Typing of field isolates of infectious bronchitis virus based on the sequence of the hypervariable region in the S1 gene
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J Vet Diagn Invest 15:344–348 (2003)
Typing of field isolates of infectious bronchitis virus based on
the sequence of the hypervariable region in the S1 gene
Chang-Won Lee, Deborah A. Hilt, Mark W. Jackwood
Abstract. A universal primer set was developed that amplifies a region covering hypervariable region
(HVR) 1 and HVR 2 in the S1 gene of the infectious bronchitis virus (IBV). The universality of this primer
set was confirmed by testing the reference strains of different serotypes or variants of the IBV present in the
United States. An approximately 450-bp region containing HVR 1 and HVR 2 of 7 untyped field isolates
obtained in 1999 and 2000 was amplified. Direct sequencing followed by phylogenetic analysis on that region
allowed us to type those field isolates that were not typable by reverse transcriptase–polymerase chain reaction
(RT-PCR) and restriction fragment length polymorphism (RFLP). Furthermore, it was found that typing by
phylogenetic analysis of that region correlates with virus neutralization results. Together with RT-PCR and
RFLP, this method will serve as a fast typing method for IBV diagnosis.
The infectious bronchitis virus (IBV) has been a ma- 2% of the field isolates could not be typed by this
jor cause of economic losses in the poultry industry.4 method. Thus, sequencing the entire S1 gene has been
IBV is a coronavirus in the new order Nidovirales, conducted to type those isolates. However, amplifica-
which has a positive-sense, single-stranded RNA ge- tion and sequencing of the entire S1 gene is not effi-
nome.1 Since the first identification in 1936, more than cient and not suitable for diagnostic methods because
50 serotypes or variants of the virus have been re- it is time consuming and costly. It has been shown that
ported worldwide.4 Thus, determining the type as well the genetic typing based on the hypervariable region
as isolation of the virus is important to select an ap- (HVR) 1 (HVR 1) (114–325) of the S1 gene could
propriate vaccine against IBV infection in the next represent the grouping method based on the whole S1
flock. Further, typing of IBV strains is necessary for gene and can be used for typing IBV isolates.24 A more
understanding the epidemiology and evolution of recent study10 utilized direct sequencing of the S1
IBVs. However, clear classification of isolates is very gene, which includes HVR 1 and HVR 2 to differen-
difficult because of high mutation frequency of the tiate IBV types. Furthermore, a new serotype of IBV
RNA genome, recombination, and multiple cross-re- was identified by analyzing an approximately 450-bp
actions among IBVs.8,15,17 As a result, standardization region of the S1 gene containing HVR 1 and HVR 2.14
of tests has been hampered, and many different meth- In the present study, a primer set that amplifies the
ods are being used worldwide.5 region covering HVR 1 and HVR 2 of the reference
Genotyping, which correlates with serotype, is fre- strains of different serotypes or variants present in the
quently used because it is extremely fast and conve- United States was designed. This primer set was used
nient compared with virus neutralization (VN) testing to analyze 7 field isolates that were untypable by RT-
or other traditional serotyping tests. Methods include PCR or RFLP. By conducting direct sequencing on that
sequencing, detection of genotype-specific parts of the region followed by phylogenetic analysis, those field
genome by multiplex reverse transcriptase–polymerase isolates could be classified. Furthermore, those clas-
chain reaction (RT-PCR), and determining the position sification results were confirmed by conducting the
of enzyme cleavage sites by restriction fragment length standard VN test.
polymorphism (RFLP) and RNase T1 fingerprint-
ing.9,12,13 In 1993, a rapid diagnostic test was devel- Materials and methods
oped,13 based on RT-PCR and RFLP analysis of the Viruses and embryonated chicken eggs. Viruses examined
gene that codes for the S1 subunit of the spike gly- in this study are listed in Table 1. These isolates were ob-
coprotein, which contains neutralizing and serotype- tained from chickens and allantoic fluid samples submitted
specific epitopes.2,7,19 Using that test, more than 500 to the Poultry Diagnostic and Research Center, University
clinical samples of the IBV were examined each year. of Georgia (Athens, GA). Specific pathogen–free embryo-
However, because IBV mutates very quickly, about nated chicken eggs (ECE)a that were 9- to 11-day-old were
used for seed stock production, titration of isolates, and virus
neutralization tests as previously described.6,21,23
From the Department of Avian Medicine, College of Veterinary RNA extraction and RT-PCR. Ribonucleic acid was iso-
Medicine, The University of Georgia, Athens, GA 30602. lated from infective allantoic fluids using a PCR template
344
Downloaded from vdi.sagepub.com by guest on August 24, 2015IBV typing by HVR sequence analysis 345
Table 1. List of IBV field isolates examined in this study, in- dwarfing, curling, clubbed down feathers, and urates repre-
cluding origin, type, and GenBank accession numbers. senting nonvirus neutralization. The Reed and Muench for-
mula20 was used to calculate endpoint titers for homologous
Isolates/date Origin Type* Accession no.
and heterologous neutralization.
VA/7996/99 VA, USA Arkansas AF363590
MS/8306/99 MS, USA Connecticut AF363591 Results
MS/8373/99 MS, USA Connecticut AF363592
CT/8814/99 CT, USA Massachusetts AF363593 RT-PCR with universal primer set. Using C2U-
NE/11162/00 NE, USA California AF363594 deg59 and Ag07239 primers, an approximately 450-bp
MN/11180/00 MN, USA Arkansas AF363595 region in the S1 gene of all the reference IBV strains
MX/7277/99 Puebla, Mexico Unclassified AF363596 as well as 7 untyped field viruses isolated in the United
* Typing based on Fig. 2. States and 1 from Mexico were amplified. The D1466
Dutch strain, which is very different from other IBV
strains in the S1 gene sequence, could also be ampli-
preparation kitb according to the manufacturer’s recommen- fied. Because this region shows a lot of nucleotide in-
dations. Ribonucleic acid from the phenol-inactivated allan- sertions and deletions, the size of the PCR product
toic fluid was extracted as previously described.16 For the ranged from 438 to 468 bp, depending on the strain
amplification of approximately 450-bp HVR, the Ag07239 (Fig. 1).
antisense primer15 and C2U sense primer24 with slight mod-
Typing based on HVR sequence. When field isolates
ifications were used. A degeneracy of the C2U primer at the
39 end was made, and the primer was renamed C2U-deg59.
and reference strains were phylogenetically analyzed
The C2U-deg59 primer sequence is as follows: 59- on the basis of deduced amino acid sequence of the
TGGTTGGCATTTACACGGRG-39,where R indicates an HVR, field isolates were grouped with different sero-
adenine (A) or guanine (G) base. The RT-PCR was per- types of IBV (Fig. 2). Field isolates MS/8373/99 and
formed using the one tube RT-PCR system.c All the reagents MS/8306/99 belonged to the Connecticut serotype,
except primers and RNase inhibitor are provided with the CT/8814/99 belonged to the Massachusetts serotype,
kit. Ten microliter of 53 RT buffer, 4 ml of 0.1 M dithio- NE/11622/00 belonged to the California serotype, and
threitol, 6 ml of MgCl2 (25 mM), 1 ml of diethylnitrophenyl VA/7996/99 and MN/11180/00 belonged to the Ar-
thiophosphate (10 mM), 1 ml of Titan Mix, 1 ml of RNase kansas serotype. The isolate from the Mexico serotype,
inhibitor (40 U/ml),d and 1 ml (50 pmol) of each primer were MX/7277/99, did not cluster with any other IBV se-
mixed to a final volume of 50 ml. The 1-step RT-PCR was
rotypes.
conducted by incubating the 1-hr RT reaction at 42 C, heat-
ing to 95 C for 5 min, followed by 35 cycles of denaturation
Sequence variability in the HVR was extremely
at 94 C for 30 sec, annealing at 50 C for 30 sec, and poly- large (Table 2). In general, less than 60% similarity in
merization at 68 C for 90 sec with a final elongation step of the amino acid sequence was found among different
10 min at 70 C using a thermal cycler.e Amplification prod- serotypes. MX/7277/99 was unique in the HVR se-
ucts were analyzed in a 1.0% agarose gel. quence. This isolate showed less than 40% similarity
Direct sequencing and phylogenetic analysis. Polymerase in amino acid sequence with other strains. However, it
chain reaction products were cut from a 1% agarose gel and shared more than 60% sequence similarity with DE072
purified.f Purified PCR products were sequenced directly by and D1466. When the sequence similarity was com-
automated sequencingg at the Molecular Genetics Instrumen- pared with the consensus sequence of these 3 isolates,
tation Facility, University of Georgia (Athens, GA). The nu- the similarity increased to 80% (Fig. 3).
cleotide sequences reported here have been deposited with
VN analysis. Field isolates were examined further
GenBank (Table 1).
Assembly of sequencing contigs, translation of nucleotide
by a 1-directional VN test with antisera against Mas-
sequence into protein sequence, and initial multiple sequence sachusetts, Connecticut, California, and Arkansas se-
alignments were performed with commercial software.h Phy- rotypes of IBV. Viruses were considered to be related
logenetic trees for HVR in the S1 gene were generated using to or to be members of the same serotype if the anti-
the neighbor-joining method with 100 bootstrap replicates.22 serum protected 50% or more of the embryos (Table
One-directional VN test. The VN test was performed in 3). Field isolates showed the highest protection rate
ECE using the diluted-serum constant-virus (beta) proce- with antisera prepared from reference IBV of same
dure.6 Monospecific antisera were prepared as previously de- group shown in Fig. 2.
scribed against the Massachusetts 41 (Massachusetts sero-
type ATCC VR-21, American Type Culture Collection, Discussion
Rockville, MD), Connecticut (Connecticut serotype, Dr. J.
Gelb Jr, University of Delaware, Newark, DE), CV56b (Cal- The precursor S protein is posttranslationally
ifornia serotype, California Veterinary Diagnostic Labora- cleaved into S1 and S2 subunits.3 In the S1 subunit, 3
tory System, Fresno, CA), and Arkansas DPI (Arkansas se- HVRs are located within amino acids 38–67, 91–141,
rotype, Dr. J. Gelb Jr) reference strains of IBV.6 Chicken and 274–387.2,11,18 Neutralizing epitopes are associated
embryos were evaluated 1 wk after inoculation with death, with the defined HVR,2,7,19 and variation in this region
Downloaded from vdi.sagepub.com by guest on August 24, 2015346 Lee, Hilt, Jackwood
Figure 1. Agarose gel electrophoresis of the RT-PCR product obtained with the universal primer set. Marker; molecular weight marker
(Biomarker-low, Bio Venture, Murfreesboro, TN).
is thought to correlate with serotype. A recent study24
typed IBV on the basis of HVR 1 sequence and dem-
onstrated that genotyping correlated with serotyping
by the VN test. However, the primer set of that study
could not be used to amplify some of the American
serotypes, like DE072 and GA98, which show wide
prevalence in the southeastern part of the United
States. A more recent study10 utilized direct sequenc-
ing of the S1 gene, which included HVR 1 and HVR
2, to identify IBV serotypes. The primer set used in
that study10 amplifies more than 600 bp and contains
relatively more conserved regions than the region an-
alyzed in the previous report.24
The primer set described in this study amplifies an
approximately 450-bp region covering HVR 1 and
HVR 2 of all the reference strains of different sero-
types or variants present in the United States tested so
far. As a diagnostic tool, a short length of PCR product
is desirable because it is easily obtained and analyzed
by direct sequencing. Furthermore, earlier work dem-
onstrates that phylogenetic analysis on this region is
very useful for differentiating closely related isolates.14
Phylogenetic analysis on the basis of sequence in-
formation has been a useful tool for epidemiological
studies. Previously, a close relatedness between DE072
and D1466 was demonstrated on the basis of sequence
analysis.16 Mexican variant, MX/7277/99, which has a
Figure 2. Phylogenetic trees on the basis of deduced amino acid
sequence of the HVR in the S1 gene. The trees were generated by
unique HVR sequence, shares 80% sequence similarity
the neighbor-joining method with 100 bootstrap replicates. Branch with the DE072 and D1466 consensus sequence. Be-
lengths and bootstrap values (in parenthesis) are provided. cause there was no crossover site identified, it is pos-
Downloaded from vdi.sagepub.com by guest on August 24, 2015IBV typing by HVR sequence analysis 347
Comparison of the nucleotide and deduced amino acid sequence of the HVR in the S1 gene of 7 field isolates with 6 different reference strains of different IBV serotypes.
95.6
59.9
59.2
72.7
40.0
39.7
38.4
94.4
59.4
56.8
59.4
74.8
11180
100
75.9
59.7
59.9
83.6
39.5
36.0
34.7
75.0
59.2
60.8
60.9
70.9
11162
100
62.3
91.9
88.7
61.4
41.2
41.1
40.6
61.9
87.8
88.5
60.3
58.1
8814
100
61.0
89.2
98.0
62.7
41.2
42.0
40.8
60.8
96.2
79.7
60.2
54.9
8373
Figure 3. Alignment of deduced amino acid sequences of MX/
100 7277/99, DE/072/92, and D1466. Dots indicate amino acids identical
to that of the consensus sequence. A dash was introduced to align
the sequences.
60.9
87.3
96.0
60.9
39.0
39.9
39.7
60.6
92.5
81.6
58.8
55.9
8306
100
sible that MX/7277/99 shares the same ancestral strain
with DE072 and D1466 and has undergone extensive
99.1
62.8
60.5
73.9
42.7
41.1
39.2
59.6
58.6
60.3
73.8
90.2
7996
100
genetic variation caused by evolution in a geographi-
Nucleotide Identity (%)
cally different location.
Phylogenetic analysis based on HVR 1 and HVR 2
39.9
39.4
39.0
36.9
62.5
62.8
32.6
31.1
33.8
34.1
28.1
31.1
7277
100
clearly differentiates different serotypes from one an-
other (Fig. 2). However, despite the advantages of this
method as a diagnostic tool, it may not always corre-
D1466
41.4
40.4
40.6
35.2
58.1
63.7
31.9
30.4
33.8
33.3
28.9
28.1
late with serotyping because there may be other re-
100
gions that play a role in determining serotypes. Fur-
thermore, despite the universality of our primers, there
DE072
43.7
40.7
41.1
39.5
59.0
62.7
32.8
31.3
34.6
34.3
32.1
30.6
is always a chance that new viruses cannot be ampli-
100
fied with this primer set. Two significant advantages
of this technique are that sequencing data are less sus-
CV56b
ceptible to ambiguity than serotyping methods, which
74.1
59.9
62.0
30.6
27.4
28.9
72.1
55.1
58.6
55.9
81.4
69.3
100
characterize isolates’ antigenicity, and that genotyping
can be done very quickly compared with serotyping.
Screening by RT-PCR and RFLP followed by sequenc-
60.7
88.7
56.1
33.3
32.6
31.1
57.6
92.4
95.5
79.5
58.3
54.5
Conn
100
ing the short HVR, if necessary, will be useful when
faced with a large number of untypable IBV field iso-
lates for diagnosis.
Mass41
63.3
77.3
54.4
34.3
32.6
31.9
59.6
78.7
78.9
83.1
58.1
55.9
100
Sources and manufacturers
a. Charles Rivers Spafas, North Franklin, CT.
ArkDPI
59.6
57.6
72.1
32.8
31.9
31.9
98.6
59.6
58.6
60.3
74.5
90.9
b. High Pure PCR Template Preparation Kit, Roche, Indianapolis,
100
IN.
c. Titany One Tube RT-PCR System, Roche, Indianapolis, IN.
d. Takara Shuzo Co., Shiga, Japan.
e. GeneAmp PCR system 2400, Perkin–Elmer, Norwalk, CT.
ArkDPI
Mass41
CV56b
DE072
D1466
11162
11180
f. QIA Quick Gel Extraction Kit, Qiagen, Santa Clarita, CA.
Conn
7277
7996
8306
8373
8814
Table 3. Virus neutralization results expressed as percentage of
protection relative to homologous protection values.
Amino acid identity (%)
Virus
Antisera 8373 8814 11162 11180
Massachusetts 11.6 80.8 14.1 7.9
Table 2.
Connecticut 100 57.8 22.1 39.1
Arkansas 20.0 36.9 21.7 100
California 13.6 36.0 65.9 12.6
Downloaded from vdi.sagepub.com by guest on August 24, 2015348 Lee, Hilt, Jackwood
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