The Requirement of Glycoprotein C for Interindividual Spread Is Functionally Conserved within the Alphaherpesvirus Genus (Mardivirus), but Not the Host (Gallid).

Marek's disease (MD) in chickens is caused by Gallid alphaherpesvirus 2, better known as MD herpesvirus (MDV). Current vaccines do not block interindividual spread from chicken-to-chicken, therefore, understanding MDV interindividual spread provides important information for the development of potential therapies to protect against MD, while also providing a natural host to study herpesvirus dissemination. It has long been thought that glycoprotein C (gC) of alphaherpesviruses evolved with their host based on their ability to bind and inhibit complement in a species-selective manner. Here, we tested the functional importance of gC during interindividual spread and host specificity using the natural model system of MDV in chickens through classical compensation experiments. By exchanging MDV gC with another chicken alphaherpesvirus (Gallid alphaherpesvirus 1 or infectious laryngotracheitis virus; ILTV) gC, we determined that ILTV gC could not compensate for MDV gC during interindividual spread. In contrast, exchanging turkey herpesvirus (Meleagrid alphaherpesvirus 1 or HVT) gC could compensate for chicken MDV gC. Both ILTV and MDV are Gallid alphaherpesviruses; however, ILTV is a member of the Iltovirus genus, while MDV is classified as a Mardivirus along with HVT. These results suggest that gC is functionally conserved based on the virus genera (Mardivirus vs. Iltovirus) and not the host (Gallid vs. Meleagrid).

Detection of Laryngotracheitis Virus in Poultry Flocks with Respiratory Disorders in Slovenia.

Infectious laryngotracheitis (ILT) is an acute, highly contagious infectious disease of the upper respiratory tract in chickens and other poultry species that causes significant economic losses in countries worldwide. Between 2017 and 2019, seven outbreaks of mild to severe respiratory disorders with high suspicion of ILT occurred in commercial and backyard poultry flocks in Slovenia. In all submissions, infection with ILT virus (ILTV) was confirmed by PCR, which is the first report of ILT in Slovenia. Circulating ILT strains were characterized by the sequence and phylogenetic analysis of two fragments of the ICP4 gene. Four strains-three detected in non-vaccinated flocks and one in a flock vaccinated against ILT-were identical or very similar to the chicken embryo-origin live virus vaccines, and the other three were closely related to Russian, Chinese, Australian, and American field strains and to tissue culture origin vaccine strains. As in other diseases, coinfections with other respiratory pathogens in confirmed ILT cases may cause a more severe condition and prolong the course of the disease. In our study, coinfections with Mycoplasma synoviae (7/7 tested flocks), infectious bronchitis virus (5/5 tested flocks), Mycoplasma gallisepticum (4/7 tested flocks), Ornithobacterium rhinotracheale (3/4 tested flocks), and avian pox virus (1/2 tested flocks) were confirmed, indicating the importance of these pathogens in the occurrence of ILT infections.

Molecular characterization and genetic diversity of the infectious laryngotracheitis virus strains circulating in Egypt during the outbreaks of 2018 and 2019.

Infectious laryngotracheitis (ILT) is a respiratory disease that causes significant economic losses in the poultry industry worldwide. In this study, ILT outbreaks were reported on 30 farms located in eight Egyptian governorates between January 2018 and May 2019. Gross examination of diseased chickens revealed congestion and hemorrhage of laryngeal and tracheal mucosa with fibrinohemorrhagic casts and/or caseous material in the lumens. Histopathological examination showed epithelial sloughing, syncytium formation, heterophilic exudation, and development of eosinophilic intranuclear inclusion bodies. Infectious laryngotracheitis virus (ILTV) antigen was detected in the tracheal epithelium, infiltrated inflammatory cells, and syncytial cells, using immunohistochemistry. PCR targeting a portion of the thymidine kinase gene was further utilized to confirm the presence of ILTV DNA. The complete coding sequences of three envelope glycoprotein genes, gG, gD, and gJ, and a partial sequence of the infected cell polypeptide 4 (ICP4) gene from samples representing all of the farms and disease outbreaks were determined. Five prototype strains with unique sequences were chosen for detailed molecular characterization. Sequence comparisons and phylogenetic analysis of the partial ICP4 gene revealed that two strains were chicken embryo origin (CEO)-vaccine-like strains, and three were tissue culture origin (TCO)-vaccine-like strains. Analysis of the gJ gene sequence indicated that all of the strains were CEO vaccine-like strains. It was predicted that the latter three strains were recombinants of CEO- and TCO-vaccine-like strains. In conclusion, immunohistochemistry coupled with multi-genomic PCR sequencing proved to be efficient for identification and typing of ILTV strains during disease outbreaks. Both CEO-vaccine-like and recombinant virus strains were circulating in Egypt during the 2018 and 2019 outbreaks.

Full genomic characterisation of an emerging infectious laryngotracheitis virus class 7b from Australia linked to a vaccine strain revealed its identity.

Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that infects chickens, causing upper respiratory tract illness and substantial economic losses to the commercial poultry industry worldwide. Due to its geographical isolation, Australia has had a unique population of ILTV genotypes, and this has provided the researchers with an excellent opportunity to examine the evolution of herpesviruses. Recent studies on the evolution of ILTV have reported the emergence of recombinant ILTVs in Australian poultry flocks. More recently, there has been an increasing number of field outbreaks caused by ILTV isolates that are indistinguishable from serva vaccine strain using current molecular tests that rely on restriction fragment analysis of selected regions of the viral genome. In this study, whole-genome analysis of one of the field isolates revealed a new class of ILTV, identified here as class 7b, emerged as a result of recombination probably between another recombinant strain and the Serva vaccine strain (now reclassified as 7a). Interestingly, the 7b virus had the highest similarity to class 9, a virus that dominates the ILTV population in Victoria, where 7b has never been reported to date. Also, sequence analysis detected sequences unique to class 10, another recombinant virus that became predominant in some states of Australia between 2013 and 2014 but disappeared since then. These results demonstrate the influence of recombination as a continuous process towards more virulent and transmissible ILTVs.

MinION sequencing to genotype US strains of infectious laryngotracheitis virus.

Over the last decade the US broiler industry has fought long-lasting outbreaks of infectious laryngotracheitis (ILTV). Previously, nine genotypes (I-IX) of ILTVs have been recognized using the polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) method with three viral alleles (gB, gM and UL47/gG). In this study, the genotyping system was simplified to six genotypes by amplicon sequencing and examining discriminating single nucleotide polymorphisms (SNPs) within these open reading frames. Using phylogenomic analysis of 27 full genomes of ILTV, a single allele (ORF A/ORF B) was identified containing SNPs that could differentiate ILTVs into genotypes congruent with the phylogenetic partitioning. The allelic variations allowed for the cataloging of the 27 strains into 5 genotypes: vaccinal TCO, vaccinal CEO, virulent CEO-like, virulent US and virulent US backyard flocks from 1980 to 1990, correlating with the PCR-RFLP genotypes I/ II/ III (TCO), IV (CEO), V (virulent CEO-like), VI (virulent US) and VII/VIII/IX (virulent US backyard flock isolates). With the unique capabilities of third generation sequencing, we investigated the application of Oxford Nanopore MinION technology for rapid sequencing of the amplicons generated in the single-allele assay. This technology was an improvement over Sanger-based sequencing of the single allele amplicons due to a booster amplification step in the MinION sequencing protocol. Overall, there was a 90% correlation between the genotyping results of the single-allele assay and the multi-allele assay. Surveillance of emerging ILTV strains could greatly benefit from real-time amplicon sequencing using the single-allele assay and MinION sequencing. RESEARCH HIGHLIGHTS A multi-allelic assay identified nine ILTV genotypes circulating in the US Single-allele genotyping is congruent with whole genome phylogenetic partitioning US ILTV strains can be grouped into five genotypes using the single-allele assay The single-allele assay can be done using MinION sequencing of barcoded amplicons.

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.

Phylogenetic Evidence of a Close Relationship between the Peruvian Strain Vfar-043 and Two U.S. Origin Iltv Field Strains.

Infectious laryngotracheitis virus (ILTV) is the causative agent of an acute respiratory avian disease known as infectious laryngotracheitis (ILT), which has been associated with economic losses in poultry. The presence of ILTV has been widely reported in South American countries; however, only one full genomic sequence (VFAR-043 strain) has been recently published, from an outbreak in Peru. The aim of this study was to determine the genetic relationship of the Peruvian strain with other ILTV strains from different geographic regions. The phylogenetic analyses revealed a close relationship between VFAR-043 and two U.S. origin strains (1874C5 and J2) using only the whole genome, Unique Long (UL), and Unique Short (US) genomic regions. Then these three genomic sequences were compared to evaluate their genetic variations using the USDAref as a reference strain. Genetic variations such as synonymous and nonsynonymous single-nucleotide polymorphisms, insertions, deletions, and nucleotide-codon variations were identified among these three strains. Moreover, the phylogenetic tree analysis using gene sequences of the US5 and ICP4 coding regions from South American isolates showed that VFAR-043 does not have a close relationship with either the Argentinian (US5) or Brazilian (ICP4) reported sequences. However, a close relationship was observed between VFAR-043 and another Peruvian isolate (USP-81) when the ICP4 gene sequence was analyzed. All these results suggest that VFAR-043 together with 1874C5 and J2 are closely related. These findings contribute to our understanding of the epidemiology of ILTV in South America.

Evidencia filogenética de una relación genética cercana entre la cepa peruana del virus de la laringotraqueítis infecciosa aviar VFAR-043 y dos cepas de campo con origen en los Estados Unidos. El virus de laringotraqueítis infecciosa aviar es el agente causal de una enfermedad aviar respiratoria aguda conocida como laringotraqueítis infecciosa que está asociada con pérdidas económicas en la industria avícola. La presencia del virus de la laringotraqueítis infecciosa ha sido ampliamente reportada en países de América del Sur; sin embargo, solamente una secuencia genómica completa (cepa VFAR-043) ha sido publicada recientemente y obtenida a partir de un brote en Perú. El objetivo de este estudio fue determinar la relación genética de la cepa peruana con otras cepas de diferentes regiones geográficas. El análisis filogenético reveló una cercana relación entre el virus VFAR-043 y dos cepas de los Estados Unidos (18746C5 y J2) usando el genoma completo y las regiones genómicas única larga (UL) y la región genómica única corta (UC). Posteriormente, estas tres secuencias genómicas fueron comparadas con la cepa de referencia del Departamento de Agricultura de los Estados Unidos (USDAref) para evaluar sus variaciones genéticas. Variaciones genéticas como polimorfismo de nucleótido único (con las siglas en inglés SNP) tanto de tipo sinónimo y no-sinónimo, inserciones, deleciones y variación de nucleótido en un codón fueron identificadas entre estas tres cepas (VFAR-043, 18746C5 y J2). Además, el análisis de los árboles filogenéticos usando secuencias genéticas de la región codificadora de US5 e ICP4 de aislamiento sudamericanos reveló que el virus VFAR-043 no mostró relación genética cercana con secuencias argentinas (US5) ni secuencias brasileras (ICP4) que están reportadas. No obstante, se observó una relación cercana entre el virus VFAR-043 y otro aislamiento peruano (USP-81) cuando se analizó la secuencia genética del gen ICP4. Todos estos resultados sugieren que los virus VFAR-043, 1874C5 y J2 están genéticamente relacionados. Estos hallazgos contribuyen al conocimiento de la epidemiologia del virus de la laringotraqueítis infecciosa aviar en América del Sur.

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.

Esophagitis and Pharyngitis Associated with Avian Infectious Laryngotracheitis in Backyard Chickens: Two Cases.

Infectious laryngotracheitis (ILT) is a contagious viral respiratory disease of great economic importance for the global poultry industry caused by Gallid herpesvirus 1 (GaHV-1). Lesions of the upper digestive tract caused by this virus have not been reported before. Two small flocks of backyard chickens experienced an outbreak of ILT, one in 2006 and the other in 2014. These birds had typical ILT lesions, characterized by a necrohemorrhagic laryngitis and tracheitis but were also affected by a severe erosive and necrotic esophagitis and pharyngitis. On microscopic examination of the esophagus and pharynx, numerous individual epithelial cells were degenerated or necrotic. Syncytial cells were present in the mucosa or sloughed in the overlying inflammatory crust, and some of these cells contained an amphophilic intranuclear viral inclusion. GaHV-1 was detected in tissues, from respiratory and digestive tracts, either by PCR, immunohistochemistry, or both diagnostic assays. This case stresses the importance for veterinarians, owners, and technicians to pay attention to different or atypical clinical manifestations of ILT given its highly contagious nature.

Development and application of a TaqMan single nucleotide polymorphism genotyping assay to study infectious laryngotracheitis virus recombination in the natural host.

To date, recombination between different strains of the avian alphaherpesvirus infectious laryngotracheitis virus (ILTV) has only been detected in field samples using full genome sequencing and sequence analysis. These previous studies have revealed that natural recombination is widespread in ILTV and have demonstrated that recombination between two attenuated ILTV vaccine strains generated highly virulent viruses that produced widespread disease within poultry flocks in Australia. In order to better understand ILTV recombination, this study developed a TaqMan single nucleotide polymorphism (SNP) genotyping assay to detect recombination between two field strains of ILTV (CSW-1 and V1-99 ILTV) under experimental conditions. Following in vivo co-inoculation of these two ILTV strains in specific pathogen free (SPF) chickens, recovered viruses were plaque purified and subjected to the SNP genotyping assay. This assay revealed ILTV recombinants in all co-inoculated chickens. In total 64/87 (74%) of the recovered viruses were recombinants and 23 different recombination patterns were detected, with some of them occurring more frequently than others. The results from this study demonstrate that the TaqMan SNP genotyping assay is a useful tool to study recombination in ILTV and also show that recombination occurs frequently during experimental co-infection with ILTV in SPF chickens. This tool, when used to assess ILTV recombination in the natural host, has the potential to greatly contribute to our understanding of alphaherpesvirus recombination.

Natural recombination in alphaherpesviruses: Insights into viral evolution through full genome sequencing and sequence analysis.

Recombination in alphaherpesviruses was first described more than sixty years ago. Since then, different techniques have been used to detect recombination in natural (field) and experimental settings. Over the last ten years, next-generation sequencing (NGS) technologies and bioinformatic analyses have greatly increased the accuracy of recombination detection, particularly in field settings, thus contributing greatly to the study of natural alphaherpesvirus recombination in both human and veterinary medicine. Such studies have highlighted the important role that natural recombination plays in the evolution of many alphaherpesviruses. These studies have also shown that recombination can be a safety concern for attenuated alphaherpesvirus vaccines, particularly in veterinary medicine where such vaccines are used extensively, but also potentially in human medicine where attenuated varicella zoster virus vaccines are in use. This review focuses on the contributions that NGS and sequence analysis have made over the last ten years to our understanding of recombination in mammalian and avian alphaherpesviruses, with particular focus on attenuated live vaccine use.

Spread of the newly emerging infectious laryngotracheitis viruses in Australia.

Infectious laryngotracheitis (ILT) is a significant viral disease of chickens in many countries around the globe. In this report the status of ILT in Australia has been used as a model to evaluate the evolution of the ILT viruses (ILTVs). Due to its geographical isolation, Australia harbored a distinct lineage of ILT viruses (ILTV) up to 2007. However examination of the ILT viruses (ILTV) involved in outbreaks between 2007 and 2009 has revealed that many of the outbreaks were caused by two new viral genotypes, class 8 and class 9. These two recombinant viruses were found to emerge as a result of recombination between previously existing live vaccine strains (SA2 and A20), and another live vaccine strain (Serva) introduced into the country in 2007. The new recombinant ILTVs were also shown to possess significantly higher virulence and replication capacity compared with a previously predominant ILTV, class 2. In the current study, examination of a large number of ILTVs isolated from outbreaks between 2009 and 2015 revealed the emergence of yet another recombinant virus (class 10) that appears to have become a predominant genotype in New South Wales. In Victoria however, the recombinant class 9 gradually became the predominant virus, replacing class 2. Therefore, there was an unusual pattern in geographical spread of the newly emerged viruses in different states of the country. These results suggest that ILTV is fast evolving towards a greater transmissibility and therefore greater capacity to spread into ILTV-free areas.

Genotyping of infectious laryngotracheitis virus using allelic variations from multiple genomic regions.

Live attenuated vaccines are extensively used worldwide to control the outbreak of infectious laryngotracheitis. Virulent field strains showing close genetic relationship with the infectious laryngotracheitis virus (ILTV) vaccines of chicken embryo origin have been detected in the poultry industry. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, a reliable molecular epidemiological method, of multiple genomic regions was performed. The PCR-RFLP is a time-consuming method that requires considerable amount of intact viral genomic DNA to amplify genomic regions greater than 4 kb. In this study, six variable genomic regions were selected and amplified for sequencing. The multi-allelic PCR-sequence genotyping showed better discrimination power than that of previous PCR-sequencing schemes using single or two target regions. The allelic variation patterns yielded 16 strains of ILTV classified into 14 different genotypes. Three Korean field strains, 550/05/Ko, 0010/05/Ko and 40032/08/Ko, were found to have the same genotype as the commercial vaccine strain, Laryngo Vac (Zoetis, Florham Park, NJ, USA). Three other Korean field strains, 40798/10/Ko, 12/07/Ko, and 30678/14/Ko, showed recombined allelic patterns. The multi-allelic PCR-sequencing method was proved to be an efficient and practical procedure to classify the different strains of ILTV. The method could serve as an alternate diagnostic and differentiating tool for the classification of ILTV, and contribute to understanding of the epidemiology of the disease at a global level.

Infection of Broilers with Two Virulent Strains of Infectious Laryngotracheitis Virus: Criteria for Evaluation of Experimental Infections.

Infectious laryngotracheitis (ILT) is a highly contagious disease of chickens and is responsible for significant economic losses in the poultry industry worldwide; it is caused by Gallid herpesvirus-1 (GaHV-1), commonly known as infectious laryngotracheitis virus (ILTV). Experimental evaluation of ILTV strains is fundamental to identify changes in virulence that can contribute to the severity and spread of outbreaks and consequently influence the efficacy of vaccination. Several criteria had been utilized to determine the degree of virulence associated with ILTV strains. The objectives of this study were to compare the levels of virulence of the standard United States Department of Agriculture (USDA) challenge strain with a contemporary outbreak-related strain (63140) and to evaluate the efficacy of individual criteria to identify changes in virulence. Broilers were inoculated with increasing infectious doses of each strain. The criteria utilized to evaluate virulence were clinical signs of the disease, mortality, microscopic tracheal lesions, trachea genome viral loads, and antibody titers. Clinical signs scores were a useful parameter to define the peak of clinical disease but did not reveal differences in virulence between strains. Similarly, trachea microscopic lesion scores or levels of serum antibody titers were parameters that did not reveal obvious differences in virulence between strains. However, mortalities and increased viral genome loads in trachea of chickens inoculated with lower (log10 1 to 2) infectious doses clearly differentiated 63140 as a more-virulent ILTV strain. This study provides the framework to compare the virulence level of emerging ILTV isolates to the now-characterized USDA and 63140 strains.

Real-time PCR quantification of infectious laryngotracheitis virus in chicken tissues, faeces, isolator-dust and bedding material over 28 days following infection reveals high levels in faeces and dust.

Infectious laryngotracheitis (ILT) is an important disease of chickens caused by ILT virus (ILTV). We used the Australian SA2 and A20 vaccine strains of ILTV to determine tissue distribution and excretion characteristics of ILTV in specific-pathogen-free chickens and to determine whether ILTV is readily detectable in environmental samples such as faeces, bedding material and dust using real-time quantitative PCR. Three groups of 10 freshly hatched chicks were placed in isolators and infected orally with high doses of the two strains of vaccine virus or left unchallenged as controls. Over a 28-day post-infection (p.i.) period, faecal and serum samples were collected at frequent intervals from six individually identified chickens in each group. Dust and litter samples from the isolators were collected less frequently. Tissue samples were collected from three to four sacrificed or dead/euthanized birds at 6, 14 and 28 days p.i. Infection resulted in clinical ILT, a pronounced antibody response and sustained qPCR detection of the viral genome in the trachea, Harderian gland, lung and kidney up to 28 days p.i. A high level of the viral genome was also detected in faeces between 2 and 7 days p.i., declining by about approximately four orders of magnitude to low, but detectable, levels at 21 and 28 days p.i. The finding of high-level shedding of ILTV in faeces warrants further investigation into the epidemiological role of this, and the sustained high levels of ILTV observed in dust suggest that it may be a useful sample material for monitoring ILTV status in flocks.

Growth kinetics and transmission potential of existing and emerging field strains of infectious laryngotracheitis virus.

Attenuated live infectious laryngotracheitis virus (ILTV) vaccines are widely used in the poultry industry to control outbreaks of disease. Natural recombination between commercial ILTV vaccines has resulted in virulent recombinant viruses that cause severe disease, and that have now emerged as the dominant field strains in important poultry producing regions in Australia. Genotype analysis using PCR-restriction fragment length polymorphism has shown one recombinant virus (class 9) has largely replaced the previously dominant class 2 field strain. To examine potential reasons for this displacement we compared the growth kinetics and transmission potential of class 2 and class 9 viruses. The class 9 ILTV grew to higher titres in cell culture and embryonated eggs, but no differences were observed in entry kinetics or egress into the allantoic fluid from the chorioallantoic membrane. In vivo studies showed that birds inoculated with class 9 ILTV had more severe tracheal pathology and greater weight loss than those inoculated with the class 2 virus. Consistent with the predominance of class 9 field strains, birds inoculated with 10(2) or 10(3) plaque forming units of class 9 ILTV consistently transmitted virus to in-contact birds, whereas this could only be seen in birds inoculated with 10(4) PFU of the class 2 virus. Taken together, the improved growth kinetics and transmission potential of the class 9 virus is consistent with improved fitness of the recombinant virus over the previously dominant field strain.

Spatial and temporal epidemiology of infectious laryngotracheitis in central California: 2000-2012.

In October of 2005 an outbreak of a vaccine-like strain of infectious laryngotracheitis (ILT), indistinguishable from the chicken embryo origin (CEO)-like vaccine strains, was detected by routine passive surveillance in the Central Valley of California, U. S. A. In response, a highly coordinated industry effort by two companies led to a significant decrease in the incidence of ILT over the same geographic region between 2008-2012. In order to understand the geographic and temporal spread of ILT in California before and after the outbreak, Global Information Systems (GIS) mapping coupled with spatial, temporal, and spatial- temporal statistics were used to identify retrospective and prospective low-rate clustering (i.e., less ILT than statistically expected) and high-rate clustering (i.e., more ILT than statistically expected) of ILT spatially and temporally. Results showed two high-rate retrospective spatial-temporal clusters and one low-rate prospective spatial-temporal cluster which were all statistically significant (P < 0.05). Overall, spatial-temporal clustering accounted for 36.9% of the positive ILT cases, while temporal clustering and spatial clustering done separately each accounted for 0% of the ILT cases, respectively. This demonstrates the utility of combining spatial and temporal clustering for ILT surveillance. Due to the risk of reversion to virulence and spread to immunologically naive broilers, future application of the CEO-based vaccine in the identified high rate spatial-temporal clusters should be avoided and other vaccine alternatives considered in order to avoid repeat outbreaks in those areas. This should especially be followed during the winter months of December, January, and February, which were found to have the highest prevalence of ILT (P < 0.05). Analysis of GIS data within the high-rate clusters showed that wind direction and farm density were minor factors in the spread of ILT. Shared roads may have played a role in the spread of ILT in one of the two high rate spatial-temporal clusters.

Chicken embryo origin-like strains are responsible for Infectious laryngotracheitis virus outbreaks in Egyptian cross-bred broiler chickens.

Infectious laryngotracheitis virus (ILTV) continues to cause respiratory disease in Egypt in spite of vaccination. The currently available modified live ILTV vaccines provide good protection but may also induce latent infections and even clinical disease if they spread extensively from bird-to-bird in the field. Four field ILTV isolates, designated ILT-Behera2007, ILT-Giza2007, ILT-Behera2009, and ILT-Behera2010 were isolated from cross-bred broiler chickens. The pathogenicity based on intratracheal pathogenicity index, tracheal lesion score, and mortality index for chicken embryos revealed that ILT-Behera2007, ILT-Behera2009 and ILT-Behera2010 isolates were highly pathogenic whereas ILT-Giza2007 was non-pathogenic. To study the molecular epidemiology of these field isolates, the infected cell protein 4 gene was amplified and sequenced. Phylogenetic analysis revealed that ILT-Behera2007, ILT-Behera2009, and ILT-Behera2010 are chicken embryo origin (CEO) vaccine-related isolates while ILT-Giza2007 is a tissue culture origin vaccine-related isolate. These results suggest that CEO laryngotracheitis vaccine viruses could increase in virulence after bird-to-bird passages causing severe outbreaks in susceptible birds.

Detection and phylogenetic characterization of a novel herpesvirus from the trachea of two stranded Common Loons (Gavia immer).

Two wild adult Common Loons (Gavia immer) were evaluated after being found stranded in mainland north-central Florida on separate occasions. On the basis of upper airway endoscopic and cytologic findings, we diagnosed severe ulcerative tracheitis antemortem in one of the birds while more subtle lesions were observed in the other. A novel herpesvirus was detected in antemortem tracheal samples using nested consensus PCR amplification of the polymerase gene and sequencing. Despite prolonged intensive medical care, the bird with severe lesions failed to improve and was euthanized 9 days after endoscopy. No viral inclusions were evident histologically in the lesions. However, an undulating tracheal mucosa in a "mountain ridge" pattern, resulting from epithelial regeneration and hyperplasia, was present, as is seen in the late stages of infectious laryngotracheitis in chickens. The second bird recovered and was released. The genetic distance between this and other characterized herpesviruses supports placement of this virus as a novel species, referred to as Gaviid herpesvirus 1 (GavHV1). Phylogenetically, GavHV1 clusters within the genus Iltovirus. The relationship between the observed lesions and the virus remains to be demonstrated.

Differentiation of field isolates and vaccine strains of infectious laryngotracheitis virus by DNA sequencing.

Two different regions of the infected cell protein 4 (ICP4) gene of infectious laryngotracheitis virus (ILTV) were amplified and sequenced for characterization of field isolates and tissue culture-origin (TCO) and chicken embryo-origin (CEO) vaccine strains. Phylogenetic analysis of the two regions showed differences in nucleotide and amino acid sequences between field isolates and attenuated vaccines. The PCR-RFLP results were identical to those obtained by DNA sequencing and validated their use to differentiate ILTV strains. The approach using the sequencing of the two fragments of the ICP4 gene showed to be an efficient and practical procedure to differentiate between field isolates and vaccine strains of ILTV.