Kinetic Model for the Heterogeneous Biocatalytic Reactions Using Tethered Cofactors.

Understanding the mechanism of interfacial enzyme kinetics is critical to the development of synthetic biological systems for the production of value-added chemicals. Here, the interfacial kinetics of the catalysis of ß-nicotinamide adenine dinucleotide (NAD+)-dependent enzymes acting on NAD+ tethered to the surface of silica nanoparticles (SiNPs) has been investigated using two complementary and supporting kinetic approaches: enzyme excess and reactant (NAD+) excess. Kinetic models developed for these two approaches characterize several critical reaction steps including reversible enzyme adsorption, complexation, decomplexation, and catalysis of the surface-bound enzyme/NAD+ complex. The analysis reveals a concentrating effect resulting in a very high local concentration of enzyme and cofactor on the particle surface, in which the enzyme is saturated by surface-bound NAD, facilitating a rate enhancement of enzyme/NAD+ complexation and catalysis. This resulted in high enzyme efficiency within the tethered NAD+ system compared to that of the free enzyme/NAD+ system, which increases with decreasing enzyme concentration. The role of enzyme adsorption onto solid substrates with a tethered catalyst (such as NAD+) has potential for creating highly efficient flow biocatalytic systems.

Codon pair bias deoptimization of essential genes in infectious laryngotracheitis virus reduces protein expression.

Infectious laryngotracheitis virus (ILTV; an alphaherpesvirus) is a respiratory pathogen of chickens and causes significant economic losses in the poultry industry globally, in addition to severe animal health and welfare concerns. To date, studying the role of ILTV genes in viral infection, replication or pathogenesis has largely been limited to genes that can be deleted from the ILTV genome and the resultant deletion mutants characterized in vitro or in vivo. However, this approach is not suitable for the study of essential genes. This study trialled two different codon deoptimization techniques that aimed to separately disrupt and downregulate the expression of two ILTV genes, ICP8 and UL12, which are essential or very important in viral replication. The target genes were partially recoded using codon usage deoptimization (CUD) and codon pair bias deoptimization (CPBD) approaches and characterized in vitro. Viruses deoptimized via CPBD showed decreased protein expression as assessed by Western blotting and/or fluorescence microscopy to measure the intensity of the fluorescent marker fused to the target protein. Viruses deoptimized by CUD showed less consistent results, with some mutants that could not be generated or isolated. The results indicate that CPBD is an attractive and viable tool for the study of essential or critically important genes in ILTV. This is the first study, to our knowledge, that utilizes CPBD and CUD techniques for the study of ILTV genes.

Innovation in precision fermentation for food ingredients.

A transformation in our food production system is being enabled by the convergence of advances in genome-based technologies and traditional fermentation. Science at the intersection of synthetic biology, fermentation, downstream processing for product recovery, and food science is needed to support technology development for the production of fermentation-derived food ingredients. The business and markets for fermentation-derived ingredients, including policy and regulations are discussed. A patent landscape of fermentation for the production of alternative proteins, lipids and carbohydrates for the food industry is provided. The science relating to strain engineering, fermentation, downstream processing, and food ingredient functionality that underpins developments in precision fermentation for the production of proteins, fats and oligosaccharides is examined. The production of sustainably-produced precision fermentation-derived ingredients and their introduction into the market require a transdisciplinary approach with multistakeholder engagement. Successful innovation in fermentation-derived ingredients will help feed the world more sustainably.

Biosensor-guided rapid screening for improved recombinant protein secretion in Pichia pastoris.

Pichia pastoris (Komagataella phaffii) is widely used for industrial production of heterologous proteins due to high secretory capabilities but selection of highly productive engineered strains remains a limiting step. Despite availability of a comprehensive molecular toolbox for construct design and gene integration, there is high clonal variability among transformants due to frequent multi-copy and off-target random integration. Therefore, functional screening of several hundreds of transformant clones is essential to identify the best protein production strains. Screening methods are commonly based on deep-well plate cultures with analysis by immunoblotting or enzyme activity assays of post-induction samples, and each heterologous protein produced may require development of bespoke assays with multiple sample processing steps. In this work, we developed a generic system based on a P. pastoris strain that uses a protein-based biosensor to identify highly productive protein secretion clones from a heterogeneous set of transformants. The biosensor uses a split green fluorescent protein where the large GFP fragment (GFP1-10) is fused to a sequence-specific protease from Tobacco Etch Virus (TEV) and is targeted to the endoplasmic reticulum. Recombinant proteins targeted for secretion are tagged with the small fragment of the split GFP (GFP11). Recombinant protein production can be measured by monitoring GFP fluorescence, which is dependent on interaction between the large and small GFP fragments. The reconstituted GFP is cleaved from the target protein by TEV protease, allowing for secretion of the untagged protein of interest and intracellular retention of the mature GFP. We demonstrate this technology with four recombinant proteins (phytase, laccase, ß-casein and ß-lactoglobulin) and show that the biosensor directly reports protein production levels that correlate with traditional assays. Our results confirm that the split GFP biosensor can be used for facile, generic, and rapid screening of P. pastoris clones to identify those with the highest production levels.

Whole genome sequence analysis of equid gammaherpesvirus -2 field isolates reveals high levels of genomic diversity and recombination.

BACKGROUND: Equid gammaherpesvirus 2 (EHV2) is a gammaherpesvirus with a widespread distribution in horse populations globally. Although its pathogenic significance can be unclear in most cases of infection, EHV2 infection can cause upper respiratory tract disease in foals. Co-infection of different strains of EHV2 in an individual horse is common. Small regions of the EHV2 genome have shown considerable genetic heterogeneity. This could suggest genomic recombination between different strains of EHV2, similar to the extensive recombination networks that have been demonstrated for some alphaherpesviruses. This study examined natural recombination and genome diversity of EHV2 field isolates. RESULTS: Whole genome sequencing analysis of 18 EHV2 isolates, along with analysis of two publicly available EHV2 genomes, revealed variation in genomes sizes (from 173.7 to 184.8 kbp), guanine plus cytosine content (from 56.7 to 57.8%) and the size of the terminal repeat regions (from 17,196 to 17,551 bp). The nucleotide sequence identity between the genomes ranged from 86.2 to 99.7%. The estimated average inter-strain nucleotide diversity between the 20 EHV2 genomes was 2.9%. Individual gene sequences showed varying levels of nucleotide diversity and ranged between 0 and 38.1%. The ratio of nonsynonymous substitutions, Ka, to synonymous substitutions, Ks, (Ka/Ks) suggests that over 50% of EHV2 genes are undergoing diversifying selection. Recombination analyses of the 20 EHV2 genome sequences using the recombination detection program (RDP4) and SplitsTree revealed evidence of viral recombination. CONCLUSIONS: Analysis of the 18 new EHV2 genomes alongside the 2 previously sequenced genomes revealed a high degree of genetic diversity and extensive recombination networks. Herpesvirus genome diversification and virus evolution can be driven by recombination, and our findings are consistent with recombination being a key mechanism by which EHV2 genomes may vary and evolve.

Construction and characterisation of glycoprotein E and glycoprotein I deficient mutants of Australian strains of infectious laryngotracheitis virus using traditional and CRISPR/Cas9-assisted homologous recombination techniques.

In alphaherpesviruses, glycoproteins E and I (gE and gI, respectively) form a heterodimer that facilitates cell-to-cell spread of virus. Using traditional homologous recombination techniques, as well as CRISPR/Cas9-assisted homologous recombination, we separately deleted gE and gI coding sequences from an Australian field strain (CSW-1) and a vaccine strain (A20) of infectious laryngotracheitis virus (ILTV) and replaced each coding sequence with sequence encoding green fluorescent protein (GFP). Virus mutants in which gE and gI gene sequences had been replaced with GFP were identified by fluorescence microscopy but were unable to be propagated separately from the wildtype virus in either primary chicken cells or the LMH continuous chicken cell line. These findings build on findings from a previous study of CSW-1 ILTV in which a double deletion mutant of gE and gI could not be propagated separately from wildtype virus and produced an in vivo phenotype of single-infected cells with no cell-to-cell spread observed. Taken together these studies suggest that both the gE and gI genes have a significant role in cell-to-cell spread in both CSW-1 and A20 strains of ILTV. The CRISPR/Cas9-assisted deletion of genes from the ILTV genome described in this study adds this virus to a growing list of viruses to which this approach has been used to study viral gene function.

ICTV Virus Taxonomy Profile: Herpesviridae 2021.

Members of the family Herpesviridae have enveloped, spherical virions with characteristic complex structures consisting of symmetrical and non-symmetrical components. The linear, double-stranded DNA genomes of 125-241 kbp contain 70-170 genes, of which 43 have been inherited from an ancestral herpesvirus. In general, herpesviruses have coevolved with and are highly adapted to their hosts, which comprise many mammalian, avian and reptilian species. Following primary infection, they are able to establish lifelong latent infection, during which there is limited viral gene expression. Severe disease is usually observed only in the foetus, the very young, the immunocompromised or following infection of an alternative host. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Herpesviridae, which is available at ictv.global/report/herpesviridae.

Mycoplasma bovis mbfN Encodes a Novel LRR Lipoprotein That Undergoes Proteolytic Processing and Binds Host Extracellular Matrix Components.

Mycoplasma bovis causes serious infections in ruminants, leading to huge economic losses. Lipoproteins are key components of the mycoplasma membrane and are believed to function in nutrient acquisition, adherence, enzymatic interactions with the host, and induction of the host's immune response to infection. Many genes of M. bovis have not been assigned functions, in part because of their low sequence similarity with other bacteria, making it difficult to extrapolate gene functions. This study examined functions of a surface-localized leucine-rich repeat (LRR) lipoprotein encoded by mbfN of M. bovis PG45. Homologs of MbfN were detected as 48-kDa peptides by Western blotting in all the strains of M. bovis included in this study, with the predicted 70-kDa full-length polypeptide detected in some strains. Sequence analysis of the gene revealed the absence in some strains of a region encoding the carboxyl-terminal 147 amino acids found in strain PG45, which could account for the variation detected by immunoblotting. In silico analysis of MbfN suggested that it may have an adhesion-related function. In vitro binding assays confirmed MbfN to be a fibronectin and heparin-binding protein. Disruption of mbfN in M. bovis PG45 significantly reduced (P = 0.033) the adherence of M. bovis PG45 to MDBK cells in vitro, demonstrating the role of MbfN as an adhesin.IMPORTANCE Experimental validation of the putative functions of genes in M. bovis will advance our understanding of the basic biology of this economically important pathogen and is crucial in developing prevention strategies. This study demonstrated the extracellular matrix binding ability of a novel immunogenic lipoprotein of M. bovis, and the role of this protein in adhesion by M. bovis suggests that it could play a role in virulence.

Koala and Wombat Gammaherpesviruses Encode the First Known Viral NTPDase Homologs and Are Phylogenetically Divergent from All Known Gammaherpesviruses.

There is a large taxonomic gap in our understanding of mammalian herpesvirus genetics and evolution corresponding to those herpesviruses that infect marsupials, which diverged from eutherian mammals approximately 150 million years ago (mya). We compare the genomes of two marsupial gammaherpesviruses, Phascolarctid gammaherpesvirus 1 (PhaHV1) and Vombatid gammaherpesvirus 1 (VoHV1), which infect koalas (Phascolarctos cinereus) and wombats (Vombatus ursinus), respectively. The core viral genomes were approximately 117 kbp and 110 kbp in length, respectively, sharing 69% pairwise nucleotide sequence identity. Phylogenetic analyses showed that PhaHV1 and VoHV1 formed a separate branch, which may indicate a new gammaherpesvirus genus. The genomes contained 60 predicted open reading frames (ORFs) homologous to those in eutherian herpesviruses and 20 ORFs not yet found in any other herpesvirus. Seven of these ORFs were shared by the two viruses, indicating that they were probably acquired prespeciation, approximately 30 to 40 mya. One of these shared genes encodes a putative nucleoside triphosphate diphosphohydrolase (NTPDase). NTPDases are usually found in mammals and higher-order eukaryotes, with a very small number being found in bacteria. This is the first time that an NTPDase has been identified in any viral genome. Interrogation of public transcriptomic data sets from two koalas identified PhaHV1-specific transcripts in multiple host tissues, including transcripts for the novel NTPDase. PhaHV1 ATPase activity was also demonstrated in vitro, suggesting that the encoded NTPDase is functional during viral infection. In mammals, NTPDases are important in downregulation of the inflammatory and immune responses, but the role of the PhaHV1 NTPDase during viral infection remains to be determined.IMPORTANCE The genome sequences of the koala and wombat gammaherpesviruses show that the viruses form a distinct branch, indicative of a novel genus within the Gammaherpesvirinae Their genomes contain several new ORFs, including ORFs encoding a ß-galactoside α-2,6-sialyltransferase that is phylogenetically closest to poxvirus and insect homologs and the first reported viral NTPDase. NTPDases are ubiquitously expressed in mammals and are also present in several parasitic, fungal, and bacterial pathogens. In mammals, these cell surface-localized NTPDases play essential roles in thromboregulation, inflammation, and immune suppression. In this study, we demonstrate that the virus-encoded NTPDase is enzymatically active and is transcribed during natural infection of the host. Understanding how these enzymes benefit viruses can help to inform how they may cause disease or evade host immune defenses.

Latency characteristics in specific pathogen-free chickens 21 and 35 days after intra-tracheal inoculation with vaccine or field strains of infectious laryngotracheitis virus.

Latency is an important feature of infectious laryngotracheitis virus (ILTV) yet is poorly understood. This study aimed to compare latency characteristics of vaccine (SA2) and field (CL9) strains of ILTV, establish an in vitro reactivation system and examine ILTV infection in peripheral blood mononuclear cells (PBMC) in specific pathogen-free chickens. Birds were inoculated with SA2 or CL9 ILTV and then bled and culled at 21 or 35 days post-inoculation (dpi). Swabs (conjunctiva, palatine cleft, trachea) and trigeminal ganglia (TG) were examined for ILTV DNA using PCR. Half of the TG, trachea and PBMC were co-cultivated with cell monolayers to assess in vitro reactivation of ILTV infection. ILTV DNA was detected in the trachea of approximately 50% of ILTV-inoculated birds at both timepoints. At 21 dpi, ILTV was detected in the TG only in 29% and 17% of CL9- and SA2-infected birds, respectively. At 35 dpi, ILTV was detected in the TG only in 30% and 10% of CL9- and SA2-infected birds, respectively. Tracheal organ co-cultures from 30% and 70% of CL9- and SA2-infected birds, respectively, were negative for ILTV DNA at cull but yielded quantifiable DNA within 6 days post-explant (dpe). TG co-cultivation from 30% and 40% of CL9-and SA2-infected birds, respectively, had detectable ILTV DNA within 6 dpe. Latency characteristics did not substantially vary based on the strain of virus inoculated or between sampling timepoints. These results advance our understanding of ILTV latency and reactivation. RESEARCH HIGHLIGHTS Following inoculation, latent ILTV infection was detected in a large proportion of chickens, irrespective of whether a field or vaccine strain was inoculated. In vitro reactivation of latent ILTV was readily detected in tracheal and trigeminal ganglia co-cultures using PCR. ILTV latency observed in SPF chickens at 21 days post-infection was not substantially different to 35 days post-infection.

An unexpected vestigial protein complex reveals the evolutionary origins of an s-triazine catabolic enzyme.

Cyanuric acid is a metabolic intermediate of s-triazines, such as atrazine (a common herbicide) and melamine (used in resins and plastics). Cyanuric acid is mineralized to ammonia and carbon dioxide by the soil bacterium Pseudomonas sp. strain ADP via three hydrolytic enzymes (AtzD, AtzE, and AtzF). Here, we report the purification and biochemical and structural characterization of AtzE. Contrary to previous reports, we found that AtzE is not a biuret amidohydrolase, but instead it catalyzes the hydrolytic deamination of 1-carboxybiuret. X-ray crystal structures of apo AtzE and AtzE bound with the suicide inhibitor phenyl phosphorodiamidate revealed that the AtzE enzyme complex consists of two independent molecules in the asymmetric unit. We also show that AtzE forms an α2ß2 heterotetramer with a previously unidentified 68-amino acid-long protein (AtzG) encoded in the cyanuric acid mineralization operon from Pseudomonas sp. strain ADP. Moreover, we observed that AtzG is essential for the production of soluble, active AtzE and that this obligate interaction is a vestige of their shared evolutionary origin. We propose that AtzEG was likely recruited into the cyanuric acid-mineralizing pathway from an ancestral glutamine transamidosome that required protein-protein interactions to enforce the exclusion of solvent from the transamidation reaction.

Infectious Laryngotracheitis Virus Viral Chemokine-Binding Protein Glycoprotein G Alters Transcription of Key Inflammatory Mediators In Vitro and In Vivo.

Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that infects chickens, causing upper respiratory tract disease and significant losses to poultry industries worldwide. Glycoprotein G (gG) is a broad-range viral chemokine-binding protein conserved among most alphaherpesviruses, including ILTV. A number of studies comparing the immunological parameters between infection with gG-expressing and gG-deficient ILTV strains have demonstrated that expression of gG is associated with increased virulence, modification of the amount and the composition of the inflammatory response, and modulation of the immune responses toward antibody production and away from cell-mediated immune responses. The aims of the current study were to examine the establishment of infection and inflammation by ILTV and determine how gG influences that response to infection. In vitro infection studies using tracheal organ tissue specimen cultures and blood-derived monocytes and in vivo infection studies in specific-pathogen-free chickens showed that leukocyte recruitment to the site of infection is an important component of the induced pathology and that this is influenced by the expression of ILTV gG and changes in the transcription of the chicken orthologues of mammalian CXC chemokine ligand 8 (CXCL8), chicken CXCLi1 and chicken CXCLi2, among other cytokines and chemokines. The results from this study demonstrate that ILTV gG interferes with chemokine and cytokine transcription at different steps of the inflammatory cascade, thus altering inflammation, virulence, and the balance of the immune response to infection.IMPORTANCE Infectious laryngotracheitis virus is an alphaherpesvirus that expresses gG, a conserved broad-range viral chemokine-binding protein known to interfere with host immune responses. However, little is known about how gG modifies virulence and influences the inflammatory signaling cascade associated with infection. Here, data from in vitro and in vivo infection studies are presented. These data show that gG has a direct impact on the transcription of cytokines and chemokine ligands in vitro (such as chicken CXCL8 orthologues, among others), which explains the altered balance of the inflammatory response that is associated with gG during ILTV infection of the upper respiratory tract of chickens. This is the first report to associate gG with the dysregulation of cytokine transcription at different stages of the inflammatory cascade triggered by ILTV infection of the natural host.

Development and application of high-resolution melting analysis for the classification of infectious laryngotracheitis virus strains and detection of recombinant progeny.

Live attenuated vaccines against infectious laryngotracheitis virus (ILTV) are widely used in the poultry industry to control disease and help prevent economic losses. Molecular epidemiological studies of currently circulating strains of ILTV within poultry flocks in Australia have demonstrated the presence of highly virulent viruses generated by genomic recombination events between vaccine strains. In this study, high-resolution melting (HRM) analysis was used to develop a tool to classify ILTV isolates and to investigate ILTV recombination. The assay was applied to plaque-purified progeny viruses generated after co-infection of chicken embryo kidney (CEK) monolayers with the A20 and Serva ILT vaccine strains and also to viruses isolated from field samples. The results showed that the HRM analysis is a suitable tool for the classification of ILTV isolates and can be used to detect recombination between ILTV vaccine strains in vitro. This method can be used to classify a broad range of ILTV strains to facilitate the classification and genotyping of ILTV and help to further understand recombination in these viruses.

Single Nucleotide Polymorphism Genotyping Analysis Shows That Vaccination Can Limit the Number and Diversity of Recombinant Progeny of Infectious Laryngotracheitis Viruses from the United States.

Infectious laryngotracheitis (ILTV; Gallid alphaherpesvirus 1) causes mild to severe respiratory disease in poultry worldwide. Recombination in this virus under natural (field) conditions was first described in 2012 and more recently has been studied under laboratory conditions. Previous studies have revealed that natural recombination is widespread in ILTV and have also demonstrated that recombination between two attenuated ILTV vaccine strains generated highly virulent viruses that produced widespread disease within poultry flocks in Australia. In the United States, natural ILTV recombination has also been detected, but not as frequently as in Australia. To better understand recombination in ILTV strains originating from the United States, we developed a TaqMan single nucleotide polymorphism (SNP) genotyping assay to detect recombination between two virulent U.S. field strains of ILTV (63140 and 1874c5) under experimental in vivo conditions. We also tested the capacity of the Innovax-ILT vaccine (a recombinant vaccine using herpesvirus of turkeys as a vector) and the Trachivax vaccine (a conventionally attenuated chicken embryo origin vaccine) to reduce recombination. The Trachivax vaccine prevented ILTV replication, and therefore recombination, in the trachea after challenge. The Innovax-ILT vaccine allowed the challenge viruses to replicate and to recombine, but at a significantly lower rate than in an unvaccinated group of birds. Our results demonstrate that the TaqMan SNP genotyping assay is a useful tool to study recombination between these ILTV strains and also show that vaccination can limit the number and diversity of recombinant progeny viruses.IMPORTANCE Recombination allows alphaherpesviruses to evolve over time and become more virulent. Historically, characterization of viral vaccines in poultry have mainly focused on limiting clinical disease, rather than limiting virus replication, but such approaches can allow field viruses to persist and evolve in vaccinated populations. In this study, we vaccinated chickens with Gallid alphaherpesvirus 1 vaccines that are commercially available in the United States and then performed coinoculations with two field strains of virus to measure the ability of the vaccines to prevent field strains from replicating and recombining. We found that vaccination reduced viral replication, recombination, and diversity compared to those in unvaccinated chickens, although the extent to which this occurred differed between vaccines. We suggest that characterization of vaccines could include studies to examine the ability of vaccines to reduce viral recombination in order to limit the rise of new virulent field strains due to recombination, especially for those vaccines that are known not to prevent viral replication following challenge.

Mapping B lymphocytes as major reservoirs of naturally occurring latent equine herpesvirus 5 infection.

Equine herpesvirus 5 (EHV5) is a commonly detected gammaherpesvirus, which, along with the closely related EHV2, constitute the only two known percaviruses that infect horses. Apart from detection in horse populations worldwide and the recent publication of the whole genome, there is little known about the biology and pathogenesis of this virus, with many assumptions made by parallels with EHV2. The long-term survival of gammaherpesviruses within infected hosts involves the establishment and maintenance of latency in selected cell and tissues types, particularly lymphocytes. A latent gammaherpesvirus infection is characterized by a limited number of genes expressing in a particular cell or tissue type. In this study, we have used in vitro co-culturing to detect EHV5 in equine PBMCs and characterize the predominant cellular site for the establishment and maintenance of a latent infection. These experiments were conducted by isolating PBMCs from 10 horses and sorting subpopulations into two T lymphocyte (CD4 and CD8), B lymphocyte and macrophage enriched or depleted fractions. These lymphocyte and macrophage fractions were examined for the presence of latent EHV5 by in vitro co-culturing with equine foetal kidney cells. The lymphocyte fraction enriched with B lymphocytes had a significantly increased (P=0.005) number of plaques formed during co-culturing, whereas the B lymphocyte depleted fraction had a significant reduction in the number of plaques formed after co-culturing. Taken together, these results demonstrate that equine gammaherpesviruses establish latency in the equine PBMCs, with the predominant site for maintenance of latent virus being B lymphocytes.

High-Resolution X-Ray Structures of Two Functionally Distinct Members of the Cyclic Amide Hydrolase Family of Toblerone Fold Enzymes.

The Toblerone fold was discovered recently when the first structure of the cyclic amide hydrolase, AtzD (a cyanuric acid hydrolase), was elucidated. We surveyed the cyclic amide hydrolase family, finding a strong correlation between phylogenetic distribution and specificity for either cyanuric acid or barbituric acid. One of six classes (IV) could not be tested due to a lack of expression of the proteins from it, and another class (V) had neither cyanuric acid nor barbituric acid hydrolase activity. High-resolution X-ray structures were obtained for a class VI barbituric acid hydrolase (1.7 Å) from a Rhodococcus species and a class V cyclic amide hydrolase (2.4 Å) from a Frankia species for which we were unable to identify a substrate. Both structures were homologous with the tetrameric Toblerone fold enzyme AtzD, demonstrating a high degree of structural conservation within the cyclic amide hydrolase family. The barbituric acid hydrolase structure did not contain zinc, in contrast with early reports of zinc-dependent activity for this enzyme. Instead, each barbituric acid hydrolase monomer contained either Na+ or Mg2+, analogous to the structural metal found in cyanuric acid hydrolase. The Frankia cyclic amide hydrolase contained no metal but instead formed unusual, reversible, intermolecular vicinal disulfide bonds that contributed to the thermal stability of the protein. The active sites were largely conserved between the three enzymes, differing at six positions, which likely determine substrate specificity.IMPORTANCE The Toblerone fold enzymes catalyze an unusual ring-opening hydrolysis with cyclic amide substrates. A survey of these enzymes shows that there is a good correlation between physiological function and phylogenetic distribution within this family of enzymes and provide insights into the evolutionary relationships between the cyanuric acid and barbituric acid hydrolases. This family of enzymes is structurally and mechanistically distinct from other enzyme families; however, to date the structure of just two, physiologically identical, enzymes from this family has been described. We present two new structures: a barbituric acid hydrolase and an enzyme of unknown function. These structures confirm that members of the CyAH family have the unusual Toblerone fold, albeit with some significant differences.

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.

Low genetic diversity among historical and contemporary clinical isolates of felid herpesvirus 1.

BACKGROUND: Felid herpesvirus 1 (FHV-1) causes upper respiratory tract diseases in cats worldwide, including nasal and ocular discharge, conjunctivitis and oral ulceration. The nature and severity of disease can vary between clinical cases. Genetic determinants of virulence are likely to contribute to differences in the in vivo phenotype of FHV-1 isolates, but to date there have been limited studies investigating FHV-1 genetic diversity. This study used next generation sequencing to compare the genomes of contemporary Australian clinical isolates of FHV-1, vaccine isolates and historical clinical isolates, including isolates that predated the introduction of live attenuated vaccines into Australia. Analysis of the genome sequences aimed to assess the level of genetic diversity, identify potential genetic markers that could influence the in vivo phenotype of the isolates and examine the sequences for evidence of recombination. RESULTS: The full genome sequences of 26 isolates of FHV-1 were determined, including two vaccine isolates and 24 clinical isolates that were collected over a period of approximately 40 years. Analysis of the genome sequences revealed a remarkably low level of diversity (0.0-0.01 %) between the isolates. No potential genetic determinants of virulence were identified, but unique single nucleotide polymorphisms (SNPs) in the UL28 and UL44 genes were detected in the vaccine isolates that were not present in the clinical isolates. No evidence of FHV-1 recombination was detected using multiple methods of recombination detection, even though many of the isolates originated from cats housed in a shelter environment where high infective pressures were likely to exist. Evidence of displacement of dominant FHV-1 isolates with other (genetically distinct) FHV-1 isolates over time was observed amongst the isolates obtained from the shelter-housed animals. CONCLUSIONS: The results show that FHV-1 genomes are highly conserved. The lack of recombination detected in the FHV-1 genomes suggests that the risk of attenuated vaccines recombining to generate virulent field viruses is lower than has been suggested for some other herpesviruses. The SNPs detected only in the vaccine isolates offer the potential to develop PCR-based methods of differentiating vaccine and clinical isolates of FHV-1 in order to facilitate future epidemiological studies.

The first genome sequence of a metatherian herpesvirus: Macropodid herpesvirus 1.

BACKGROUND: While many placental herpesvirus genomes have been fully sequenced, the complete genome of a marsupial herpesvirus has not been described. Here we present the first genome sequence of a metatherian herpesvirus, Macropodid herpesvirus 1 (MaHV-1). RESULTS: The MaHV-1 viral genome was sequenced using an Illumina MiSeq sequencer, de novo assembly was performed and the genome was annotated. The MaHV-1 genome was 140 kbp in length and clustered phylogenetically with the primate simplexviruses, sharing 67% nucleotide sequence identity with Human herpesviruses 1 and 2. The MaHV-1 genome contained 66 predicted open reading frames (ORFs) homologous to those in other herpesvirus genomes, but lacked homologues of UL3, UL4, UL56 and glycoprotein J. This is the first alphaherpesvirus genome that has been found to lack the UL3 and UL4 homologues. We identified six novel ORFs and confirmed their transcription by RT-PCR. CONCLUSIONS: This is the first genome sequence of a herpesvirus that infects metatherians, a taxonomically unique mammalian clade. Members of the Simplexvirus genus are remarkably conserved, so the absence of ORFs otherwise retained in eutherian and avian alphaherpesviruses contributes to our understanding of the Alphaherpesvirinae. Further study of metatherian herpesvirus genetics and pathogenesis provides a unique approach to understanding herpesvirus-mammalian interactions.

Analysis of the complete genomic sequences of two virus subpopulations of the Australian infectious bronchitis virus vaccine VicS.

Although sequencing of the 3' end of the genome of Australian infectious bronchitis viruses (IBVs) has shown that their structural genes are distinct from those of IBVs found in other countries, their replicase genes have not been analysed. To examine this, the complete genomic sequences of the two subpopulations of the VicS vaccine, VicS-v and VicS-del, were determined. Compared with VicS-v, the more attenuated VicS-del strain had two non-synonymous changes in the non-structural protein 6 (nsp6), a transmembrane (TM) domain that may participate in autocatalytic release of the 3-chymotrypsin-like protease, a polymorphic difference at the end of the S2 gene, which coincided with the body transcription-regulating sequence (B-TRS) of mRNA 3 and a truncated open reading frame for a peptide encoded by gene 4 (4b). These genetic differences could be responsible for the differences between these variants in pathogenicity in vivo, and replication in vitro. Phylogenetic analysis of the whole genome showed that VicS-v and VicS-del did not cluster with strains from other countries, supporting the hypothesis that Australian IBV strains have been evolving independently for some time, and analyses of individual polymerase peptide and S glycoprotein genes suggested a distant common ancestor with no recent recombination. This study suggests the potential role of the TM domain in nsp6, the integrity of the S2 protein and the B-TRS 3, and the putative accessory protein 4b, as well as the 3' untranslated region, in the virulence and replication of IBV and has provided a better understanding of relationships between the Australian vaccine strain of IBV and those used elsewhere.