Genetic Diversity of Infectious Laryngotracheitis Virus during In Vivo Coinfection Parallels Viral Replication and Arises from Recombination Hot Spots within the Genome Carlos A. Loncoman,a Carol A. Hartley,a Mauricio J. C. Coppo,a Paola K. Vaz,a Andrés Diaz-Méndez,a Glenn F. Browning,a Maricarmen García,b Stephen Spatz,c Joanne M. Devlina 2017

Genetic Diversity of Infectious Laryngotracheitis Virus during In Vivo Coinfection Parallels Viral Replication and Arises from Recombination Hot Spots within the Genome Carlos
A. Loncoman,a Carol A. Hartley,a Mauricio J. C. Coppo,a Paola K. Vaz,a Andrés Diaz-Méndez,a Glenn F. Browning,a Maricarmen García,b Stephen Spatz,c Joanne M. Devlina
Asia-Pacific Centre for Animal Health, Melbourne Veterinary School, Faculty of Veterinary and Agricultural Sciences, University of Melbourne, Parkville, Victoria, Australiaa ; Poultry Diagnostic and Research Center, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USAb; Southeast Poultry Research Laboratory, Agricultural Research Service, United States Department of Agriculture, Athens, Georgia, USAc

ABSTRACT 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.

KEYWORDS herpesvirus, recombination, replication, diversity, SNP genotyping assay, vaccine, infectious laryngotracheitis virus (ILTV), recombination hot spot, gallid herpesvirus 1, ILTV Received 12 July 2017 Accepted 12 September 2017 Accepted manuscript posted online 22 September 2017 Citation Loncoman, CA, Hartley CA, Coppo MJC, Vaz PK, Diaz-Méndez A, Browning GF, García M, Spatz S, Devlin JM. 2017. Genetic diversity of infectious laryngotracheitis virus during in vivo coinfection parallels viral replication and arises from recombination hot spots within the genome. Appl Environ Microbiol 83:e01532-17. https://doi.org/10 .1128/AEM.01532-17. Editor Harold L. Drake, University of Bayreuth Copyright © 2017 American Society for Microbiology. All Rights Reserved. Address correspondence to Carlos A. Loncoman, cloncoman@student.unimelb.edu.au. MICROBIAL ECOLOGY crossm December 2017 Volume 83 Issue 23 e01532-17 Applied and Environmental Microbiology aem.asm.org 1 on November 16, 2017 by guest http://aem.asm.org/

Downloaded from I nfectious laryngotracheitis virus (ILTV; Gallid alphaherpesvirus 1) is an alphaherpesvirus that causes mild to severe respiratory disease in chickens. The virus causes major economic losses due to mortality and decreases in weight gain and egg production in poultry industries worldwide (1). Recombination between different strains of ILTV has been recognized as a problem for the poultry industry, particularly because natural recombination between attenuated vaccine strains of ILTV has been shown to generate virulent viruses (2). The importance of recombination as a mechanism of viral genome diversification and evolution in ILTV and other alphaherpesviruses is increasingly being recognized (3). Viruses can acquire genetic changes through several mechanisms, including point mutation and recombination, with the latter particularly important in many alphaherpesviruses (4–6). Viruses that belong to the order Herpesvirales have double-stranded linear DNA genomes and have complex viral DNA replication machinery, comprising a DNA polymerase with a highly efficient proofreading capacity, resulting in very low rates of spontaneous mutation (7–9). Therefore, rather than random changes or point mutations throughout the genome, recombination is considered to be a major evolutionary driving force enabling many alphaherpesviruses to persist, evolve and eventually become more virulent over time (2, 10, 11). Four types of recombination have been described, based on the structure of the crossover site: site-specific recombination, transposition recombination, nonhomologous or illegitimate recombination, and homologous recombination (12). Homologous and illegitimate recombination, specifically intraspecific recombination, are the most commonly described recombination mechanisms in alphaherpesviruses (12). Intraspecific recombination under in vitro conditions has been extensively described for several alphaherpesvirus species, including Human alphaherpesvirus 1, also known as herpes simplex virus 1 (HSV-1) (13), Bovine alphaherpesvirus 1, also known as bovine herpesvirus 1 (BoHV-1) (14), Human alphaherpesvirus 3, also known as varicella-zoster virus (VZV) (15), Felid alphaherpesvirus 1, also known as felid herpesvirus 1 (FeHV-1) (16) and Suid alphaherpesvirus 1, also known as pseudorabies virus (PRV) (17). Full-genome sequencing and sequence analysis are the most suitable methods for detecting and describing alphaherpesvirus recombination events that occur under natural (field) conditions, but even nowadays these techniques have their limitations, including intensive labor and costs. In contrast, under experimental conditions, other techniques to detect recombination are still more efficient and cost-effective, as well as more suitable for testing large numbers of viruses (14, 18). We have recently described the development and validation of a TaqMan single nucleotide polymorphism (SNP) genotyping assay to study recombination under experimental conditions in specific-pathogen-free (SPF) chickens (18), the natural host of ILTV. Use of the natural host to study recombination may reveal aspects of herpesvirus biology that may not be apparent in studies that utilize laboratory animal models of infection (19). The aim of this study was to apply this ILTV SNP genotyping assay to examine in vivo recombination between two ILTV strains over time. This was performed in order to describe recombination patterns and to examine genetic diversity among the viral progeny during the course of an ILTV infection. Additionally, we aimed to identify any recombination hot spots in the ILTV genome by performing full-genome sequencing of selected recombinants and analyzing them along with other ILTV genomes that were already available from different geographical regions, including Asia, Australia, Europe, and the United States. RESULTS Bird survival rates and virus genome quantification. Groups of chickens were coinoculated with the V1-99 and CSW-1 strains of ILTV at two different doses or were inoculated with only either V1-99 or CSW-1. The survival rates in groups of birds that were inoculated only with low or high doses of V1-99 ILTV were around 70% and 45% throughout the experiment, respectively (Fig. 1). Groups that received only CSW-1 had 100% survival rates throughout the experiment (Fig. 1). In the coinoculated groups, the Loncoman et al. Applied and Environmental Microbiology December 2017 Volume 83 Issue 23 e01532-17 aem.asm.org 2 on November 16, 2017 by guest http://aem.asm.org/ Downloaded from FIG 2 Replication of ILTV in SPF chickens based on genome copy numbers in tracheal swabs measured using qPCR. Medians are indicated by lines for each group. Birds were inoculated with either CSW-1 or V1-99 or coinoculated (co-inoc) with 103 PFU (low dose) (A to D) or with 104 PFU (high dose) (E to H) of the V1-99 and (Continued on next page) Loncoman et al. Applied and Environmental Microbiology December 2017 Volume 83 Issue 23 e01532-17 aem.asm.org 4 on November 16, 2017 by guest http://aem.asm.org/ Downloaded from lated with the low dose of viruses, 14, 26, 15, and 9 genotype patterns were detected at 2, 4, 6, and 8 days postinoculation, respectively (Tables 2 and 3). Some of these genotype patterns were detected consistently over time in both low- and high-doseinoculated groups. In the singly inoculated groups, no recombination was detected within the 10 viruses that were isolated and purified from each infected chicken at day 4 from the groups inoculated with the higher viral dose of 1 104 PFU/ml (see Fig. S1 in the supplemental material). Viral-diversity analysis. The higher level of viral replication in the low-dosecoinoculated group at day 4 than day 2 (Fig. 2A and B) coincided with a higher level of viral diversity, as assessed using Renyi diversity profiles (Fig. 5A) and as indicated by significantly higher values for diversity as measured by richness (0), ShannonWeaver (1), 1/Simpson (2), 1/Berger Parker (infinite [Inf]), and Hill (0.25, 0.5, 4, 8, 16, 32, and 64) scales, as well as evenness values at these time points (see Table S1 in the supplemental material). The Renyi diversity profile was higher at day 6 than at day 8 (Fig. 5B); however, the diversity measurements were not significantly different between these days (see Table S2). In the high-dose-coinoculated group, the Renyi diversity profile was higher at day 4 than at day 2 (Fig. 5C), coinciding with the higher levels of viral replication at day 4 than day 2 in this group (Fig. 2E and F). Significant differences were detected only in the Shannon-Weaver (1), 1/Simpson (2), and Hill (4) values for these days (see Table S3). Full-genome sequencing and identification of recombination hot spots. Six progeny viruses were selected for full-genome sequencing and analysis (see Table S4) in order to identify all recombination events and compare these to those detected with the SNP genotyping assay and to examine the distribution of recombination breakpoints along the genome. These recombination patterns were also searched for among 35 other full ILTV genome sequences of isolates from Asia, Australia, Europe, and the United States in order to identify recombination hot spots in ILTV from distinct geographical regions. In addition to the distribution of recombination breakpoints detected by RDP4, reticulate network and phi test analyses were performed as previously described (20) (see Fig. S2). The six SNPs targeted by the TaqMan SNP genotyping assay were confirmed by full-genome analysis. Additional events were detected outside the genome regions targeted by the TaqMan assay. The full genomes of the two genotype pattern 9 viruses shared 99.9% pairwise sequence identity. Between these two genotype pattern 9 viruses, 11 SNPs were detected within genes UL[ 1], US5, US6, US7, US8, and US9, located between bp 110000 and bp 140000 in the aligned sequences. Two SNPs were identified in the protein IF and UL49 genes located between bp 8000 to bp 18000 in the aligned sequences, and two SNPs were identified in the UL43 and UL17 genes between bp 74000 and bp 86000. When CSW-1 was used as the reference strain, a total of 55 polymorphisms were found in the six viruses sequenced in this study (see Table S5 in the supplemental material). A high number of recombination breakpoints were identified in two locations extending from bp 20000 to bp 50000 and from bp 106000 to bp 137000 in the six genome sequences from viruses isolated in this study (Fig. 6B). When the full-genome sequences from other ILTV isolates from varied geographical regions were examined, the recombination hot spots detected in those genomes were consistent with those found in the six genomes sequenced from the in vivo study (Fig. 6C, D, and E). Recombination events within genes UL[ 1] and ICP4 were detected in ILTV sequences FIG 2 Legend (Continued) CSW-1 strains of ILTV. (A and B) Low-dose-coinoculated birds F, G, H, I, and J (in the circles) had higher numbers of viral genome copies at day 4 than at day 2 (P 0.05, Mann-Whitney test). These birds did not survive to day 6. (C and D) Low-dose-coinoculated birds K, L, and M (in the rectangles) did not have significantly different numbers of viral genome copies between day 6 and day 8 (P 0.05, Mann-Whitney test). (E and F) High-dosecoinoculated birds A, B, C, D, and E (in the triangles) had higher numbers of viral genome copies at day 4 than at day 2 (P 0.05, Mann-Whitney test). (G and H) High-dose-coinoculated birds did not survive to days 6 and 8. ILTV Recombination in the Natural Host Applied and Environmental Microbiology December 2017 Volume 83 Issue 23 e01532-17 aem.asm.org 5 on November 16, 2017 by guest http://aem.asm.org/ Downloaded from FIG 3 Recombination patterns, as determined using SNP genotyping, in progeny viruses isolated 2 and 4 days after coinoculation of SPF chickens with 104 PFU of each of CSW-1 and V1-99 ILTV. Each row corresponds to a virus isolate, with the CSW-1 SNPs indicated by gray boxes and the V1-99 SNPs by black boxes. Each distinct recombination pattern was given a unique genotype code (final column). Loncoman et al. Applied and Environmental Microbiology December 2017 Volume 83 Issue 23 e01532-17 aem.asm.org 6 on November 16, 2017 by guest http://aem.asm.org/ Downloaded from from Australia and the United States and in our in vivo recombinants (Table 4). Recombination events among the Asian ILTV strains were detected at sites similar to those detected in the Australian strains, with recombination breakpoints found in genes UL4, UL3.5, UL28, UL29, UL30, UL31, UL32, UL33, and UL34 (Table 4). Only one recombination breakpoint was detected and confirmed by several methods in the ILTV sequences from Europe (Italy) (Table 4). Hence, because of the low number of recombination events in the European group, no recombination hot spots were detected. DISCUSSION Our results show that higher levels of diversity in the recombinant progeny occurred at the same time as higher levels of virus replication (as measured by viral titers). A direct relationship between viral replication and recombination diversity would have important implications for disease control, as it suggests that measures taken to reduce viral replication, such as administration of vaccines to limit virus replication following challenge (21), may be able to reduce the diversity of recombinant viruses, thus potentially reducing the capacity of ILTV to evolve to higher levels of virulence. Vaccines are used FIG 4 Recombination patterns, as determined using SNP genotyping, in progeny viruses isolated 2, 4, 6, and 8 days after coinoculation of SPF chickens with 103 PFU of each of CSW-1 and V1-99. Each row corresponds to a virus isolate, with the CSW-1 SNPs indicated by gray boxes and the V1-99 SNPs by black boxes. Each distinct recombination pattern was given a unique genotype code (final column). ILTV Recombination in the Natural Host Applied and Environmental Microbiology December 2017 Volume 83 Issue 23 e01532-17 aem.asm.org 7 on November 16, 2017 by guest http://aem.asm.org/ Downloaded from regularly in poultry industries to control ILT. As previously described, most vaccines reduce, but do not prevent, ILTV replication (as measured by viral titers) after challenge, due to cell-mediated immune responses that develop in the host in response to vaccination (21). Many vaccine efficacy studies examine ILTV replication after challenge, but some focus only on clinical signs of disease and mortality (21, 22). Furthermore, studies to meet vaccine registration requirements remain focused mainly on mortality and clinical signs of disease (23). Expanding standard vaccine characterization studies to include examination of a vaccine’s capacity to reduce viral replication after subsequent challenge (as measured by viral titers), and potentially viral recombination, would help to further control disease caused by this virus. Similar approaches could be taken for other attenuated live vaccines that are used to control alphaherpesviruses, such as FeHV-1, BoHV-1, and PRV, in veterinary medicine (24, 25) and for varicella-zoster virus (VZV) vaccines in human medicine (26), as these viruses all have capacity for recombination (3). In this study, a high proportion (67%) of viruses detected following in vivo ILTV coinoculation were recombinants. This is consistent with findings from an HSV-1 study that found that 59% of progeny viruses from the cornea and 74% of progeny viruses from the trigeminal ganglia of mice coinoculated with two strains of HSV-1 were identified as recombinants using restriction fragment length polymorphism (RFLP) analysis (13). Additionally, in vitro studies have also shown abundant recombination following alphaherpesvirus coinoculation, although lower proportions of recombinants are typically detected in cell culture than in in vivo models. For example, approximately 26% of progeny viruses obtained after coinoculation of two HSV-1 strains into cultured cells were identified as recombinants using RFLP analysis (13), and 13% of the viral progeny obtained after coinoculation of two VZV strains into cell culture were identified TABLE 1 Genotypes of viruses detected in high-dose-coinoculated (104 PFU) chickens at 2 and 4 days after coinoculationa Day 2 Day 4 Genotype pattern code Chicken(s) from which virus was isolated No. (%) of isolates Genotype pattern code Chicken(s) from which virus was isolated No. (%) of isolates 4  aFor day 2, there were a total of 42 (46.2%) parental viruses and 49 (53.8%) recombinants, for a total of 91 (100%) viruses; for day 4, there were a total of 16 (20.8%) parental viruses and 61 (79.2%) recombinants, for a total of 77 (100%) viruses. Loncoman et al. Applied and Environmental Microbiology December 2017 Volume 83 Issue 23 e01532-17 aem.asm.org 8 on November 16, 2017 by guest http://aem.asm.org/ Downloaded from as recombinants by RFLP analysis (15). Similarly, 10% to 21% of the viral progeny obtained after coinoculation of an FeHV-1 wild-type strain and two vaccine strains were identified as recombinants by restriction endonuclease digestion of PCR products (16). More recently a TaqMan SNP genotyping assay was developed to better detect TABLE 2 Genotypes of viruses detected in low-dose-coinoculated (103 PFU) chickens at 2 and 4 days after coinoculationa Day 2 Day 4 Genotype pattern code Chicken(s) from which virus was isolated No. (%) of isolates Genotype pattern code Chicken(s) from which virus was isolated No. (%) of isolates 1  there were a total of 38 (53.5%) parental viruses and 33 (46.5%) recombinants, for a total of 71 (100%) viruses; for day 4, there were a total of 29 (35.4%) parental viruses and 53 (64.6%) recombinants, for a total of 82 (100%) viruses. TABLE 3 Genotypes of viruses detected in low-dose-coinoculated (103 PFU) chickens at 6 and 8 days after coinoculationa Day 6 Day 8 Genotype pattern code Chicken(s) from which virus was isolated No. (%) of isolates Genotype pattern code Chicken(s) from which virus was isolated  aFor day 6, there were a total of 15 (34.9%) parental viruses and 28 (65.1%) recombinants, for a total of 43 (100%) viruses; for day 4, there were a total of 31 (57.4%) parental viruses and 23 (42.6%) recombinants, for a total of 54 (100%) viruses. ILTV Recombination in the Natural Host Applied and Environmental Microbiology December 2017 Volume 83 Issue 23 e01532-17 aem.asm.org 9 on November 16, 2017 by guest http://aem.asm.org/ Downloaded from TABLE 4 Recombination breakpoint analysis of full ILTV genome sequences and field isolates from Asia, Australia, and the United States and in vivo recombinants Origin of isolates Breakpoint (in alignment), 99% CIa Gene(s) at the beginning of the breakpoint locationb Gene(s) at the end of the breakpoint locationb Possible viruses involved in recombination eventc Method(s) by which breakpoint was detected in RDP4 Beginning End Asia (China) 106423–109054 125644–129391  ILTV Recombination in the Natural Host Applied and Environmental Microbiology December 2017 Volume 83 Issue 23 e01532-17 aem.asm.org 13 on November 16, 2017 by guest http://aem.asm.org/ Downloaded from TABLE 4 (Continued) Origin of isolates Breakpoint (in alignment), 99% CIa Gene(s) at the beginning of the breakpoint locationb Gene(s) at the end of the breakpoint locationb Possible viruses involved in recombination eventc Method(s) by which breakpoint was detected in RDP4 Beginning End In vivo recombinants 53623–68876 120517–121888 UL35, UL36, UL37, UL38, UL39 No gene between UL [ 1] and ICP4 R, sample 23; M, unknown; m, CSW-1 RDP, GENECONV, Bootscan, MaxChi, Chimaera, SiScan, 3SEQ 121902–124017 134435–137336 US10, SORF3 US6, US7, US8 R, sample 27; M, V1-99; m, unknown RDP, GENECONV, Bootscan, MaxChi, Chimaera, SiScan, 3SEQ 71911–78480 112397–120377 UL42, UL43, UL44, UL21, UL20 ICP4 R, sample 15; M, V1-99; m, CSW-1 RDP, GENECONV, Bootscan, MaxChi, Chimaera, SiScan, 3SEQ 121904–125713 129197–137424 US10, protein SORF3, US2, US3 US4, US5, US6, US7, US8 R, sample 15; M, V1-99; m, sample 23 GENECONV, Bootscan, 3SEQ 74976–97499 111841–114434 UL44, UL21, UL20, UL19, UL18, UL15, UL17, UL14, UL13, UL12, UL11, UL10, UL9 No gene Between UL [-1] and ICP4 R, sample 12; M, sample 1; m, V1-99 RDP, GENECONV, Bootscan, MaxChi, Chimaera, 3SEQ 2256–97499 106970–137424 Entire UL region Entire US region R, sample 12; M, unknown; m, V1-99 GENECONV, MaxChi, Chimaera 1–32193 105020–137424 UL56, protein IF, UL54, UL53, UL52, UL51, UL50, UL49.5, UL49, UL48, UL46, UL45, protein IA-IB-IC-ID-IE, UL22 Entire US region R, sample 12; M, unknown; m, V1-99 GENECONV, MaxChi, Chimaera, 3SEQ 1–1909 5870–17769 No gene UL56, protein IF, UL54, UL53, UL52, UL51, UL50 R, sample 23; M, sample 1; m, sample 12 SiScan 1–53621 107559–120122 UL56, protein IF, UL54, UL53, UL52, UL51, UL50, UL49.5, UL49, UL48, UL46, UL45, protein IA-IB-IC-ID-IE, UL22, UL24, UL25, UL26, UL26.5, UL27, UL28, UL29, UL30, UL31, UL32, UL33, UL34 Entire US region R, V1-99; M, unknown; m, sample 8 GENECONV, MaxChi, SiScan, 3SEQ 1–137424 1–137424 Entire UL region Entire UL region R, sample 12; M, CSW-1; m, sample 27 GENECONV, Bootscan, MaxChi, 3SEQ 1–113123 1–113123 Entire UL region Entire UL region R, sample 27; M, V1-99; m, sample 23 RDP, GENECONV, MaxChi, Chimaera 1–17769 19248–137424 UL56, protein IF, UL54, UL53, UL52, UL51, UL50 UL48, UL46, UL45, protein IA-IB-IC-ID-IE, UL22, UL24, UL25, UL26, UL26.5, UL27, UL28, UL29, UL30, UL31, UL32, UL33, UL34 R, CSW-1; M, sample 15; m, sample 27 MaxChi, Chimaera, 3SEQ 1–137424 1–137424 Entire UL and US regions Entire UL and US regions R, sample 8; M, sample 15; m, unknown GENECONV, MaxChi, 3SEQ 11775–17921 17923–137424 UL53, UL52, UL51, UL50, UL49.5 UL49, UL48, UL46, UL45, protein IA-IB-IC-ID-IE, UL22, UL24, UL25, UL26, UL26.5, UL27, UL28, UL29, UL30, UL31, UL32, UL33, UL34 R, sample 27; M, sample 8; m, unknown MaxChi, 3SEQ 1–137424 1–137424 Entire UL and US regions Entire UL and US regions R, sample 15; M, sample 1; m, unknown 3SEQ aCI, confidence interval. bUL, unique long; US, unique short. cR, recombinant; M, major parent; m, minor parent. Loncoman et al. Applied and Environmental Microbiology December 2017 Volume 83 Issue 23 e01532-17 aem.asm.org 14 on November 16, 2017 by guest http://aem.asm.org/
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