Origin and evolution of Georgia 98 (GA98), a new serotype of avian infectious bronchitis virus

Virus Research 80 (2001) 33 – 39 www.elsevier.com/locate/virusres

Origin and evolution of Georgia 98 (GA98), a new serotype of avian infectious bronchitis virus Chang-Won Lee, Mark W. Jackwood * Department of A6ian Medicine, College of Veterinary Medicine, The Uni6ersity of Georgia, 953 College Station Road, Athens, GA 30602, USA Received 23 April 2001; received in revised form 22 June 2001; accepted 22 June 2001

Abstract We previously identified GA98, a new serotype of infectious bronchitis virus (IBV), which is closely related to the DE072 serotype of IBV genetically, but not antigenically. Herein, we analyzed the 421bp sequence of a hypervariable region (HVR) (position 114–534, counting from the ATG start site) of the S1 subunit of GA98 IBVs to further examine the evolution of these viruses. These viruses were isolated between the years 1997 and 2000. Phylogenetic analysis of the deduced amino acid sequence on that region indicated that GA98 isolates from different regions of Georgia were the result of a single introduction of the S1 gene of the DE072 serotype progenitor. Most of the mutations were nonsynonymous and had become fixed in a progressive manner. The evolutionary and mutation rates in the HVR was calculated as 2.5 and 1.5% per year, respectively. This new serotype of IBV appears to be evolving very fast compared with other serotypes of IBV. We further determined the complete coding sequence of the S1 gene of seven isolates obtained from one selected region in North Georgia. Together with virus neutralization data, it appears that GA98 arose from immune selection caused by DE072 vaccine use. Reasons for this conclusion are discussed. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Infectious bronchitis virus; Georgia 98 serotype; Viral evolution; Mutation rate

Infectious bronchitis virus (IBV) is classified as a member of the family coronaviridae in the new order Nidovirales (Cavanagh, 1997). The enveloped virus has a positive-sense, single-stranded RNA genome of approximately 27 kilo-bases, which codes for an RNA-dependent RNA poly* Corresponding author. Tel.: + 1-706-542-5475; fax: +1706-542-5630. E-mail address: [email protected] (M.W. Jackwood).

merase, spike (S), gene 3a, 3b, envelope (E), membrane (M), gene 5a, 5b, and nucleocapsid protein (NP) in the 5%–3% direction (Sutou et al., 1988; Siddell, 1995). IBV, like many other RNA viruses, has a high error rate during the transcription of its genomes (Lai and Cavanagh, 1997). This is mainly because of the fact that RNA polymerase lacks the 3% – \ 5% exonuclease activity (editing function) of DNA polymerase (Garrett and Grisham, 1999). The high error rate produces a quasispecies phe-

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nomenon where many different viral genotypes will cocirculate in the host, with each virus potentially having different levels of fitness for the host environment (Domingo et al., 1985). Mutation rates during viral replication have been estimated in the range of 10 − 3 – 10 − 5 substitutions per nucleotide. Because the IBV genome is approximately 30,000 nucleotides, an average of three mutations per template copied will result in mutation rates of 10 − 4 substitutions per nucleotide. When chickens are infected with IBV, many genetically different variants can be produced. Transmission of only a few genetically different virus particles to other susceptible chickens will result in genetic drift. If point mutations occur in the antigenic domain of the viral proteins, viruses with slightly changed antigenic structure will appear. This process is called antigenic drift and is well characterized in influenza viruses (Bean et al., 1992). Because widespread vaccination has been conducted to control IBV, this immune selection pressure together with high mutation rate of the genome, may explain the existence of many serotypes and variants of IBV. Between 1990 and 1992 in the Delmarva region, chickens suffered respiratory disease caused by a serotype of IBV not previously detected in the United States (Gelb et al., 1997). The serotype was designated DE072 after the prototype strain DE/072/92. It differed from all known IBV serotypes by at least 40% of S1 amino acid sequences (Gelb et al., 1997). Further analysis on a different part of the genome indicated that DE072 was related to the D1466 Dutch vaccine strain (Lee and Jackwood, 2000b, 2001a). An attenuated live vaccine, made from the DE072 strain, was introduced in 1993 and disease associated with this serotype of IBV was not a major problem until 1996. Since 1996, the incidence of isolation of DE072-like viruses increased every year particularly in the southeastern United States. We previously conducted genetic and antigenic analysis of recent DE072-like field isolates and identified GA98, a new serotype of IBV, which might have arisen from the DE072 serotype by antigenic drift (Lee et al., 2001b). To further examine the evolution of this new serotype of IBV, we conducted molecular and antigenic anal-

ysis of GA98 field isolates obtained from 1997 through 2000. The GA98 serotype of IBV presents us with a unique opportunity to examine the evolution of this serotype because its sequence is extremely different from other serotypes of IBV in the USA, and the virus has persisted in the field long enough to determine evolutionary trends. Viruses used in this study are listed in Table 1. Isolates of IBV were replicated in 9–11-day-old embryonated chicken eggs (Senne, 1998), and RNA was extracted directly from allantoic fluid as previously described (Lee and Jackwood, 2001a). A single-tube reverse transcriptase-polymerase chain reaction (RT-PCR) for genomic IBV RNA was completed as described (Lee and Jackwood, 2000b). Primer sequence and location, for the amplification of the HVR and entire S1 gene, was reported (Lee et al., 2000a, 2001b). Amplification products were purified using the QIA quick Gel Extraction Kit (Qiagen, Santa Clarita, CA). Automated sequencing with the Prism™ DyeDeoxy terminator cycle sequencing kit (Perkin Elmer, Foster City, CA) was conducted at the Molecular Genetics Instrumentation Facility, University of Georgia. Nucleotide sequence editing, analysis, prediction of amino acid sequences, and alignments were conducted using DNAStar™ (Madison, WI) software. Phylogenetic relationships among IBV isolates presented were constructed with the Phylogenetics Analysis Using Parsimony (PAUP* 4.0C) software (Swofford, 1998). The amino acid sequence of the HVR (position 39–178) in the S1 subunit of the spike protein gene of 37 isolates were aligned followed by neighbor-joining analysis (Saitou and Nei, 1987) to generate the resultant phylogram (Fig. 1). The trees demonstrated the previously described groups of isolates including DE072, GA98, and unclassified IBVs (Lee et al., 2001b). At the nucleotide level, the sequence identity between the DE072 (DE/072/92) strain and GA98 lineage isolates ranged from 91.2 to 96.0%. When the amino acid sequences were compared, lower identities were found (88.6–92.1%), which indicates most of the nucleotide changes were nonsynonymous. The 20 isolates from the GA98 group are likely from the same lineage, and the nucleotide differences

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among them appear to be the result of mutations accumulating over time. We found 17 nucleotide sites in the 421 bp HVR, which indicates mutations had become fixed in a progressive manner from DE072 to GA/13055/00 (data not shown). The sequence of the DE072 strain was used as the point of origin in regression analysis to determine the sequence changes of different GA98 isolates (Fig. 2). The number of nucleotide differences was determined using pairwise distance

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comparisons between the DE072 strain and GA98 serotype isolates. The number of substitutions was then plotted with the Excel (Micosoft) program in a scattergram against the number of months after isolation from the DE072. Linear regression analysis was used to determine a trend line, and the slope was used to determine the rate of nucleotide and amino acid change over time. The trend line from the regression analysis and the equation for the line were added to the graph. A positive

Table 1 IBV examined in this study Isolates

Date isolated

State of origin

Genebank c

DE/492/90 DE/072/92 DE072 vaccine DE/174/92 GA/6305/97 GA/6334/97 GA/9794/97 GA/0470/98 GA/1159/98 GA/1315/98 GA/2787/98 GA/3058/98 GA/5381/99 GA/5416/99 GA/5658/99 GA/5791/99 GA/5808/99 GA/7994/99 GA/8077/99 GA/8234/99 GA/8367/99 GA/9600/99 GA/9719/99 GA/10498/00 GA/10627/00 GA/10718/00 GA/11468/00 GA/11951/00 GA/11998/00 GA/12574/00 GA/12996/00 GA/13055/00 NC/5339/97 MN/6370/97 AR/6386/97 IL/2831/98 KS/5425/99

1990 1992 Derived from DE/072/92 strain 1992 June-1997 May-1997 November-1997 January-1998 March-1998 March-1998 June-1998 July-1998 January-1999 January-1999 February-1999 March-1999 February-1999 May-1999 June-1999 July-1999 August-1999 November-1999 December-1999 January-2000 January-2000 February-2000 February-2000 March-2000 March-2000 April-2000 May-2000 June-2000 March-1997 May-1997 May-1997 June-1998 February-1999

Delaware Delaware

AF274424 U77298 AF274435 AF274423 AF338716 AF274431 AF274433 AF274437 AF274426 AF274427 AF274438 AF274428 AF274439 AF274440 AF206256 AF274430 AF274429 AF338717 AF338718 AF274432 AF274420 AF274421 AF274422 AF338720 AF338721 AF338722 AF338723 AF338724 AF338725 AF338726 AF338727 AF338719 AF274434 AF274443 AF274436 AF274441 AF274442

Delaware Georgia Georgia Georgia Georgia Georgia Georgia Georgia Georgia Georgia Georgia Georgia Georgia Georgia Georgia Georgia Georgia Georgia Georgia Georgia Georgia Georgia Georgia Georgia Georgia Georgia Georgia Georgia Georgia North Carolina Minnesota Arkansas Illinois Kansas

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Fig. 1. Phylogenetic tree generated by the neighbor joining method base on amino acid sequence of the HVR of the S1 gene. Trees were midpoint rooted and branch lengths are provided. Isolates with bold characters were isolated from the same region in North Georgia.

mutation rate of 1.5% nucleotide changes and the evolutionary rate of 2.5% amino acid changes per year were observed. The complete coding sequence of the S1 subunit of the spike protein gene was further determined for seven viruses isolated from one region in North Georgia. This region was of interest because the first GA98 serotype of IBV was isolated from and continues to persist on this region. The deduced amino acid sequence of the GA98 lineage viruses were compared with the DE072 and DE072 vaccine strains. With the sequence of the

DE072 strain as a reference point, the GA98 lineage viruses showed progressive and fixed substitution changes (Table 2). These progressive changes were identified by comparing the accumulating nucleotide changes from the 1992 DE072 isolate to the most recent isolate in the lineage, 13055, isolated in the year 2000. The changes affected more than two members of the lineage and correlated with the date of isolation of the virus. We further serologically analyzed seven isolates from this farm by conducting virus neutralization

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(6305, 0470, 7994, 8077, and 13055) had a similar amount of amino acid differences as the DE072 vaccine strain in the HVR and entire S1 gene, isolates 5381 and 9794 were serologically more closely related (70.1 and 50.7%, respectively) to the DE072 vaccine than GA98 lineage viruses (range 3.7–27.0%) (Table 3). It is possible that these two isolates arose as viral quasispecies, but because of their close relatedness to the DE072 vaccine, those isolates were negatively selected resulting in the GA98 lineage of IBV becoming dominant in Georgia. Isolate 8077 is very different from both the DE072 and GA98 serotype of IBV in HVR and S1 sequence and in antigenic relatedness (Table 3). Predicting whether IB viruses will or will not persist in the environment is difficult. It is possible that the uniqueness of the 8077 lineage virus will allow it to persist in Georgia, or the single isolation of this virus may indicate the unfitness of this virus for the host environment resulting in 8077 lineage viruses eventually disappearing. In agreement with a previous report, our results also indicate that small differences in the S1 sequence can cause large antigenic changes (Cavanagh et al., 1992; Moore et al., 1998). To study the evolution of virus, it is necessary to analyze several isolates that have descended from a single introduction of the virus and which have persisted in the environment long enough to determine trends. There are a few reports in the literature that address this issue because, due to widespread vaccination in the field, it is very difficult to determine the mutational or evolution-

Fig. 2. Evolutionary and mutation rates of the GA98 serotype of IBV. The number of nucleotide and amino acid changes from the index isolate, DE/072/92, are given on the y-axis. The number of months after each virus was isolated is compared with the index case given on the x-axis.

(VN) tests with antisera against the DE072 vaccine and GA/0470/98 (CWL0470), which is a prototype strain of GA98 serotype IBV. The VN test was performed in chicken eggs using the dilutedserum, constant-virus (beta) procedure (Gelb et al., 1991). Monospecific antisera were prepared as previously described (Gelb and Jackwood, 1998). The VN results were used to calculate the percentages of relatedness values (Archetti and Horsfall, 1950) to two prototype strains. Those values were compared with the HVR and entire S1 sequencing results (Table 3). Most of the isolates shared less than 27% serologic relatedness with the DE072 vaccine except isolates 5381 and 9794. Although isolates 9794, 5381, and GA98 lineage viruses

Table 2 Comparison of amino acid sequence at discriminative sites in the whole S1 protein Isolatea

DE/072/92 GA/6305/97 GA/0470/98 GA/7994/99 GA/13055/00 a

Amino acid position 3

23

32

58

67

85

115

123

141

144

278

281

282

283

331

387

389

418

479

530

A V V G G

E V V V V

E G G G G

N N N D D

G R R R R

I S S S S

S N N N N

T L L L L

R R R I I

V G G N N

E A A A A

G Q Q Q Q

K K N E E

H H Y Y Y

P L L L L

N T T T T

K S S R R

T S S S S

H N N N N

S G G G G

Four GA98 lineage viruses isolated in one region in North Georgia were compared with the DE/072/92 strain.

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Table 3 Comparison of the amino acid similarity in the HVR and S1 gene with VN results Virusa

DE072 vac 9794 5381 6305 CWL0470 7994 13055 8077

HVRb

VNc

S1

DE072 vac

CWL0470

DE072 vac

CWL0470

DE072 vac

CWL0470

100 90.0 91.4 91.4 91.4 90.0 90.0 89.3

91.4 91.4 91.4 98.6 100 97.1 96.4 90.7

100 93.8 94.4 94.8 94.0 93.1 93.5 92.7

94.0 94.6 93.8 99.1 100 97.9 97.9 92.7

100 50.7 70.1 25.1 27.0 15.1 3.7 15.2

17.5 12.5 B6.3 78.0 100 98.4 100 57.8

a

Seven viruses isolated in one region in North Georgia were compared with DE072 vac (DE072 vaccine) strain. Position 39–178 in the S1 protein. c VN results were expressed as a percentage of relatedness values to two prototype strains (Archetti and Horsfall, 1950). b

ary rate of IBV. One serotype, 793/B, of IBV was analysed previously (Adzhar et al., 1997; Cavanagh et al., 1998). The 1.5% mutation rate per year in the HVR of GA98 is very high compared with the work done with 793/B serotype of IBV, which showed a 0.3% nucleotide change per year in the same region. Furthermore, the GA98 lineage viruses showed progressive and fixed substitution change. However, there was no evidence that mutations had become fixed in a progressive manner in 793/B lineage, which indicates that this serotype was not evolving. We believe that our data clearly shows that GA98 is the result of positive immune selection driven by DE072 vaccine use. At the time of the Adzhar and Cavanagh study, no vaccine had been used against the 793/B serotype of IBV. In contrast, the DE072 vaccine was introduced in 1993 and has been used in Georgia since 1997. As a result, the GA98 serotype of IBV has evolved much faster than 793/B serotype of IBV. Herein, we present the most comprehensive sequence analysis of isolates related to a single serotype of IBV to date. The data provided here can provide a benchmark and will be used as a reference for studying the evolution of other IBV serotypes. This work also highlights the effect of vaccine use on the evolutionary rate of IBV and serves as a reminder to use IBV vaccines responsibly.

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