2014 origin and characteristics of the recombinant novel avian infectious bronchitis coronavirus...

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Infection, Genetics and Evolution xxx (2014) xxx–xxx

MEEGID 1892 No. of Pages 7, Model 5G

11 March 2014

Contents lists available at ScienceDirect

Infection, Genetics and Evolution

journal homepage: www.elsevier .com/locate /meegid

Origin and characteristics of the recombinant novel avian infectiousbronchitis coronavirus isolate ck/CH/LJL/111054

http://dx.doi.org/10.1016/j.meegid.2014.02.0151567-1348/� 2014 Elsevier B.V. All rights reserved.

⇑ Corresponding author. Tel.: +86 451 85935065; fax: +86 451 82734181.E-mail address: [email protected] (S. Liu).

Please cite this article in press as: Liu, S., et al. Origin and characteristics of the recombinant novel avian infectious bronchitis coronavirus isolateLJL/111054. Infect. Genet. Evol. (2014), http://dx.doi.org/10.1016/j.meegid.2014.02.015

Shengwang Liu ⇑, Qianqian Xu, Zongxi Han, Xiaoli Liu, Huixin Li, Hongbo Guo, Nana Sun, Yuhao Shao,Xiangang KongDivision of Avian Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences,Harbin 150001, People’s Republic of China

2728293031323334353637

a r t i c l e i n f o

Article history:Received 4 September 2013Received in revised form 12 February 2014Accepted 14 February 2014Available online xxxx

Keywords:Avian infectious bronchitis virusRecombinant eventConnecticut serotypeMassachusetts serotype

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a b s t r a c t

Recombination among infectious bronchitis viruses (IBVs), coupled with point mutations, insertions, anddeletions that occur in the genome, is thought to contribute to the emergence of new IBV variants. In thisstudy an IBV, ck/CH/LJL/111054, was isolated from a H120-vaccinated chicken, which presented with asuspected IBV infection. Phylogenetic analysis of the S1 subunit sequence confirmed that strain ck/CH/LJL/111054 is of the Connecticut-type; however, further extensive full-length genomic analysis identifiedthe occurrence of recombination events. Therefore, strain ck/CH/LJL/111054 may have originated fromrecombination events between Conn- and Mass-like strains at three recombination breakpoints: twolocated within the nsp3 gene sequence and one in the nsp12 gene sequence. Further, the uptake of the50 untranslated regions, nsp2, parts of nsp3, nsp4–11, and parts of nsp 12 from Mass-like virus by ck/CH/LJL/111054 might have resulted in changes in viral replication efficiency rather than antigenicchanges, via cross-neutralization analysis with the H120 strain. Recombination events coupled withthe accumulation of mutations in the ck/CH/LJL/111054 genome may account for its increased virulencein specific-pathogen free chickens.

� 2014 Elsevier B.V. All rights reserved.

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1. Introduction

Avian infectious bronchitis virus (IBV), a gamma-coronavirus inthe Coronaviridae family (Carstens, 2010), is an enveloped, posi-tive-sense, single-stranded, RNA virus with a genome size of approximately 27 kb and causes a highly contagious upper-respiratorytract disease in domestic chickens, which is characterized by cough-ing, sneezing and tracheal rales. The 50 two-thirds of the IBV genomeencodes two polyproteins (1a and 1ab) that contain proteins neces-sary for RNA replication, whereas the 30 one-third encodes severalstructural proteins: the surface spike glycoprotein (S), and smallenvelope (E), membrane (M), and nucleocapsid (N) proteins. It isgenerally believed that the S1 subunit of S glycoprotein containshypervariable regions that play a role in attachment to host recep-tors, membrane fusion, and entry into the host cell. The S1 subunitof S glycoprotein carries conformationally dependent virus-neutral-izing and serotype-specific determinants, and is a determinant of

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cell tropism. Additional accessory protein genes are located in the30 region of the genome in an IBV species-specific position.

Since first described in 1936, IBV had undergone a high rate ofgenetic change that has contributed to the appearance of diversesubpopulations of the virus that continually emerge to form newvariants of existing strains as well as completely new types. TheMassachusetts (Mass) and Connecticut (Conn) strains are two ofthe most well characterized and important IBV types and were firstisolated in the 1930s and 1956, respectively, in the United States ofAmerica (Cavanagh and Gelb, 2008). In addition, Mass-typeviruses, which cause heavy economic losses to the poultry indus-try, have also been detected up to the present time in severalEuropean and Asian countries, including China, although at differ-ent frequencies (Jackwood, 2012). In contrast, Conn-type viruses,which show only 95.4% of nucleotides homology in the S1 subunitsequence with Mass-type viruses and are in different serotypesfrom the Mass-type viruses, circulate mainly in the United Statesof America and Central America (Jackwood, 2012).

Generally, point mutations, insertions, and deletions that occurin the S1 subunit of the spike protein in IBV strains contribute tothe existence of many serotypes and the emergence of variants

ck/CH/

http://dx.doi.org/10.1016/j.meegid.2014.02.015

mailto:[email protected]


http://www.sciencedirect.com/science/journal/15671348

http://www.elsevier.com/locate/meegid


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2 S. Liu et al. / Infection, Genetics and Evolution xxx (2014) xxx–xxx


11 March 2014

that have been isolated worldwide (Cavanagh and Gelb, 2008).Consequently, genotyping and phylogenetic analysis of IBV aretypically focused on the S1 subunit sequence. However, drawingconclusions based on a single gene sequence, and sometimes evena partial gene sequence, requires caution, as the correct molecularphylogeny of viruses can only be demonstrated by analyzing com-plete genomic sequences because IBV mutates at a high frequencydue to the high error rate of RNA polymerization. Like that of othercoronaviruses (CoVs), a unique feature of IBV genetics is the highfrequency of RNA recombination events in its natural evolution(Liu et al., 2013b). Recombination among CoVs is an attribute ofthe genus and is thought to contribute to the emergence of newIBV variants (Masters, 2006; Kusters et al., 1989; Wang et al.,1993; Liu et al., 2013b). Recombination events can occur betweenfield strains and also between field and vaccine strains (Masters,2006; Kusters et al., 1989; Wang et al., 1993; Liu et al., 2013b).In the present study, we isolated an IBV strain, ck/CH/LJL/111054,and performed genomic sequence analysis that revealed evidenceof multiple recombination events between Mass- and Conn-likestrains. Coupled with the tendency for mutations, our results raiseconcerns of the likelihood of future outbreaks of new IBV serotypesor variants due to incorrect usage of multiple vaccines and high-lights the need for continuous viral surveillance.

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2. Materials and methods

2.1. Viral isolation and H120 vaccine

Tissue samples of swollen proventriculus were collected from a26-day-old layer in Jilin province, China, in 2011. The chickenflocks were vaccinated against IBV with a commercially available,live attenuated, H120 vaccine at 2 days-old and received a boostervaccination at 16 days-old. Some of the chickens showed earlysigns of respiratory disease at 24 days-old. Post-mortem examina-tions were performed and the gross lesions were evaluated. Thegross examination showed mild to severe tracheitis and proven-triculitis. The morbidity was 20% and the mortality was nearly 10%.

For virus isolation, the samples were prepared as 10% w/v tissuesuspensions in 0.1% phosphate-buffered saline (PBS), clarified bycentrifugation at 1500g at 4 �C for 10 min, and filtered through0.22-lm membrane filters (Millipore products division, Bedford,MA, USA) before inoculation into the allantoic cavities of 9- to11-day-old embryos of specific pathogen-free (SPF) eggs chickens.Two passages were performed and characteristic embryo changes,such as dwarfing, stunting, or curling of the embryos, were ob-served between post-inoculation days 2 and 7. The virus isolatewas designated as ck/CH/LJL/111054. In addition, the vaccinestrain, H120, was used in this study. The embryo-propagated viralstocks of the ck/CH/LJL/111054 and H120 strains were produced byinoculating the virus into embryonated SPF chicken eggs via theallantoic cavity and collecting the infectious allantoic fluid 72 hpost-inoculation as previously described (Liu et al., 2013b).

2.2. Eggs and chickens

White Leghorn SPF chickens and fertile chicken eggs wereobtained from the Laboratory Animal Center, Harbin VeterinaryResearch Institute, China. All experimental procedures were ap-proved by the Ethical and Animal Welfare Committee of Heilongji-ang province, China.

2.3. Cross virus neutralization tests

Cross virus neutralization tests were performed using anti-seraagainst strains ck/CH/LJL/111054 and H120 to determine their anti-

Please cite this article in press as: Liu, S., et al. Origin and characteristics of theLJL/111054. Infect. Genet. Evol. (2014), http://dx.doi.org/10.1016/j.meegid.201

genic relationship. The viral titers of the ck/CH/LJL/111054 andH120 strains were determined by inoculation at 10-fold dilutionsinto groups of five 10-day-old embryonated chicken eggs asdescribed previously (Liu et al., 2013a). The 50% embryo infectiousdose (EID50) was calculated using the methods of Reed and Muench(1938).

Cross virus neutralization tests were performed using constant(102 EID50) viral titers and diluted serum against ck/CH/LJL/111054and H120, respectively, in SPF chickens embryos for serotyping(Liu et al., 2013b). The end-point of each serum sample was calcu-lated using the methods of Reed and Muench (1938).

2.4. Sequencing and phylogenic analysis of IBV strain ck/CH/LJL/111054

Fifteen specific primer sets spanning the entire viral genomewere used for amplifying the complete genome of ck/CH/LJL/111054, as described previously (Liu et al., 2013b). The 30 and 50

ends of the viral genomes were confirmed by rapid amplificationof cDNA ends (RACE) using a 30/50 RACE kit (Takara Bio Inc., Shiga,Japan) according to the manufacturer’s instructions (Liu et al.,2013b). Viral RNA was extracted from 400 lL of ck/CH/LJL/111054viral stock, using TRIzol reagent (Invitrogen, Carlsbad, CA, USA)according to the manufacturer’s instructions. The PCR productswere cloned into the pMD 18-T vector (Takara Bio Inc.) followingthe manufacturer’s instructions and each fragment of the viral gen-ome was sequenced at least five times to determine a consensus se-quence. Prediction of the open reading frames (ORFs) wasperformed using the Vector NTI Advanced 10 bioinformatics soft-ware package (Invitrogen) and the sequences were analyzed usingLasergene DNAStar DNA & Protein Sequence Analysis software (ver-sion 7; Lasergene Corp, Madison, WI, USA). The pairwise nucleotideidentity was determined using Vector NTI Advanced 10 softwareand multiple sequence alignments were generated using Clustal-W nucleotide sequence alignment algorithm (Liu et al., 2013a).

Twenty reference strains which the S1 subunit sequences wereavailable and 53 IBV reference strains which the entire genomicsequences were available in the GenBank database(www.ncbi.nlm.nih.gov/genbank/) had been selected for genomicpairwise comparison and phylogenetic analysis by using the neigh-boring-joining method of DNAStar software. The results were vali-dated and confirmed by constructing maximum likelihoodphylogenetic trees with 100 bootstrap replicates under the trans-version model (TVM)+I+U4, using PhyML 3.0 software (Guindonet al., 2010) and using MEGA4.0 version at bootstrap value 1000 rep-licates (Liu et al., 2003b). The selected IBV reference strains and theiraccession numbers are shown in Fig. 1A and B, respectively.

To identify recombinant event, the complete genomic sequenceof ck/CH/LJL/111054 were pairwise compared with those ofConnecticut-type strains (Conn46 1972, Conn46 1983, Conn461991 and Conn46 1996) and Massachusetts-type strains (H120,M41, Beaudette, ck/CH/LNM/091017 and ck/CH/LHLJ/100902). Inaddition, the data sets were scanned using a Recombination Detec-tion Program (RDP) v2, and similarity plot and bootscan analyseswere performed using the Simplot program (Lole et al., 1999) toidentify recombination events and recombination breakpoints(McKinley et al., 2011). Furthermore, a phylogenetic tree wasreconstructed for each recombinant fragment to avoid phyloge-netic biases derived from ignoring recombination (Schierup andHein, 2000; Arenas and Posada, 2010). In addition, the populationrecombination rate was estimated by using the coalescent-likeli-hood programs in the LAMARC package (version 2.1.9, Kuhner,2006; Kuhner and Smith, 2007). Rho (the population recombina-tion rate) is presented as 4NrL, where N is the population size, ris the recombination rate per site and L is the sequence length(number of nucleotides).

recombinant novel avian infectious bronchitis coronavirus isolate ck/CH/4.02.015

http://www.ncbi.nlm.nih.gov/genbank/


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ck/CH/LDL/091022(JX195175)

YX10(JX840411)

DY07(HM245923)

ck/CH/LZJ/111113(JX195176)

GX/Y15(HQ848267)

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Conn46 1983(FJ904718)

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Beaudette(NC 001451)

ck/CH/LGD/120723(KC013541)

ck/CH/LGD/120724(KC119407)

Mass41 vaccine(GQ504725)

partridgeGDS142003(AY646283)

H52(EU817497)

H120(GU393335)

ck/CH/LNM/091017(JF330899)

ZJ971(EU714028)

ck/CH/LHLJ/07VII(JF274479)

SUN8067(JQ977697)

Iowa 97(GU393337)

Pea/fowl/GD/KQ6/2003(AY641576)

NGA/A116/E7/2006(FN430415)

YN(JF893452)

SAIBK(DQ288927)

SC021202(EU714029)

A2(EU526388)

TW/2575/98(DQ646405)

ck/CH/LHB/100801(JF330898)

BJ(AY319651)

ck/CH/LSD/05I(EU637854)

CK/SWE/0658946/10(JQ088078)

ITA/90254/2005(FN430414)9697

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H120(GU393335)

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Beaudette(NC 001451)

ck/CH/LHLJ1/00902(JF828980)

Conn46 1966(FJ904716)

Conn46 1983(FJ904718)

Conn46 1972(FJ904717)

Conn46 1991(FJ904719)53

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Conn-type strains Mass-type strains

A B

Fig. 1. Relationships among the ck/CH/LJL/111054, Conn-type, Mass-type and reference strains are illustrated with a maximum likelihood phylogeny unrooted tree, based onS1 nucleotides 1–1626 (A) and the full-length genomic sequences (B), respectively. The ck/CH/LJL/111054 isolate is shown in bold. The Conn- and Mass-type strains areshown in green and pink, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

S. Liu et al. / Infection, Genetics and Evolution xxx (2014) xxx–xxx 3


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2.5. Accession number

The genomic sequence of the IBV strains ck/CH/LJL/111054 wassubmitted to the GenBank database and assigned the accessionnumber KC506155.

2.6. Pathogenicity studies

Thirty 1-day-old SPF White Leghorn chicks were used to assessthe pathogenicity of the ck/CH/LJL/111054. Three groups of tenchickens were kept in isolators with negative pressure. Chickensin groups 1 and 2 were inoculated oculonasally with, respectively,a dose of log105.5 ck/CH/LJL/111054 and log105.3 H120 EID50 perchick. Birds in group 3 were mock-inoculated with sterile allantoicfluid and served as the control. Birds in each group were examineddaily for signs of infection through post-inoculation day 30. Bloodsamples were collected from all birds in each treatment group atpost-inoculation days 4, 8, 12, 16, 20, and 24. Serum samples were


assayed using a commercial total antibody enzyme-linked immu-nosorbent assay kit (IDEXX Laboratories Inc., Westbrook, ME,USA) according to the manufacturer’s instructions. Each samplewas tested in triplicate.

3. Results

3.1. Isolate ck/CH/LJL/111054 belongs to Conn type IBV

The S1 subunit sequence of isolate ck/CH/LJL/111054 had 99.6%,99.6%, 99.9%, and 99.9% identities to those of the IBV/Conn/66, IBV/Conn/72, IBV/Conn/83, and IBV/Conn/91 strains (McKinley et al.,2011), respectively. Thus, the ck/CH/ LJL/111054 isolate was clearlyof the Conn genotype, although it also shared 95.5% and 94.5%identities with those of Mass-type M41 and H120, respectively.In addition, IBV Mass-type M41 and H120 had not more 94.5%and 94.4% identities with those of Conn-type strains.



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3.2. Isolate ck/CH/LJL/111054 possibly originated from recombinationbetween the Conn- and Mass-type strains

To examine sequence characteristics not only in S1 subunit, butalso over the entire length of the genome, we sequenced the full-length genome sequence of isolate ck/CH/LJL/111054, which wasassembled into one contiguous sequence to represent the entireviral genome. A sequence of 27,632 nucleotides was obtained fromck/CH/LJL/111054, excluding the poly-A tail at the 30 end. The cod-ing capacity and organization of the ck/CH/LJL/111054 genomewas similar overall to those of other IBVs. However, the phyloge-netic analysis using S1 subunit sequence of the ck/CH/LJL/111054was different than the phylogenetic analysis using the full-lengthgenomic sequences (Fig. 1B). The nucleotide identities betweenisolate ck/CH/LJL/111054 and strains IBV/Conn/66, IBV/Conn/72,IBV/Conn/83, and IBV/Conn/91 were 96.3%, 96.4%, 96.4%, and96.3%, respectively, which was lower than those of the S1 subunitsequence, which reportedly exhibited great genetic variation in theIBV genome and used to determine IBV type (Cavanagh and Gelb,2008). However, isolate ck/CH/LJL/111054 shared genomic nucleo-tide identities of 91.1% and 95.5% with strains M41 and H120,respectively. Therefore, the similarity between ck/CH/LJL/111054and H120 was close to that between isolate ck/CH/LJL/111054and Conn-type strains.

We conducted a pairwise comparison of the genomic sequenceof the isolate ck/CH/LJL/111054 with four Conn-type strains,Conn46 1966, Conn46 1972, Conn 46 1983, and Conn46 1991,and two Mass-types, M41 and H120. From the 50 untranslatedregion (UTR) to genome position 2754 in the non-structural pro-tein 3 (nsp3) sequence of strain ck/CH/LJL/111054 showed a muchcloser relationship and greater nucleotide sequence identity to thatof Mass-type H120 than to the Conn strains (Supplementary file 1).However, the ck/CH/LJL/111054 isolate fell into the same group asConn strains from location 2772 to 3649 in the phylogenetic treeand shared 100% identity with those of the Conn-type, in contrastto 77.4% and 87.6% identities with those of Mass-type H120 vac-cine and M41 strains, respectively. It is worth noting that theck/CH/LJL111054 isolate from position 3657 to 13,332 had a nearlyidentical sequence (99.9%) to that of H120 and was different fromthose of the M41 and Conn strains (89.6% and 91.3%, respectively).Interestingly, the ck/CH/LJL/111054 isolate had more than 99.5%nucleotide identity with the Conn strains from nt 13346 to the 30

end of the genome, including the 30 end of genes 1, S, 3, M, the5th accessory protein gene, N, and 30 UTR. Here, we investigatedthe presence of three recombination breakpoints, two in nsp3and one in nsp12 (Supplementary files 2 and 3), in which the firstrecombination breakpoint was confirmed by Simplot analyses(Fig. 2). The phylogenetic analysis also showed the same resultsas the sequence identity analysis using the corresponding genefragments (Fig. 3). These data strongly suggested that ck/CH/LJL/111054 arose from a homologous RNA recombination event frommultiple template switches between Mass- and Conn-like viruses.These results implied that template switches occurred within thensp3 and nsp12 sequences.

The population recombination rate was estimated using theLAMARC package. The maximum likelihood estimates (MLE) of hand r were 2.377914 and 0.000590, respectively, and the sequencelength of ck/CH/LJL/111054 is 27636 bp, therefore, The Rho calcu-lated by Lamarc is 38.77, which is actually a high recombinationrate.

3.3. The genetic similarities of ck/CH/LJL/111054 with other Conn typeviruses

The percent nucleotide similarities between strains ck/CH/LJL/111054 and H120 at the 50 end of the genome (from the 50 UTR


to genome position 2754) were 99.4% and 99.8%, respectively, indi-cating that other than recombination events, the ck/CH/LJL/111054isolate experienced multiple mutations in the genome over time. Inaddition, the Conn-like ck/CH/LJL/111054 sequences in both the S1subunit sequence (Table 1) and full-length genome was closer tothose of Conn 46 1983 and 1991, especially a 3-bp nucleotideinsertion at genome position 20,521–20,525 among ck/CH/LJL/111054, Conn 46 1983, and Conn 46 1991, by comparison to thoseof Conn 46 1966 and 1973.

3.4. The ck/CH/LJL/111054 isolate is serologically different from Mass-type IBV

The last dilution of each serum against ck/CH/LJL/111054 andH120, which protected 50% of the embryos against 102 EID50 ofstrains ck/CH/LJL/111054 and H120, was tested at 1:158.68 and1:81.00, respectively. However, serum against strain ck/CH/LJL/111054 could not neutralize 102 EID50 of H120 and vice versa, indi-cating that the ck/CH/LJL/111054 isolate was antigenically distinctfrom the IBV Mass serotype. Due to the unavailability of theConn-type virus and the serum against this type of virus in our lab-oratory, we could not serologically identify the antigenic changesof the ck/CH/LJL/111054 isolate after recombination and evolutionover time, by comparison with the Conn-type virus.

3.5. Virulence and seroconversion

Mild clinical signs were observed in some of the ck/CH/LJL/111054-infected chicks from post-inoculation days 3 to 10, includ-ing listlessness, huddling, and ruffled feathers. None of the chicksdied during the experiment and no overt disease was observed inchicks of the H120-inoculated and control groups. In addition,chicks inoculated with strains ck/CH/LJL/111054 and H120 werenegative for IBV antibodies until post-inoculation day 8. At eachtime point from post-inoculation day 8 onwards, the ck/CH/LJL/111054-inoculated chicks had comparable, but slightly higherseroconversion rates than those inoculated with strain H120(Table 2).

4. Discussion

By phylogenetic analysis of the S1 subunit sequence and full-length genomic sequence, and extensive pairwise comparisons ofthe genomic sequences of strain ck/CH/LJL/111054 with those ofthe Conn- and Mass-type strains, multiple recombinant eventsand three recombination breakpoints were identified during theevolution of the virus. The first recombination breakpoint was con-firmed by the Simplot analyses. In addition, we surveyed the wholegenome of the three virus strains by using the LAMARC package forcalculating the recombination rate. The population recombinationrate is 38.77, which is actually a high recombination rate, suggest-ing the recombinant events happened in the genome of IBV ck/CH/LJL/111054 strain. Like most other RNA viruses, template switchingby RNA-dependent RNA polymerases is a mechanism that contrib-utes to genetic recombination events and sequence diversity incoronaviruses (Lai, 1992). In this study, three recombination break-points were identified in the genome of ck/CH/LJL/111054 strain,two located within the nsp3 sequence and one within the nsp12sequence. The recombination events leading to formation of theck/CH/LJL/111054 sequence can be explained by two models. Inthe simplest scenario, recombination may have involved onlytwo parental viral strains with RNA replication initiating in aMass-like template of either negative or positive polarity and thenswitching of the polymerase-nascent cRNA complex to a Conn-likevirus template, followed by double switches in the nsp3 and 12



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Fig. 2. Simplot analysis to detect recombination and estimate recombination breakpoints within the ck/CH/LJL/111054 genome. Similarity plot using ck/CH/LNM/091017(Accession number: JF330899) as the query sequence. The arrows showed the deduced recombination breakpoint (2755–2771).



11 March 2014

sequences, respectively. The first switch may have occurred at thebeginning of the nsp3 sequence. Alternatively, a more complicatedscheme was proposed. In this scheme, a Mass–Conn hybrid arosefrom a single switch at either of the three or two recombinationbreakpoints and spread among the chicken flocks and in turn expe-rienced additional recombination events with another Conn-likevirus.

The Mass-like sequences in the ck/CH/LJL/111054 genomeshowed high genetic similarities and were closely related to a MassH120 vaccine, compared to that of strain M41, implicating a possi-ble H120-related vaccine origin. Mass-type vaccines are very com-monly used in chicken flocks in China and can persist in the fieldfor long periods. Until now, no Conn-like viruses have been foundin chicken flocks in China, however, Mass–Conn bivalent vaccinesis used extensively throughout China, although it is not govern-ment approved. Due to the unavailability of the sequence ofConn-type vaccine strains so far, we cannot compare the sequencesin the genome of isolate ck/CH/LJL/111054 with those of a Conn-type vaccine strain.

The 50 UTRs in the IBV genome, like other CoVs, usually harborimportant structural elements involved in replication and/or trans-lation (Chang et al., 1994; Raman et al., 2003; Raman and Brian,2005). The nsp3 and 5 sequences downstream of the 50 UTR con-tain papain-like protein 2 proteases and the main protease, respec-tively, which cleaves nsp2, 3, 4, and 5–16, respectively (Masters,2006). Nsp2, 4, and 6 contain hydrophobic residues predicted toplay a role in anchoring the replication/transcription complex tothe Golgi apparatus. Nsps 7, 8, 9, and 10 are reported to haveRNA-binding activity, whereas nsp11/12 contains the RNA-depen-dent RNA-polymerase (Masters, 2006). Perhaps the acquisition ofthe 50 UTR, nsp2, parts of nsp3, nsp4–11, and parts of nsp12 from


Mass-like virus by ck/CH/LJL/111054 alter the viral replicationefficiency, and thus alter its pathogenity in chickens because it isreported that the replicase gene of avian coronavirus, infectiousbronchitis virus, is a determinant of pathogenicity (Hodgsonet al., 2004; Armesto et al., 2009). Therefore, investigations usingreverse genetic systems should provide further insight into thisissue and increase our understanding of IBV pathogenesis.

IBV pathogenicity was shown to be polygenic, involving thespike as well as replicase proteins (Brooks et al., 2004; Cavanaghet al., 1992; Fang et al., 2005; Jackwood et al., 2010) and the recom-bination between vaccine strains might result in a virus thatacquired attenuated vaccine viral sequences, thus the outcomemay likely be a relatively benign virus regardless of the type ofparental viruses involved. It appeared that the emergence of mostvariants or new IBV types and outbreaks of new diseases are due tothe accumulation of mutations in the regions that contain genesimportant for pathogenicity, especially the spike glycoprotein gene(McKinley et al., 2011). The ck/CH/LJL/111054 isolate likely origi-nated from two vaccines by recombination events; however, theviral genome experienced mutations during its evolution. Althoughit is difficult to determine which mutation(s) play key roles inpathogenicity, some chickens showed clinical signs after challengewith strain ck/CH/LJL/111054, indicating altered virulence com-pared with the two parental vaccine viruses. However, this obser-vation requires further investigation.

The ck/CH/LJL/111054 isolate was shown to be antigenicallydistinct from the Mass-type IBVs via cross neutralization analysisin this study. This was not unexpected because analysis of the S1subunit sequence, which contains conformationally dependentvirus-neutralizing and serotype-specific epitopes (Cavanagh et al.,1998), showed that strain ck/CH/LJL/111054 was a Conn-type,



416

417

418

419

420

421

422

423

424

425

426

427

428

429

430

431

432

433Q3

Conn46 1972

Conn46 1996

Conn46 1991

Conn46 1983

Beaudette

ck/CH/LHLJ/100902

M41 99

93

ck/CH/LJL/111054

H120

ck/CH/LNM/091017 61

100

74

39

100

0.005

1-2754

3657-13332 Conn46 1972

Conn46 1996

Conn46 1983

Conn46 1991

Beaudette

ck/CH/LHLJ/100902

M41

ck/CH/LNM/091017

H120.

ck/CH/LJL/111054

100

943 100

100

100

100

53

0.01

2772-3649 Conn46 1983

Conn461996

Conn46 1972

Conn46 1991

ck/CH/LJL/111054

ck/CH/LHLJ/100902

M41

Beaudette

H120

ck/CH/LNM/091017 100

100

73

100

0.02

13346-27636 Conn46 1972

Conn46 1996

ck/CH/LJL/111054

Conn46 1983

Conn46 1991

Beaudette

H120

ck/CH/LNM/091017

ck/CH/LHLJ/100902

M41 100

100

74

98

0.005

Fig. 3. Phylogenetic analysis of genome positions 1–2754, 2772–3649, 3657–13,332, and 13,346–27,636 among ck/CH/LJL/111054 and four Conn-type and five Mass-typestrains. The trees were constructed using the neighbor-joining method.

Table 1Pairwise comparison of nucleotide sequences of the S gene between Conn46 strainsand isolate ck/CH/LJL/111054.

Strain 374a 986 3316 3322 3328 3333 3336 3348

Conn46 1966 G T T C A T C AConn46 1972 G T T C A T C Ack/CH/LJL/

111054A G C T G G T T

Conn46 1983 A G C T G G T TConn46 1991 A G C T G G T T

a Position in S1 gene. The nucleotides identical to those in strain ck/CH/LJL/111054 are highlighted in bold.



11 March 2014

which is serotypically different from the Mass-type strains(Cavanagh and Gelb, 2008). Due to the unavailability of theConn-type virus and serum against this type of viruses in our

Table 2Results of the pathogenicity study using four IBV strains.

Groupa Dose, median embryo infectious doses (log10)b Morbidity (%

CK/CH/LJL/111054 5.5 40H120 5.3 0Control – 0

a Ten chicks per group.b Dose per chick, 100 lL.c Number seroconverted/number inoculated.d Days after challenge.


laboratory, we could not serologically identify the antigenicchanges in isolate ck/CH/LJL/111054 after recombination and evo-lution over time, by comparisons with that of Conn-type viruses.

Recombination events contribute to the genetic diversity ofCoVs and can lead to the emergence of new viruses and subsequentoutbreaks of new diseases (Woo et al., 2009). High frequencies ofrecombination events between IBV vaccine and field strains havebeen reported (Masters, 2006; Kusters et al., 1989; Wang et al.,1993; Liu et al., 2013b). Herein, we isolated an IBV strain whichmay have originated from recombinant events betweenMass- and Conn-like strains. The implication of our resultsemphasizes the importance of IBV surveillance in chicken flocksvaccinated with IBV live vaccines.

5. Uncited reference

Liu et al. (2013c).

) Mortality (%) Antibody (%)c

4d 8 12 16 20

0 0/10(0) 3/10(30) 7/10(70) 8/10(80) 10/10(100)0 0/10(0) 2/10(20) 6/10(60) 7/10(70) 10/10(100)0 0/10(0) 0/10(0) 0/10(0) 0/10(0) 0/10(0)



434

435

436

437

438

439

440

441

442

443444445446447448449450451452453454455456457458459460461462463464465466467468469470471472473474475476

477478479480481482483484485486487488489490491492493494495496497498499500501502503504505506507508509510511512513514515516517518519520521522523524525526

527



11 March 2014

Acknowledgments

This work was supported by Grants from the China AgricultureResearch System (No. CARS-41-K12) and Special Fund forAgro-scientific Research in the Public Interest (No. 201203056).

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.meegid.2014.02.015.

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