized. In order to delineate the synthesis and processing pathway of ILTV glycoprotein B (gB), rabbit polyclonal antibodies were raised against a Cro-gB-beta-galactosidase fusion protein. Through immunoprecipitation analysis of ILTV-infected chicken embryo liver cells it was determined that ILTV gB is initially synthesized as a 110 kDa monomeric precursor protein which rapidly assembles into homodimers composed of 100 kDa subunits. The dimer form of ILTV gB is rapidly cleaved to form two disulphide-linked species of 58 kDa. The apparent reduction in mass (from 110 to 100 kDa) of the mature form of gB during processing in the Golgi apparatus appears to be a common feature of avian herpesvirus gB proteins.
... One-step growth analyses in primary chicken kidney cells revealed that first infectious progeny viruses are formed 8 to 12 h after infection, and reach a maximum after 24 to 30 h [25] . Metabolic labeling of proteins in ILTV-infected cells in combination with inhibitors of translation or DNA replication indicated a cascadelike regulation of viral gene expression as is typical for herpesviruses [72], and transfection experiments with expression plasmids for the ILTV homologues of the major HSV-1 transcription activators VP16 and ICP4 revealed similar transactivating functions [25, 41]. Ultrastructural analyses of ILTVinfected cells showed the typical steps of herpesvirus morphogenesis (Fig. 3) [30, 35, 67]. ...
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.
Little is known about the structure and physicochemical properties of the Infectious Laryngotracheitis Virus (ILTV) genome until recently a few years. To date, there is no protocol that is suitable for preparation of pure cell-associated herpesvirus DNA isolates from infected tissues or from tissue culture. The products of traditional methods are often found to be contaminated with host cellular DNA, especially for preparations of the large ILTV DNA genome. In addition, there is a need to develop methods for the isolation of highly purified viral DNA from cell culture or tissue samples to be used for high-throughput nucleotide sequencing. In this study, the isolation of ILTV from Chorioallantoic Membranes (CAM) was chosen as the model to test a method for purification of cell-associated herpesvirus DNA. The protocol was a combination of Triton X-l 00 lysis, nuclease treatment, nuclease denaturation, phenol-chloroform extraction and subsequent selective removal of small DNA fragments. The results showed that the cell background contamination was reduced significantly to a level that was suitable for 454 pyrosequencing and downstream applications such as cloning of viral DNA fragments or transfection of genomic viral DNA. This novel protocol, therefore has several advantages compared with existing methods.
The DNA sequence of 4005 nucleotides from the Kpnl O and part of Kpnl K fragments in the short unique region of infectious laryngotracheitis virus (ILTV) was determined. The sequence contained two complete and one partial open reading frames (ORFs). The partial ORF was open at the 5' end of the sequence and represented the NH2-terminal 118 amino acids (aa) of a polypeptide. Its partial predicted protein product exhibited significant homology to the US2 gene product of HSV-1 (herpes simplex virus type 1) and it homologs in other herpesviruses. ORF 2 is 471 aa long and could encode a protein of 53.8 kDa which shared aa homology with the protein kinases encoded by HSV-1 US3 and its gene homologs. Analysis of the ORF 2 aa sequence revealed domains characteristic of protein-serine/threonine (S/T) kinases of cellular and viral origin. The ORF 3 encoded a predicted protein of 601 aa (M(r) 67.5 kDa) which exhibited limited homology (18% overall identity) with the UL47 protein (major tegument protein) of HSV-1. Northern (RNA) blot hybridization and metabolic inhibitors were used to characterize the ILTV protein kinase and the 67K mRNAs. The data revealed that protein kinase is a gamma-1 gene encoding a 1.6 mRNa, while the 67K ORF is a gamma-2 gene encoding a 2 kb mRNA.
The aim of this study was to evaluate the replication of live attenuated infectious laryngotracheitis virus vaccines in selected tissues and their ability to transmit to contact-exposed birds. Four-week-old specific-pathogen-free chickens were eye drop-inoculated with tissue culture origin (TCO) and chicken embryo origin (CEO) vaccines. Contact-exposed chickens were housed in direct contact with eye drop-inoculated chickens from the first day postinoculation. Virus isolation and real-time polymerase chain reaction were used to detect the presence of live virus and viral DNA, respectively, in the trachea, trigeminal ganglia, eye conjunctiva, cecal tonsils, and cloaca from eye drop-inoculated and contact-exposed birds at days 2, 4, 5 to 10, 14, 18, 21, 24, and 28 postinoculation. No differences were observed in the ability of the TCO and CEO vaccines to replicate in the examined tissues. Both vaccines presented a localized replication in the eye conjunctiva and the trachea. Both vaccines were capable of transmitting to contact-exposed birds, attaining peaks of viral DNA as elevated as those observed in inoculated birds. The CEO vaccine replicated faster and reached higher viral genome copy number than the TCO vaccine in the conjunctiva and trachea of eye drop-inoculated and contact-exposed birds. The viral DNA from both vaccines migrated to the trigeminal ganglia during early stages of infection. Although the CEO and TCO vaccines were not recovered from the cecal tonsils and the cloaca, low levels of viral DNA were detected at these sites during the peak of viral replication in the upper respiratory tract.
In herpes simplex virus type 1 (HSV-1)-infected HEp-2 cells, amanitin added before or at various times after infection always reduced viral multiplication. Also, the three waves of transcription of HSV-1 DNA, which led to the synthesis of alpha, beta-, and gamma-polypeptides, were all sensitive to amanitin in HEp-2 cells, and the amanitin-sensitive RNA polymerase activities of isolated nuclei were equally sensitive to the inhibitor before and during the infection. On the contrary, HSV-1 DNA transcription was totally unaffected by amanitin in AR1/9-5B cells, a mutant subline of CHO cells that possesses an amanitin-resistant RNA polymerase B. Together, these results strongly suggest that HSV-1 DNA utilizes for its transcription a polymerase undistinguishable from host cell RNA polymerase B with respect to its sensitivity to amanitin.
We have sequenced a 5.4 kb region of the genome from the avian herpesvirus infectious laryngotracheitis virus (ILTV) and identified genes homologous to the thymidine kinase (TK) gene, the capsid p40 gene of herpes simplex virus type 1 (HSV-1), a gene encoding a protein antigenic in Epstein-Barr virus infections plus one other highly conserved gene encoding a protein implicated in cell fusion in HSV-1. Computer analysis of the TK gene and the upstream overlapping gene sequence is used to produce trees showing potential evolutionary relationships between the herpesvirus subfamilies.
We have determined the DNA sequence of the long unique region (UL) in the genome of herpes simplex virus type 1 (HSV-1) strain 17. The UL sequence contained 107,943 residues and had a base composition of 66.9% G + C. Together with our previous work, this completes the sequence of HSV-1 DNA, giving a total genome length of 152,260 residues of base composition 68.3% G + C. Genes in the UL region were located by the use of published mapping analyses, transcript structures and sequence data, and by examination of DNA sequence characteristics. Fifty-six genes were identified, accounting for most of the sequence. Some 28 of these are at present of unknown function. The gene layout for UL was found to be very similar to that for the corresponding part of the genome of varicella-zoster virus, the only other completely sequenced alphaherpesvirus, and the amino acid sequences of equivalent proteins showed a range of similarities. In the whole genome of HSV-1 we now recognize 72 genes which encode 70 distinct proteins.
We report on the properties of a temperature-sensitive mutant produced by transfection of cells with intact DNA and a specific DNA fragment mutagenized with low levels of hydroxylamine. The plating efficiency of the mutant at 39 degrees C relative to that at 33.5 degrees C was 5 X 10(-6). The pattern of polypeptides produced at the nonpermissive temperature was similar to that seen with wild-type virus in infected cells treated with inhibitory concentrations of phosphonoacetic acid in that alpha and beta polypeptides were produced, whereas most gamma polypeptides were either reduced or absent. Consistently, the mutant did not make viral DNA, although temperature sensitivity of the viral DNA polymerase could not be demonstrated. Marker rescue studies with herpes simplex virus type 2 (HSV-2) DNA mapped the mutant in the L component within map positions 0.385 and 0.402 in the prototype (P) arrangement of the HSV-1 genome. Analysis of the recombinants permitted the mapping of the genes specifying infected cell polypeptides 36, 35, 37, 19.5, 11, 8, 2, 43, and 44, but only the infected cell polypeptide 8 of HSV-2 was consistently made by all recombinants containing demonstrable HSV-2 sequences. Marker rescue studies with cloned HSV-1 DNA fragments mapped the temperature-sensitive lesion within less than 10(3) base pairs between 0.383 and 0.388 map units. Translation of the RNA hybridizing to cloned HSV-1 DNA, encompassing the smallest region containing the mutation, revealed polypeptide 8 (128,000 molecular weight), which was previously identified as a beta polypeptide with high affinity for viral DNA, and a polypeptide (25,000 molecular weight) not previously identified in lysates of labeled cells.
Infection of cultured permissive animal cells with herpes simplex virus typically results in the initiation of the synthesis of the first (“α” or “immediate-early”) viral mRNA within 1–2 hr. This is soon followed by production of virus-specific proteins in a regulated sequence of stages (Honess and Roizman, 1974, 1975; Spear and Roizman, 1981). The α-mRNA is transcribed from the infecting genome by the cellular RNA polymerase II, which is normally concerned with the synthesis of cellular mRNA. In the presence of cycloheximide, an inhibitor of protein synthesis, a-mRNA accumulates and is translated as soon as the inhibitor is removed. Functional α-polypeptides are required to initiate and maintain the synthesis of β (“early”) polypeptides and, thereafter, the production of α-polypeptides decreases. The γ or “late” polypeptides are largely, though not exclusively, made after the replication of viral DNA has begun, from about 4 hr after infection. Mature progeny may accumulate from about 6 hr, reaching a maximum 10–12 hr after infection. These events usually occur against a declining background of host macromolecular synthesis. The sharpness of the decline varies with different virus strains and possibly with different host cells but substantial effects on host synthesis within 2–4 hr of infection are common.
The widely used tetrazolium dye, MTT, has several advantages as a vital stain in the identification of viral plaques. First, since the yellow colored dye MTT stains live cells dark blue, viral plaques can be counted without removal of the phenol red containing agar overlay. Secondly, the high contrast between live and dead cells afforded by MTT permits one to readily detect small plaques at an early time after infection. Thirdly, since cells intensely stained with MTT remain viable, MTT vital staining can be used to isolate cells which are either transformed or resistant to viruses. Thus, MTT vital staining should be useful in several types of viral plaque assays.
The clinical responses of SPF White Leghorn chickens to graded levels of infection with virulent (wild-strain) infectious laryngotracheitis (ILT) herpesvirus, administered by the tracheal route, were investigated in chickens from 1 day to 8 weeks of age. In 1-day-old chickens 40 plaque forming units (PFU) of ILT virus caused 55% mortality within 8 days. At least 500 PFU was needed to achieve comparable mortality at 3 weeks of age and this increased to 4,500 PFU of ILT virus by 6 to 8 weeks of age. A combination of surgical bursectomy at 1 day old and cyclophosphamide treatment ablated the ability of chickens to generate humoral antibody to ILT virus, but did not impair the level of protection induced by commercial ILT vaccine. Further, the passive transfer to ILT antibody-positive serum to 2-day-old or 4-week-old chickens did not significantly alter their susceptibility to tracheal challenge with virulent virus. Serum antibody was therefore discounted as a major immune mechanism in resistance to ILT virus infection. An experimental inactivated vaccine to ILT virus was also investigated. One intramuscular injection induced low serological responses, but no significant protection to ILT. A second injection of inactivated vaccine only marginally increased the titre of humoral antibody, but seemed to reduce the degree of respiratory distress. However, the levels of protection afforded by the inactivated vaccine were not significant compared with a live commercial ILT vaccine.
Nuclear and cytoplasmic viral RNAs, synthesized in cells productively infected with herpes simplex virus type 1 at early and late times post-infection and in the presence of DNA and protein synthesis inhibitors, have been analyzed by blot hybridization to viral DNA fragments generated by single and double digests with the Hind III, Bgl II and Hpa I restriction endonucleases. RNA transcribed in the presence of cycloheximide (immediate early, made using a cell RNA polymerase) hybridizes to restricted portions of the HSV-1 genome, and the hybridization patterns of both nuclear and cytoplasmic immediate early RNAs are similar. This finding suggests that synthesis of immediate early RNA within the nucleus may be restricted; alternatively, there may be rapid processing of primary transcripts. Virus-induced protein synthesis is a prerequisite for the switch from the restricted immediate early to the early pattern of transcription in which there is hybridization to all the DNA fragments. The hybridization pattern of RNA labeled in the presence of cytosine arabinoside resembles that of early RNA. Late RNA also hybridizes to all the DNA fragments, but the relative hybridization to the individual DNA fragments is different between early and late times. When late nuclear and late cytoplasmic RNAs are compared, there is greater relative hybridization to several different regions of the genome with nuclear RNA. This implies that either translocation of transcripts to the cytoplasm is regulated or that certain RNAs are degraded more rapidly within the cytoplasm.
Examination by electron microscopy of self-annealed single strands of pseudorabies virus DNA revealed the following structure: a sequence, (molecular weight, 9.9 × 106) which is repeated in a complementary, inverted form on the other side of a short unique sequence (molecular weight, 6.0 × 106), is present at one end of the molecule. The extreme end of the inverted repeat, the terminal region, is not repeated internally. A long unique sequence (molecular weight, 65 × 106) comprises the remainder of the molecule. A sequence homologous to the end of the long unique sequence is also found approximately 350 bases from the end. In addition, examination of self-annealed exonuclease-treated DNA revealed the presence of double-stranded lariats, as well as smaller-than-unit-size circles, indicating that other sequences homologous to the end of the molecule are present at various distances from the ends. After exonuclease digestion of the ends and self-annealing, circular unit-size molecules were not observed. The number of nicks and gaps on individual pseudorabies viral DNA molecules varied between 1 and 10 per molecule; on the average, each molecule contained approximately 3.5 nicks or gaps. The nicks or gaps were found to be located at random sites along the molecule. No alkali-sensitive bonds were detected in the DNA.
Clones representing 90% of the genome of Gallid herpesvirus 1 (infectious laryngotracheitis virus; ILTV) were obtained and used in hybridization experiments to construct EcoRI, KpnI amd SmaI physical maps. The genome was 155 kilobase pairs (kbp) and comprised of a long unique sequence (120 kbp) and a short unique sequence (17 kbp) bounded by repeat sequences each of 9 kbp. An unrelated second pair of repeat sequences was located at 0.67 and 0.88 map untis. A terminal repeat of the unique long region (UL) was also detected, but no isomerization of UL was detected.
DNA from infectious laryngotracheitis virus (ILTV) was randomly sheared and cloned into the M13 bacteriophage. Clones containing ILTV DNA were sequenced and the predicted amino acid sequences were compared to the known sequences of other herpesviruses using computer analysis. Twenty-one ILTV genes were identified, 20 by comparison to varicella-zoster virus and 19 by comparison to herpes simplex virus type 1; only 12 genes, giving consistently lower homology scores, were found by comparison with the gammaherpesvirus Epstein-Barr virus, indicating that ILTV sequences bear greater similarity to other alpha- than to gammaherpesvirus sequences.
The polypeptides associated with fowlpox virus (FPV) infection of chicken embryo skin (CES) cells were examined by metabolic labelling with [35S]-methionine and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Polypeptide synthesis was followed over the first 48 hours post infection, as this was shown to be the period of viable virus production in CES cells. In contrast to infection with vaccinia virus (VV), which leads to a rapid total inhibition of host polypeptide synthesis in a number of cell lines, FPV infection of CES cells failed to cause a complete shut down of host polypeptide synthesis, with only a small number of host polypeptides being inhibited. A total of 21 FPV coded or induced polypeptides were resolved by metabolic labelling. As with VV, these polypeptides can be divided into two groups, the pre-replicative polypeptides containing a single member of 70,000 daltons, synthesised before viral DNA replication, and the post-replicative polypeptides, synthesised only after viral DNA replication has commenced. FPV DNA replication was shown to commence between 12 and 16 hours post-infection and to continue up to 48 hours post-infection. As also observed with VV, two temporally distinct classes of post-replicative polypeptides were identified based on their time of synthesis post-infection.
The examination of purified FPV and VV by SDS-PAGE and coomassie blue staining allowed the resolution of 57 FPV particle associated polypeptides and 27 VV associated polypeptides.
The viral glycoproteins produced in cells infected with either vaccine strain or virulent isolates of infectious laryngotracheitis virus, an avian herpesvirus, were identified by in vitro labeling using [14C]glucosamine and [14C]mannose. Chicken antisera to the vaccine strain and to a virulent isolate, and rabbit antisera to the vaccine strain, immunoprecipitated four major viral glycoproteins of 205, 115, 90, and 60K mol wt. Additional glycoprotein bands were recognized by immune chicken and rabbit sera in Western blotting using a glycoprotein fraction purified from extracts of virus-infected cells. Monoclonal antibodies to the immunogenic glycoproteins were produced and characterized by immunoprecipitation and Western blotting. One group of monoclonal antibodies reacted only with the 60K glycoprotein, by both techniques, while a second group reacted with the 205, 115, and 90K glycoproteins in immunoprecipitation and with additional bands of 85 and 160K in Western blotting.
We have determined the complete DNA sequence of the short unique region in the genome of herpes simplex virus type 1, strain 17, and have interpreted it in terms of messenger RNAs and encoded proteins. The sequence contains variable regions whose length differs between DNA clones. The clones used for most of the analysis gave a short unique length of 12,979 base-pairs. We consider that this region contains 12 genes, which are expressed by mRNAs which have separate promoters, but may share 3'-termination sites, so that all but two mRNAs belong to one of four 3'-coterminal "families": 79% of the sequence is considered to be polypeptide coding. One pair of genes has an extensive out-of-frame overlap of coding sequences. The proteins encoded in the short unique region include two immediate-early species, two virion surface glycoproteins, and a DNA-binding species. Six of the genes have little or no previous characterization. From the nature of the amino acid sequences predicted for their encoded proteins, we deduce that several of these proteins may be membrane-associated.
EHV-1 polypeptide synthesis was examined in productively infected rabbit kidney and hamster embryo cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of extracts from [35S]methionine- and 3H-amino acid-labeled-infected and mock-infected cultures revealed the presence of 30 infected cell-specific polypeptides (ICPs) which ranged in apparent molecular weights from 16.5K to 213K. Twenty-two of these ICPs comigrated with virion structural proteins. Four ICPs (203K, 176K, 151K, 129K) were detected in extracts of infected cultures labeled in the presence or absence of actinomycin D (Act D) immediately after release from a 4-hr treatment with cycloheximide (CH). These polypeptides, which were designated as EHV-1 immediate early (alpha) ICPs, were not detected in unblocked (non-CH-treated) infected cells. The most abundant ICP was a 31.5K nonstructural protein which, in addition to a 74K protein, was detected in unblocked infected cells at 2-3 hr postinfection. These proteins appeared to be regulated as early (beta) ICPs, since neither protein was observed in Act D-treated cultures released from CH block. Twelve ICPs were classified as late (gamma) polypeptides on the basis of their reduced synthesis in cultures in which viral DNA replication was inhibited by phosphonoacetic acid. All but one (40K) of these late ICPs corresponded to virion structural proteins.
Analyses of the function of specific genes and sequences of large DNA viruses such as herpesviruses and poxviruses present special problems because of the size of their genomes (120 to 250 kilobase pairs). Various methods for engineering site-specific insertions or deletions based on the use of selectable markers have been developed and applied for the elucidation of the function of specific DNA sequences, the identification of genes nonessential for virus growth in cell culture, and the expression of foreign genes. These methods should also make possible the construction of viral vectors capable of delivering genes specifying antigens for the prevention of infectious diseases in humans and animals.
Eleven isolates of infectious laryngotracheitis virus (nine European and two American) were compared by restriction endonuclease analysis of their DNA after radiolabeling with 32P. Digestion with KpnI gave identical cleavage patterns for all the European isolates, but the two American viruses (one field and one vaccine) showed some differences from them and from each other. In the case of the American vaccine strain, however, these differences were only minor. After BamHI digestion, only the American field isolate appeared to be different, whereas with HindIII, all 11 isolates were identical.
Based on evidence that 50% of herpes simplex 1 DNA is transcribed in HEp 2 cells in the absence of protein synthesis the order and rates of synthesis of viral polypeptides in infected cells after reversal of cycloheximide or puromycin mediated inhibition of protein synthesis were examined. These experiments showed that viral polypeptides formed three sequentially synthesized, coordinately regulated groups designated α, β, and γ. Specifically: the α group, containing one minor structural and several nonstructural polypeptides, was synthesized at highest rates from 3 to 4 hr postinfection in untreated cells and at diminishing rates thereafter. The β group, also containing minor strucutural and nonstructural polypeptides, was synthesized at highest rates from 5 to 7 hr and at decreasing rates thereafter. The γ group containing major structural polypeptides was synthesized at increasing rates until at least 12 hr postinfection.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
Herpes simplex virus 1 (HSV-1) specifies in the infected cell a set of polypeptides (alpha) whose mRNAs are made in the absence of protein synthesis. The individual mRNAs specifying alpha polypeptides 0, 4, 22, and 27 were purified by hybridization to strand-separated or total HSV-1 DNA fragments cloned in pBR322 plasmids, translated in vitro to verify their specificity, then mapped by hybridization to separated strands and digests of cloned DNA fragments. To map the transcription initiation sites, chemically decapped individual mRNAs were recapped with [alpha-32P]GTP and vaccinia virus guanylyltransferase and hybridized to digests of the cloned DNA fragments. Each of the labeled 5' termini hybridized to a specific site, two on one strand, and two on the other. The 5' ends of the transcripts do not share sequence homology, suggesting that they are transcribed from independent promoters.
By immunoprecipitation of human cytomegalovirus-infected cell-specific polypeptides (ICPs) with a variety of human cytomegalovirus-positive sera and analysis by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, we can identify at least 20 ICSP bands from lytic infections by 6 hr postinfection (pi). Three of these polypeptide bands (78K, 73K, and 68K) may represent more than one species of polypeptide. Four polypeptide bands (78K, 77K, 73K, and 31K) can be identified as immediate-early based on their synthesis in the presence of actinomycin-D after removal from a protein synthesis block mediated by cycloheximide (CH). An immediate-early 78K polypeptide and an early 49K polypeptide are synthesized only transiently during the first 4 hr pi. Most immediate-early polypeptide synthesis is enhanced after removal of a 5 hr CH block. Taken together, these results identify many previously undetected immediate-early and early ICSPs and suggest that several regulatory events are occurring during the early phase of the lytic cycle.
Operationally, alpha genes of herpes simplex virus 1 were defined on the basis of the observations that they are the earliest genes expressed in the infected cell and that the transcription, processing, accumulation of the mRNA's in the infected cell cytoplasm can take place in the presence of inhibitors of protein synthesis, such as cycloheximide. In these studies, we translated in vitro the viral mRNA's extracted from cells infected maintained in the presence of cycloheximide, emetine, or anisomycin. Inasmuch as all the major alpha proteins (no. 0, 4, 22, and 27) were translated, we conclude that the transcription of all previously defined alpha genes is independent of the stringency of inhibition of protein synthesis and that pre-alpha genes cannot be detected in such experiments.
The transcription of the human cytomegalovirus genome was investigated at immediate early, early, and late times after infection. Viral RNAs associated with either the whole cell, the nucleus, the cytoplasm, or the polyribosomes were analyzed. At immediate early times, i.e., in the absence of de novo viral protein synthesis, the viral RNA in high abundance originated from a region of the long unique section of the prototype arrangement of the viral genome (0.660 to 0.770 map units). The viral RNA in low abundance originated from the long repeat sequences (0.010 to 0.035 and 0.795 to 0.825 map units) and a region in the long unique section (0.201 to 0.260 map units). Viral RNAs associated with the polyribosomes as polyadenylated RNA were mapped to these restricted regions of the viral genome and characterized according to size class in kilobases. At 24 h after infection in the presence of an inhibitor of viral DNA replication, i.e., at early times, the stable viral RNAs in highest abundance mapped in the long repeat sequences. Viral RNAs at intermediate abundance under these conditions mapped in two regions of the long unique section of the viral genome (0.325 to 0.460 and 0.685 to 0.770 map units). Stable viral RNAs that were associated with the polyribosomes in high abundance as polyadenylated RNA orginated from the long repeat sequences, but not from the long unique section of the viral genome. An analysis of whole-cell RNA at late times (72 h) indicated that the abundant transcription was in the regions of the long unique sequences (0.325 to 0.460 and 0.660 to 0.685 map units), and transcription of intermediate abundance was from the long repeat sequences. However, stable viral mRNA's derived from the long repeat sequences were associated with the polyribosomes at late times after infection. In addition, mRNA's originating from the long and short unique sequences were found associated with the polyribosomes at higher relative concentration than at early times after infection. It is proposed that expression of the immediate early viral genes is required to transcribe the early viral genes in the long repeat and adjacent sequences. These sequences are also transcribed at late times after infection while viral DNA synthesis continues. The expression of viral genes in most of the long and short unique sequences appears to require viral DNA replication.
The addition of TPA (phorbol-12-myristate-13-acetate) to cultures during the lytic infection with herpesvirus saimiri led to an enhanced and accelerated production of polypeptides induced by H. saimiri and to a rapid shut-down of host cell protein synthesis and allowed a detailed analysis of the protein patterns. Analysis of sequential protein synthesis in owl monkey kidney cells lytically infected with H. saimiri 11 permitted the identification of 31 virus-induced polypeptides. The use of the amino acid analogues canavanine (for arginine) and azetidine (for proline) in parallel allowed experiments on the identification of proteins synthesized early and late during lytic infection.
Strains of infectious laryngotracheitis virus (ILTV) were examined using an indirect immunofluorescent test (IIF) and with restriction endonucleases for detecting intratypic differences. Electrophoretic analysis of ILTV DNA fragments cleaved with restriction endonuclease Hind 111 clearly distinguished between strains. The IIF test did not discriminate between strains. A molecular weight estimate of ILTV DNA was made by summation of restriction endonuclease fragments cleaved with BamH1 (102.1 X 10(6)) and Hind111 (97.35 X 10(6)). Differences between the estimates may indicate the presence of submolar fragments.
The immediate-early transcripts of pseudorabies virus have been located in a region of the genome situated internally within the inverted repeat between map positions 0.99 and 0.95. A single immediate-early transcript (approximately 6 kb) can be detected both in the cytoplasmic and nuclear fractions of infected, cycloheximide-treated cells. Analysis of the proteins synthesized after removal of cycloheximide from infected cells or after translation in vitro of the RNA isolated from these cells revealed the presence of a single protein (180K) not present in similarly treated, uninfected cells. That this is a virus protein and is specified by the immediate-early region of the genome was shown by selection and translation of mRNA hybridizing with virus DNA from the appropriate region of the genome. The effects of infection of cells with a temperature-sensitive mutant (tsG1) defective in the 180K protein were studied. At the nonpermissive temperature only immediate-early RNA was transcribed and only one virus protein, the 180K protein was synthesized. Inhibition of cellular protein and DNA synthesis was also observed. After shift down of tsG1-infected cells from the nonpermissive to the permissive temperature at 3 hr post infection, a transition to early RNA transcription occurred. However, if the shift down was delayed until 5 hr post infection, transcription of the virus genome was completely inhibited and an abortive infection ensued. Shift of the mutant-infected cells from the permissive to the nonpermissive temperature resulted in a decrease in the rate of accumulation of early and late transcripts, and a resumption of the synthesis of immediate-early RNA and protein. From these as well as from previous results, it is concluded that pseudorabies virus codes for a single multifunctional immediate-early protein which is essential for the transcription of immediate-early to early RNA and is required for the continuous transcription of early (and late) RNA. The immediate-early protein is also self-regulatory; the presence of the functional immediate-early protein represses the transcription of its RNA. In addition, the immediate-early protein of pseudorabies virus appears to play a direct role, under certain conditions, in the inhibition of cellular macromolecular synthesis.
The widely used tetrazolium dye, MTT, has several advantages as a vital stain in the identification of viral plaques. First, since the yellow colored dye MTT stains live cells dark blue, viral plaques can be counted without removal of the phenol red containing agar overlay. Secondly, the high contrast between live and dead cells afforded by MTT permits one to readily detect small plaques at an early time after infection. Thirdly, since cells intensely stained with MTT remain viable, MTT vital staining can be used to isolate cells which are either transformed or resistant to viruses. Thus, MTT vital staining should be useful in several types of viral plaque assays.
Avian adenoviruses. In: Advances in veterinary virology, Proceedings No. 60, Post-Graduate Committee in Veterinary Science
- Tj Bagust
Post-Graduate Committee in Veterinary Science
- T J Bagust
- TJ Bagust