Biological Property of Recombinant Hemagglutinin-Neuraminidase Protein of Avian Paramyxovirus Type 6 Expressed by Recombinant Baculovirus

Ji-Ye Kim; Hyun-Jeong Lee; Soo-Jeong Kye; Saeromi Kim; Hee-Jung Seul; Sang-Eun Kim; Hee-Soo Lee; Suk-Chan Jung; Kang-Seuk Choi

doi:10.4167/jbv.2015.45.4.319

Journal List > J Bacteriol Virol > v.45(4) > 1034190

Go to TopGo to Top Go to BottomGo to Bottom

TOOLS

Kim, Lee, Kye, Kim, Seul, Kim, Lee, Jung, and Choi: Biological Property of Recombinant Hemagglutinin-Neuraminidase Protein of Avian Paramyxovirus Type 6 Expressed by Recombinant Baculovirus

Original Article

J Bacteriol Virol 2015;45(4):319-327.

Published online: 9 February 2015

DOI: https://doi.org/10.4167/jbv.2015.45.4.319

Biological Property of Recombinant Hemagglutinin-Neuraminidase Protein of Avian Paramyxovirus Type 6 Expressed by Recombinant Baculovirus

Ji-Ye Kim¹, Hyun-Jeong Lee¹, Soo-Jeong Kye², Saeromi Kim¹, Hee-Jung Seul¹, Sang-Eun Kim⁴, Hee-Soo Lee³, Suk-Chan Jung¹, Kang-Seuk Choi^1,^∗

¹Avian Diseases Division, Animal and Plant Quarantine Agency, Gyeonggi, Korea

²Food and Mouth Disease Diagnosis Division, Animal and Plant Quarantine Agency, Gyeonggi, Korea

³Bacterial Disease Division, Animal and Plant Quarantine Agency, Gyeonggi, Korea

⁴R&D Center, Komipham Co., Gyeonggi, Korea

^∗Corresponding author: Kang-Seuk Choi, DVM, PhD. Avian Diseases Division, Animal and Plant Quarantine Agency, 175 Anyangro, Anyang, Gyeonggi, 14089, Korea. Phone: +82-31-467-1821, Fax: +82-31-467-1814, e-mail: kchoi0608@korea.kr

^∗∗ This work was fully supported by the Animal and Plant Quarantine Agency, Republic of Korea (Grant No. B-AD15-2011-14-02).

Received 12 August 20159 November 2015 Accepted 12 November 2015

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Hemagglutination inhibition (HI) test employing whole virus antigen is a prescribed serological test for serotyping, diagnosis and surveillance for avian paramyxoviruses (APMVs). For use as alternative to the virus antigen, hemagglutinin-neuraminidase (HN) protein gene of the wild duck isolate APMV-6/WB12-163FS of APMV serotype 6 (APMV-6) was amplified, cloned and expressed in Spodoptera frugiperda insect cells. The HN gene of 1,842 bps in length showed nucleotide and amino acid homology of 93.4% and 97.1%, respectively with that of APMV-6 prototype strain. Putative sialic acid binding motif and potential N-linked glycosylation sites were conserved. In Western blot analysis, the expressed protein had a molecular mass of 66 kDa and reacted specifically with antiserum to APMV-6. In addition, the recombinant HN protein showed biological properties such as hemagglutination (HA) and elution. The recombinant HN protein produced from infected cells showed high HA titers (approximately 2¹³ HA unit/ml). The HA activity of the recombinant HN protein was inhibited by antisera to APMV-6. In cross HA inhibition test, the recombinant HN protein had the highest titers with antisera to homologous APMV serotype, although there was weak cross reaction with some of antisera to other APMV serotypes. Our results indicated that recombinant APMV-6 HN protein would have the potential as alternative to the APMV-6 antigen in HI assays.

Keywords: Avian paramyxovirus-6, Hemagglutinin-neuraminidase protein, Baculovirus expression system, Hemagglutination inhibition test

REFERENCES

1). Alexander DJ. Newcastle disease and other avian paramyxoviridae infections. Calnek BW, editor. Diseases of Poultry. 10th ed.IOWA: Iowa state university press;1997. p. 541–69.

2). Miller PJ, Afonso CL, Spackman E, Scott MA, Pedersen JC, Senne DA, et al. Evidence for a new avian paramyxovirus serotype 10 detected in rockhopper penguins from the Falkland Islands. J Virol. 2010; 84:11496–504.

3). Briand FX, Henry A, Massin P, Jestin V. Complete genome sequence of a novel avian paramyxovirus. J Virol. 2012; 86:7710.

4). Terregino C, Aldous EW, Heidari A, Fuller CM, De Nardi R, Manvell RJ, et al. Antigenic and genetic analyses of isolate APMV/wigeon/Italy/3920-1/2005 indicate that it represents a new avian paramyxovirus (APMV-12). Arch Virol. 2013; 158:2233–43.

5). Warke A, Stallknecht D, Williams SM, Pritchard N, Mundt E. Comparative study on the pathogenicity and immunogenicity of wild bird isolates of avian paramyxovirus 2, 4, and 6 in chickens. Avian Pathol. 2008; 37:429–34.

6). Warke A, Appleby L, Mundt E. Prevalence of antibodies to different avian paramyxoviruses in commercial poultry in the United States. Avian Dis. 2008; 52:694–7.

7). Zhang GZ, Zhao JX, Wang M. Serological survey on prevalence of antibodies to avian paramyxovirus serotype 2 in China. Avian Dis. 2007; 51:137–9.

8). Chang PC, Hsieh ML, Shien JH, Graham DA, Lee MS, Shieh HK. Complete nucleotide sequence of avian paramyxovirus type 6 isolated from ducks. J Gen Virol. 2001; 82:2157–68.

9). Xiao S, Subbiah M, Kumar S, De Nardi R, Terregino C, Collins PL, et al. Complete genome sequences of avian paramyxovirus serotype 6 prototype strain Hong Kong and a recent novel strain from Italy: evidence for the existence of subgroups within the serotype. Virus Res. 2010; 150:61–72.

10). Schaper UM, Fuller FJ, Ward MD, Mehrotra Y, Stone HO, Stripp BR, et al. Nucleotide sequence of the envelope protein genes of a highly virulent, neurotropic strain of Newcastle disease virus. Virology. 1988; 165:291–5.

11). Nagy E, Derbyshire JB, Dobos P, Krell PJ. Cloning and expression of NDV hemagglutinin-neuraminidase cDNA in a baculovirus expression vector system. Virology. 1990; 176:426–38.

12). Lee YJ, Sung HW, Choi JG, Lee EK, Yoon H, Kim JH, et al. Protection of chickens from Newcastle disease with a recombinant baculovirus subunit vaccine expressing the fusion and hemagglutininneuraminidase proteins. J Vet Sci. 2008; 9:301–8.

13). Choi KS, Kye SJ, Jeon WJ, Park MJ, Kim S, Seul HJ, et al. Preparation and diagnostic utility of a hemagglutination inhibition test antigen derived from the baculovirus-expressed hemagglutinin-neuraminidase protein gene of Newcastle disease virus. J Vet Sci. 2013; 14:291–7.

14). Spalatin J, Hanson RP, Beard PD. The hemagglutination-elution pattern as a marker in characterizing Newcastle disease virus. Avian Dis. 1970; 14:542–9.

15). Lee EK, Jeon WJ, Kwon JH, Yang CB, Choi KS. Molecular epidemiological investigation of Newcastle disease virus from domestic ducks in Korea. Vet Microbiol. 2009; 134:241–8.

16). Choi KS, Kye SJ, Kim JY, Seul HJ, Lee HS, Kwon HM, et al. Baculovirus expression of the avian paramyxovirus 2 HN gene for diagnostic applications. J Virol Methods. 2014; 198:12–7.

17). OIE (World Organisation for Animal Health). Newcastle disease. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (Mammals, Birds and Bees), standards for diagnostic tests and vaccines. 7th ed.Paris: OIE;2012. p. 555–73.

18). Park MJ, Kye SJ, Kim JY, Kim S, Seul HJ, Park CK, et al. Generation and biological characterization of a neutralization-resistant mutant of Newcastle disease virus. J Bacteriol Virol. 2012; 42:330–8.

19). Saif YM, Mohan R, Ward L, Senne DA, Panigrahy B, Dearth RN. Natural and experimental infection of turkeys with avian paramyxovirus-7. Avian Dis. 1997; 41:326–9.

20). Shortridge KF, Alexander DJ, Collins MS. Isolation and properties of viruses from poultry in Hong Kong which represent a new (sixth) distinct group of avian paramyxoviruses. J Gen Virol. 1980; 49:255–62.

21). Bui VN, Mizutani T, Nguyen TH, Trinh DQ, Awad SS, Minoungou GL, et al. Characterization of a genetic and antigenic variant of avian paramyxovirus 6 isolated from a migratory wild bird, the red-necked stint (Calidris ruficollis). Arch Virol. 2014; 159:3101–5.

22). Karamendin K, Kydyrmanov A, Seidalina A, Jenckel M, Starick E, Grund C, et al. Complete Genome Sequence of Avian Paramyxovirus Strain APMV-6/red-crested pochard/Balkhash/5842/2013 from Kazakhstan. Genome Announc. 2015; 3:e00158–15.

Figure 1.

Agarose gel electrophoresis of recombinant plasmids digested with restriction enzymes of EcoR I and Hind III. M, DNA marker; Lane 1, pCR/APMV6HN; lane 2, pFB/APMV6HN.

Figure 2.

Predicted amino acid sequence of HN protein gene of APMV-6/WB12-163FS and comparison with APMV-6 prototype strain (APMV-6/duck/Hong Kong/199/77). Black dot (●) represents putative N-linked glycosylation site. Putative sialic acid binding motif was expressed in a box.

Figure 3.

Western blot analisis of recombinant APMV-6 HN protein using APMV-6 reference antiserum. P. protein markar; lane 1, SPF ECE fluid; lane 2, normal Sf9 cell lysate; lane 3, APMV-6/WB12-163Fs; lane 4, rAPMV-6 HN. Allow represents HN protein of APMV-6.

Figure 4.

Measurement of NA activity of the rAPMV-6 HN protein expressed in insect cells.

Table 1.

Heat stability and hemagglutination elution pattern of th rAPMV-6 HN protein

	HA titeer
	Heat treatment^a		Incubation at 4°C
	Before	After	1 h	24h
APMV-6^b	256	128	256	256
rAPMV6 HN	256	4	256	256

^a Heat treatment represents treatment of test sample for 30 min a 56℃.

^b APMV-6 isolate (APMV-6/WB12-163FS) used as genetic templat for rAPMV-6 HN.

Table 2.

Results of cross HI test using recombinant APMV-6 HN protein and representative APMV serotypes

Antiserum	Antigen
Antiserum	rAPMV-6HN	APMV-1	APMV-2	APMV-3	APMV-4	APMV-6	163FS^c	APMV-7	APMV-8	APMV-9
rAPMV-6 HN	256^a	2	8	<2	2	256	256	2	4	<2
APMV-1	<2	128	NT^b	NT	NT	<2	<2	NT	NT	NT
APMV-2	<2	NT	512	NT	NT	8	32	NT	NT	NT
APMV-3	<2	NT	NT	512	NT	16	<2	NT	NT	NT
APMV-4	<2	NT	NT	NT	32	<2	<2	NT	NT	NT
APMV-6	128	<2	16	<2	<2	512	64	4	2	<2
163FS	512	8	32	16	2	1024	256	8	16	2
APMV-7	16	NT	NT	NT	NT	<2	16	256	NT	NT
APMV-8	<2	NT	NT	NT	NT	2	4	NT	256	NT
APMV-9	<2	NT	NT	NT	NT	8	8	NT	NT	256
Negative serum	<2	<2	<2	<2	<2	<2	<2	<2	<2	<2

^a HI titers

^b NT, not tested

^c APMV-6 isolate (APMV-6/WB12-163FS) used as genetic template for rAPMV-6 HN.

TOOLS

Similar articles