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
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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
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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)
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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.
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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).
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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).
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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.
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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.
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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
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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.
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SUPPLEMENTAL MATERIAL
Supplemental material for this article may be found at https://doi.org/10.1128/AEM
.01532-17.
SUPPLEMENTAL FILE 1, PDF file, 2.5 MB.
ACKNOWLEDGMENTS
We gratefully acknowledge Mahathi Nadimpalli, Nino Ficorilli, and Mesula Korsa for
their help with cell culture and Jose Quinteros, June Daly, Jenece Wheeler, and Cheryl
Colson for their help with the in vivo studies.
This work was supported by the Australian Research Council (FT140101287). Carlos
A. Loncoman is supported by Becas Chile, CONICYT, Gobierno de Chile.
The funders had no role in study design, data collection and interpretation, or the
decision to submit the work for publication.
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viernes, 17 de noviembre de 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 2017
viernes, 22 de septiembre de 2017
Identification of a divergent genotype of equine arteritis virus from South American donkeys J. Rivas1 | V. Neira2* | J. Mena2 | B. Brito2 | A. Garcia3 | C. Gutierrez3 | D. Sandoval1 | R. Ortega1* 2017
Identification of a divergent genotype of
equine arteritis virus from South American donkeys
J. Rivas1 | V. Neira2*
| J. Mena2 | B. Brito2 | A. Garcia3 | C. Gutierrez3 |
D. Sandoval1 | R. Ortega1*
1Facultad de Ciencias
Veterinarias, Departamento de
patolog ıa y medicina preventiva, Universidad
de Concepcion, Chill an, Chile
2Facultad de Ciencias
Veterinarias y Pecuarias, Universidad
de Chile, Santiago, Chile
3Laboratorio y Estacio n Cuarentenaria
Pecuaria, Complejo Lo Aguirre, Servicio Agr ıcola y Ganadero, Santiago, Chile
Correspondence
V. Neira, Facultad de Ciencias Veterinarias y Pecuarias, Universidad
de Chile, Santiago, Chile and R. Ortega,
Facultad
de Ciencias
Veterinarias, Departamento de patolog ıa y medicina preventiva,
Universidad
de Concepcion , Chill an, Chile.
Emails: victorneira@u.uchile.cl;
Present address
B. Brito, Foreign Animal Disease Research
Unit, Plum Island
Animal Disease Center,
ARS, USDA, NY, USA
Summary
|
A novel
equine
arteritis
virus (EAV) was isolated and
sequenced from feral donkeys
in Chile. Phylogenetic analysis indicates that
the
new
virus
and
South
African asi- nine
strains
diverged
at
least
100
years
from
equine
EAV strains. The
results indi-
cate that asinine strains belonged
to a different EAV genotype.
KEY W
ORD S
donkey,
equine,
equine
arteritis
virus, equine viral
arteritis
1 | INTRODUCTI ON
Equine viral arteritis (EVA) is a viral disease in equids, namely horses, donkeys, mules and zebras. The
causative agent is the
equine
arteri- tis
virus
(EAV), genus Equartevirus from
the
Arteriviridae family (Adams et al., 2017).
EAV
strains have been classified based on the ORF5 phylogeny
into three genotypes, the
North
American (NA) and the European 1
(EU1) and 2 (EU2) lineages (Zhang
et al., 2007).
Clinical disease is characterized by
fever and respiratory symp- toms; however, economic losses
are mostly
due to its ability
to cause abortion in mares and
severe disease or death in young
foals (Balasuriya, Go, & MacLachlan, 2013).
EAV increased global reporting during more recent years has been attributed
to more frequent international horse
movement (Dominguez,
Mu€nstermann, de Guin- dos,
& Timoney, 2016). EVA is not only transmitted through
direct
*These authors should
be considered joint senior authors.
contact during
clinical respiratory disease, but it can also
be trans-
mitted through the
venereal route. Stallions can become persistently
infected (carriers) and transmit the disease
during
breeding
(Guthrie et al., 2003).
2
| MATERIAL S A ND METHODS
In Chile, the EAV has not been detected in horses. In 2013,
during surveillance activities, samples collected from feral
donkeys
ranging in
small herds in hills and
plains nearby the Atacama Desert were positive to
neutralizing antibodies against EAV (Moreira, Garc ıa,
Valencia, & Moreno,
2016).
Following results from this study,
two male
adult donkeys, clinically healthy,
were
captured in the annual
rodeo event in October 2013. The rodeo
was conducted at Car- rizalillo, Freirina,
Chile
( 29.099469, 71.406169). Donkeys were
sent
to a slaughterhouse for human consumption.
Transbound Emerg Dis. 2017;1–6.
wileyonlinelibrary.com/journal/tbed © 2017
Blackwell Verlag GmbH | 1
2.1 | Sample
collection
Tissue samples
including
heart, lung, kidney, testes, vas deferens, epididymis, prostate and seminal vesicle
were
collected.
One
gram
from each organ was scraped and homogenized with
10 ml of mini- mum essential media (MEM).
The mix was centrifuged at 2,823 g for
20 min, and
the
supernatant was
used
for
RT-PCR and virus isola-
tion.
|
(a)
(b)
FI GU RE 1 Cytopathogenic effects
of RK-13
cells: (a) RK-13 cells mock-infected
at 7 days post-inoculation. (b) RK-13 cells infected with Atacama-2014 equine arteritis virus (EAV) isolate at 7 days post-
inoculation
European 1
European 2
North American
Asinine lineage
U38593.1/Horse/AZ87/Arizona-U.S.A/1987
AF099839.1/Horse/S-436/Poland/1988
AF099850.1/Horse/S1512/U.S.A/1995
EF102379.1/Horse/PLP00-1/Lesser_Poland-Poland/2000
EF102382.1/Horse/PLP02-4/Lesser_Poland-Poland/2002
AY453313.1/Horse/I16/Italy/1995
EF492547.1/Horse/F9/Loire-France/2002
AF099830.1/Horse/3308V-96/Italy/1995
AY359193.1/Horse/H1S/Bekes-Hungary/2001
AY359209.1/Horse/H20F/Heves-Hungary/2000
AY359208.1/Horse/H19F/Pest-Hungary/2000
EF102355.1/Horse/PLH05-1/Silesian-Poland/2005
AY359207.1/Horse/H269S/Zala-Hungary/2003
U38592.1/Horse/AUT68/Vienna-Austria/1968
AF099813.1/Horse/S-1128/Canada/1992
U46952.1/Horse/Vienna/Vienna-Austria/1968
AF099811.1/Horse/Vienna/Austria/1964
EF492558.1/Horse/F20/Ile_de_France-France/2001
AF099814.1/Horse/EAV-86-R/France/1986
GQ903863.1/Horse/S4222/California-U.S.A/2008
AY359197.1/Horse/H147S/Heves-Humgary/2001
AF099815.1/Horse/EAV-86-P/France/1986
AF099823.1/Horse/1192VE4-91/Italy/1990
AF099824.1/Horse/135VE2-95/Italy/1994
AF099838.1/Horse/Wroclaw-2/Poland/1978
AY359201.1/Horse/H197S/Fejer-Hungary/2002
AF099812.1/Horse/Fallat/Canada/1986
U46949.1/Horse/19933/Ontario-Canada/1992
U38594.1/Horse/CAN86/Alberta-Canada/1986
U46948.1/Horse/11958/Ontario-Canada/1990
AF099835.1/Horse/NEAV-1/Norway/1988
AF099836.1/Horse/NEAV-2/Norway/1989
AY359199.1/Horse/H172S/Komarom-Esztergom-Hungary/2002
U38607.1/Horse/PA76/Pennsylvania-U.S.A/1976
EF492549.1/Horse/F11/Ile_de_France-France/2004
EF492543.1/Horse/F5/Basse_Normandie-France/2004
EF492562.1/Horse/F24/Basse_Normandie-France/2004
EF492551.1/Horse/F13/Basse_Normandie-France/2004
JX868590.1/Horse/EAV_HSY/China/2011
U38608.1/Horse/PLD76/Wroclaw-Poland/1976
AF099849.1/Horse/TAQ/U.S.A/1994
U38611.1/Horse/VBS53/Bucyrus-Ohio-U.S.A/1953
AF099842.1/Horse/S-2506/Sweden/1989
U38609.1/Horse/SWZ64/Bibuna-Switzerland/1964
AF099843.1/Horse/Bibuna/Switzerland/1964
AF099828.1/Horse/1330VE-95/Italy/1995
AF099825.1/Horse/470VE1-95/Italy/1994
AF099826.1/Horse/73VE2-95/Italy/1994
AY453340.1/Horse/S2/Sweden/1999
AF099832.1/Horse/1908V-97/Italy/1996
AY349167.1/Horse/CW96/U.S.A/1996
AF118783.1/Horse/CA97/California-U.S.A/1997
AY349168.1/Horse/CW01/U.S.A/2001
AF099816.1/Horse/EAV-86-110/France/1986
AF099822.1/Horse/D526/Germany/1996
AY453344.1/Horse/S6/Sweden/2000
AY453278.1/Horse/A4/Austria/1998
* *
AY359203.1/Horse/H213S/Hungary/1999
AY359202.1/Horse/H204S/Heves-Hungary/1994
AY359204.1/Horse/H215S/Zala-Hugary/2000AY359210.1/Horse/H72F/Zala-Hungary/1998
AY359200.1/Horse/H189S/Heves-Hungary/2002
AY453335.1/Horse/RSA4/R.S.A/1996
LC000003.1/Horse/GB_Glos_2012/Gloucestershire-U.K/2012
U38599.1/Horse/KY63/Kentucky-U.S.A/1963
AY956598.1/Donkey/J3-931209/R.S.A/1993
AY956601.1/Donkey/J6-940309/R.S.A/1994
AY956597.1/Donkey/J2-931125/R.S.A/1993
AY956600.1/Donkey/J5-940309/R.S.A/1994
AY956599.1/Donkey/J4-931209/R.S.A/1993
*
Donkey/Atacama-2014/Chile/2014
–700 –600 –500 –400 –300 –200 –100 0
FI GU RE 2 Maximum clade credibility collapsed tree of equine
arteritis
virus using
170 ORF5
reference sequences. The Atacama-2014 and
South African Donkey sequences belong
to a single monophyletic group, the asinine
cluster (red).
The time to most
recent common
ancestor (tMRCA) of the asisine cluster with
other equine arteritis virus (EAV) sequences and the tMRCA
of Atacama-2014 with the closest reference are indicated with * and **, respectively
2.2
| RT-PCR
RNA was extracted using the commercial kit MagMAXTM-96 AI/ND Viral
RNA Isolation Kit (Ambion Cat# AM1835, Austin, TX, USA).
ORF6 and ORF7 were amplified by RT-PCR
using the protocols rec- ommended by the World Organisation for Animal Health (Timoney,
2012). ORF5
was
amplified
using
the
primers
and
protocols
previ- ously described (Stadejek et
al., 1999). PCR products were
submitted for Sanger sequencing. The positive samples were selected for virus isolation.
2.3
| Viral isolation
Viral isolation was attempted in
RT-PCR-positive samples.
First, monolayers of
RK-13
cells
(ATCC CCL-37TM) were grown in 12- well plates
with cell growth media,
which includes minimum essential
medium Eagle’s (MEM),
supplemented with 10% foetal
bovine serum,
10,000 IU/ml penicillin
(1%), 10,000 lg/ml streptomycin (1%) and
25 lg/ml amphotericin B (1%). Monolayers with 80%
of cell conflu- ence were
inoculated with
200
ll of filtrated positive samples
and
incubated for 1 hr
at 37°C and 5% CO2. After the incubation, the
inoculum was discarded and cells were incubated for 10 days using cell growth
medium
previously described. The
monolayers were observed daily during 10 days
inspecting for evidence of cytopatho- genic effects. Positive cultures were
tested by
RT-PCR to confirm
the presence of
the EAV.
2.4 | Phylogeny
ORF5 was used
to reconstruct the EAV phylogeny. ORF5 is the most
variable
region
of the virus
and commonly
used
for EAV phy- logeny.
ORF5 sequence generated for this study and
reference
sequences covering the known spectrum of
ORF5 genetic diversity were aligned using MUSCLE
(Edgar, 2004).The codon
partition
and nucleotide
substitution
model was selected
using
partition finder
based on the Bayesian information criterion
(BIC) (Lanfear,
Calcott, Ho, & Guindon, 2012). The best scheme
consisted in one partition
for each codon position: HKY+I+G for codon
position
1,
TrN+I+G for codon
position
2 and
GTR+I+G for codon position 3. We used a Bayesian approach for time divergence estimation
implemented in BEAST 1.8.2 (Drummond &
Rambaut, 2007). Initially, a strict clock
model and an uncorrelated relaxed
lognormal
clock, in combination
GQ903859/2007/USA/S3901
GQ903809/2005/USA/S3583
GQ903901/2005/USA/M405
GQ903811/2008/USA/S4216
EF492559/2002/France/F21
AF118780/1998/USA/G4
AF118777/1995/USA/G1
AF118779/1997/USA/G3
AF118778/1997/USA/G2
AF118782/1996/USA/RQ
AF118781/1996/USA/BT-PA96
AF118776/1997/USA/P2
AF118775/1996/USA/P1
AF118774/1998/USA/R2
AF118773/1996/USA/R1
AF118769/1995/USA/A1
AF118770/1996/USA/A2
AF118771/1996/USA/A3
AF118772/1997/USA/A4
EF492556/2001/France/F18
EF492557/2001/France/F19
EF492555/2001/France/F17
EF492560/2002/France/F22
EF492554/2001/France/F16
EF492561/2002/France/F23
EF492548/2003/France/F10
EF492553/2001/France/F15
JN211317/2000/France/F60
JN211316/2007/France/F27
AY349167/1996/USA/CW96
AY349168/2001/USA/CW01
European 1
European 2
North American
Atacama-2014
HK25
Hela-EAVP35
EF492564/2004/France/F26
EF492563/2004/France/F25
EF492562/2004/France/F24
U81013/1953/USA/VBS53
U81020/NA/USA/ATCC
EF492552/2003/France/F14
EF492550/2004/France/F12
EF492549/2004/France/F11
EF492545/2004/France/F7
EF492544/2004/France/F6
U81019/NA/USA/ARVAC
pEAVrMLV
EF492543/2004/France/F5
EF492546/2004/France/F8
EF492551/2004/France/F13
DQ846750/1953/USA/Bucyrus
U81021/1986/Canada/CAN86
U81018/1976/USA/PA76
AH007128/1999/USA/CA95G
GQ903862/2007/USA/S3955
AF107267/1999/USA/D85
AF107271/1999/USA/D89
AF107269/1999/USA/D87
AF107270/1999/USA/D88
AF107273/1999/USA/D92
AF107274/1999/USA/D94
AF107272/1999/USA/D91
AF107276/1999/USA/E86
AF107268/1999/USA/D86
AF107277/1999/USA/E88
AF107278/1999/USA/E89
AH007129/1999/USA/CA95I
U81015/1977/USA/KY77
U81017/1993/USA/KY93
AF107275/1999/USA/E85
AF107279/1984/USA/KY84
AF107266/1999/USA/D84
Donkey/Atacama-2014/Chile/2014
0.08
FI GU RE 3 Maximum likelihood ORF6 phylogenetic tree using 93 equine
arteritis
virus reference sequences. Atacama-2014 isolate is a singleton genetically
distant
from reference sequences
coloured in red
with a constant population and a Bayesian skyline
tree prior, were run. We selected
the model
based
upon
the
AICm method-of-
moments estimator (Baele et al., 2012;
Raftery,
Newton, Satagopan,
& Krivitsky, 2007) implemented
in Tracer v1.6 (Rambaut, Suchard,
Xie, & Drummond,
2014).
Based
on the lower AICm,
the uncorre-
lated exponential clock model and
a coalescent constant
population tree prior
were selected. The analysis
was run for 200,000 iterations.
Convergence and mixing of the
simulations
was assessed using Tra- cer. The maximum clade
credibility tree (MCC) was visualized in Fig-
tree version 1.4.2
(Rambaut, 2014).
Additionally, phylogenetic analyses of ORF6 and ORF7, which are more conserved genes, were performed using
MUSCLE for sequence alignment and maximum
likelihood to reconstruct the
phylogeny
in MEGA7
(Kumar, Stecher,
& Tamura, 2016).
3 | RESULTS A ND DI SCUSSI ON
Viral RNA was identified in samples from both animals. From a vas deferens sample, cytopathogenic effects (CPE)
were observed at
5 days
post-inoculation. CPE was characterized by rounding of cells and cell detachment from the monolayer (Figure 1). The isolated
virus was named Atacama-2014.
Viral RNA was
amplified,
and
partial
sequences of ORF5 (630 nucleotides- nt), ORF6 (193 nt) and ORF7 (316 nt) were obtained
by Sanger sequencing
method (Accession numbers MF543058, MF543059 and MF573786, respectively).
The
phylogeny
of
ORF5 revealed that the Atacama-2014 belonged to a monophyletic clus- ter that
included viruses
collected from
donkeys
in 1993–1994
samples (Stadejek, Mittelholzer, Oleksiewicz, Paweska, & Belak,
GQ903809/2005/USA/S3583
GQ903811/2008/USA/S4216
GQ903901/2005/USA/M405
GQ903859/2007/USA/S3901
EF492556/2001/France/F18
AF118779/1997/USA/G3
AF118780/1998/USA/G4
AF118778/1997/USA/G2
AF118777/1995/USA/G1
EF492557/2001/France/F19
EF492559/2002/France/F21
EF492555/2001/France/F17
AF118769/1995/USA/A1
AF118773/1996/USA/R1
AF118774/1998/USA/R2
AF118782/1996/USA/RQ
AF118781/1996/USA/BT-PA96
AF118770/1996/USA/A2
AF118771/1996/USA/A3
AF118772/1997/USA/A4
AF118775/1996/USA/P1
AF118776/1997/USA/P2
EF492554/2001/France/F16
EF492561/2002/France/F23
EF492560/2002/France/F22
EF492553/2001/France/F15
JN211317/2000/France/F60
EF492548/2003/France/F10
JN211316/2007/France/F27
AY349167/1996/USA/CW96
AY349168/2001/USA/CW01
GQ903862/2007/USA/S3955
AF107266/1999/USA/D84
AF107267/1999/USA/D85
AF107277/1999/USA/E88
AF107276/1999/USA/E86
AF107275/1999/USA/E85
AF107279/1984/USA/KY84
AF107278/1999/USA/E89
AF107269/1999/USA/D87
AF107270/1999/USA/D88
AF107268/1999/USA/D86
AF107272/1999/USA/D91
AF107273/1999/USA/D92
AF107274/1999/USA/D94
AF107271/1999/USA/D89
U81015/1977/USA/KY77
AH007128/1999/USA/CA95G
AH007129/1999/USA/CA95I
U81018/1976/USA/PA76
U81017/1993/USA/KY93
DQ846750/1953/USA/Bucyrus
U81020/NA/USA/ATCC
EU252113/NA/USA/Hela-EAVP35
U81013/1953/USA/VBS53
EF492564/2004/France/F26
European 1
European 2
North American
Atacama-2014
Donkey/Atacama-2014/Chile/2014
0.03
FI GU RE 4 Maximum likelihood ORF7 phylogenetic tree using 93 equine
arteritis
virus reference sequences. Atacama-2014 isolate is a singleton genetically
distant
from reference sequences
coloured in red
2006); we named
this
group
the
asinine
cluster.
The time to most recent common
ancestor (tMRCA) between the
asinine cluster and other EAV genotypes was
estimated at
1695 (95%
highest poste-
rior density
(HPD) interval 1424–1892) (Figure 2, Appendix S1). Additionally, the
tMRCA of the
Atacama-2014 sequence
and
the African donkey strains
was estimated
at
1914 (95% HPD 1779–
1983). The
ORF5
sequence with the highest
identity to the Ata- cama-2014 virus
(78.9%) that was public available was J2-931125
#AY9565 (South
African asinine cluster). The ORF5 genetic
dis- tance between
groups (NA, EU1, EU2 and
asinine) was higher between
the asinine
group
and
all other
clusters (79.5%–81.9%) compared to the remaining
distances between
the European and North American clusters (90.1%–89.1%). In South
America, only EAV sequences from Argentina are available
from
public reposito- ries.
These
Argentinean sequences were collected
during
the
early
2000s and in 2011 and have been classified within the EU1
geno- type, genetically
distant
to
the
asinine
cluster
(Metz, Serena, Panei,
Nosetto, & Echeverria, 2014).
No ORF6 and ORF7 sequences from the
donkey South Afri- can isolates were
available for phylogenetic
analysis.
The
ORF6 and ORF7 phylogeny shows the Atacama-2014 virus phylogeny as a
singleton, distant from all
other EAVs (Figures
3 and 4). How- ever, we
were
able
to sequence only three ORFs of the
virus
(10% genome),
which we
consider the
major
limitation of
this study.
The phylogeny indicates that
the
asinine cluster
represents
a
new genotype present
in South America and
Africa, both related
to carrier
donkeys. The presence of this genotype in two different con- tinents may underlie a widely distributed unreported existence of this viral strain,
different from the known
and well-characterized
EAV prevalent in horses.
It is not clear how the virus
was introduced into
the
feral
don- key
population in Chile. Historical records indicate
that
the
original population of donkeys arrived into the country
at least 500 years
ago. However, it is likely
that subsequent importations occurred. tMRCA
of
the
Chilean
and
1993 South African donkey was esti-
mated between 1779 and 1983,
suggesting
that
the
introduction of
the virus
may have occurred during imports after the original intro- duction of donkeys in Chile. However, because of
the lack of avail- ability of viral sequences collected at earlier time points
of the asinine cluster, the
tMRCA estimates
should be
carefully inter- preted.
Although EAV has
only been detected in
donkeys in Chile, the South
African asinine strains have also detected in
horses (Stadejek et
al., 2006); therefore, the EAV Chilean donkey viruses may repre- sent
a risk for different equine populations.
The characterization of this virus
in South America provides a novel perspective of the global
distribution
of EAV.
The isolation
and genetic characterization of
this new virus provides
vital informa-
tion for future EAV
surveillance. It further contributes to
understand the divergence of
the
virus
and
to
the
proper
design
of
diagnostic test
for more
accurate detection in horses and as well as other
equids.
AC KN O W L E D G EM E N T S
We thank the staff of Chilean Agricultural and Livestock Services (SAG) for all their support during the sampling, necropsy and
virology work.
CONFLI CT OF INT E RE ST
The authors declare
no conflict
of interest.
OR CI D
V. Neira http://orcid.org/0000-0001-9062-9969
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