JGV Papers in Press. Published January 27, 2015 as doi:10.1099/vir.0.000067

Journal of General Virology Development of a Novel Thermostable Newcastle Disease Virus Vaccine Vector for Expression of a Heterologous Gene --Manuscript Draft-Manuscript Number:

JGV-D-14-00214R1

Full Title:

Development of a Novel Thermostable Newcastle Disease Virus Vaccine Vector for Expression of a Heterologous Gene

Short Title:

Thermostable Newcastle Disease Virus Vaccine Vector

Article Type:

Standard

Section/Category:

Animal - Negative-strand RNA Viruses

Corresponding Author:

Qingzhong Yu USDA-ARS, Southeast Poultry Research Laboratory Athens, GA UNITED STATES

First Author:

Guoyuan Wen

Order of Authors:

Guoyuan Wen Chen Chen Jing Guo Zhenyu Zhang Yu Shang Huabin Shao Qingping Luo Jun Yang Hongling Wang Hongcai Wang Tengfei Zhang Rongrong Zhang Guofu Cheng Qingzhong Yu

Abstract:

Thermostable Newcastle disease virus (NDV) vaccines have been used widely to control Newcastle disease (ND) for village flocks, due to their independence of cold chains for delivery and storage. To explore the potential use of the thermostable NDV as a vaccine vector, an infectious clone of thermostable avirulent NDV strain TS09-C was developed using reverse genetics technology. The green fluorescence protein (GFP) gene, along with the self-cleaving 2A gene of foot-and-mouth disease virus and Ubiquitin monomer (2AUbi), were inserted immediately upstream of the NP, M, or L gene translation start codon in the TS09-C infectious clone. Detection of GFP expression in the recombinant virus-infected cells showed that the recombinant virus, rTS-GFP/M, with the GFP inserted into the M gene expressed the highest level of GFP. The rTS-GFP/M virus retained the same thermostability, growth ability, and pathogenicity as its parental rTS09-C virus. Vaccination of specific pathogen free (SPF) chickens with the rTS-GFP/M virus conferred complete protection against virulent NDV challenge. Taken together, the data suggested that the rTS09-C virus could be used as a vaccine vector to develop bivalent thermostable vaccines against ND and the target avian diseases for village chickens, especially in the developing and least-developed countries.

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1

Development of a Novel Thermostable Newcastle Disease Virus Vaccine Vector

2

for Expression of a Heterologous Gene

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Guoyuan Wen 1,2,4, †, Chen Chen 1,3, †, Jing Guo 1,3, †, Zhenyu Zhang 2, †, Yu Shang 1,3,

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Huabin Shao 1, Qingping Luo 1, Jun Yang 1, Hongling Wang 1, Hongcai Wang 1,

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Tengfei Zhang 1, Rongrong Zhang 1, Guofu Cheng 3, Qingzhong Yu 2,*

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Agricultural Sciences, Wuhan 430070, China

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Southeast Poultry Research Laboratory, Agricultural Research Services, United States Department of Agriculture, Athens, GA 30605, USA

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Institute of Animal Husbandry and Veterinary Sciences, Hubei Academy of

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Veterinary Pathology Laboratory, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, China

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China

Hubei Key Laboratory of Animal Embryo and Molecular Breeding, Wuhan 430070,

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Running title: Thermostable Newcastle Disease Virus Vaccine Vector

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† These authors contributed equally to this work

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* Corresponding author. Southeast Poultry Research Laboratory, Agricultural

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Research Services, United States Department of Agriculture, Athens, 30605, USA

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Tel: +1 706 546 3628; Fax: +1 706 546 3161.

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E-mail address: [email protected]

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SUMMARY

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Thermostable Newcastle disease virus (NDV) vaccines have been used widely to

26

control Newcastle disease (ND) for village flocks, due to their independence of cold

27

chains for delivery and storage. To explore the potential use of the thermostable NDV

28

as a vaccine vector, an infectious clone of thermostable avirulent NDV strain TS09-C

29

was developed using reverse genetics technology. The green fluorescence protein

30

(GFP) gene, along with the self-cleaving 2A gene of foot-and-mouth disease virus and

31

Ubiquitin monomer (2AUbi), were inserted immediately upstream of the NP, M, or L

32

gene translation start codon in the TS09-C infectious clone. Detection of GFP

33

expression in the recombinant virus-infected cells showed that the recombinant virus,

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rTS-GFP/M, with the GFP inserted into the M gene expressed the highest level of

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GFP. The rTS-GFP/M virus retained the same thermostability, growth ability, and

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pathogenicity as its parental rTS09-C virus. Vaccination of specific pathogen free

37

(SPF) chickens with the rTS-GFP/M virus conferred complete protection against

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virulent NDV challenge. Taken together, the data suggested that the rTS09-C virus

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could be used as a vaccine vector to develop bivalent thermostable vaccines against

40

ND and the target avian diseases for village chickens, especially in the developing and

41

least-developed countries.

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INTRODUCTION

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Newcastle disease (ND) is one of the most important infectious diseases of poultry

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due to the potential for devastating losses (Miller & Guus, 2013). The causative agent

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of the disease, Newcastle disease virus (NDV), has been classified into lentogenic,

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mesogenic, and velogenic pathotypes based on the pathogenicity for chickens. Birds

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infected with lentogenic NDV showed little or even no clinical signs. Velogenic NDV

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can produce severe disease, characterized as typical neurological and respiratory signs

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with high mortality, and poses a considerable threat to the poultry industry worldwide

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(Pedersen et al., 2004). Reporting of velogenic ND outbreaks is required for the

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member nations of the World Organization for Animal Health (OIE).

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NDV is an enveloped, non-segmented, negative-stranded RNA virus, and belongs

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to the Avulavirus genus in the Paramyxoviridae family (Lamb et al., 2005). The NDV

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genome is approximately 15.2 kb in size and consists of six genes flanked by the 3’

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Leader and 5’ Trailer in the order of 3’-nucleocapsid protein (NP)-phosphoprotein

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(P)-matrix protein (M)-fusion protein (F)-hemagglutinin-neuraminidase (HN)-large

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polymerase protein (L) (de Leeuw & Peeters, 1999; Peeters et al., 2000). Each gene is

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flanked by conserved gene start (GS) and gene end (GE) sequences. Two additional V

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and W proteins are derived from P gene by RNA editing (Steward et al., 1993).

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Control of ND by vaccination is the common strategy in both intensively raised

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commercial flocks and scavenging village flocks. The ND vaccine strains, such as

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LaSota and Hitchner B1, have been used widely in commercial flocks. However,

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these vaccines are not generally suitable in village flocks (Aini et al., 1990). The main

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problem associated with these vaccines is their thermo-instability and subsequent

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requirement of a cold chain for the delivery of viable vaccines to villages.

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Refrigeration is essential for the storage and delivery of vaccines to maintain their

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quality. The cold chain consumes approximately 80% of total cost for the vaccination

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programs (Das, 2004). The problem is even worse for the developing and

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least-developed countries, because of the lack of reliable and extensive refrigeration

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infrastructure. Thermostable NDV strains, such as V4 and I2, offer a solution to the

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problem (Spradbrow, 1993/94). When coated into the carrier food, the V4-UPM

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vaccine was found to be stable for a minimum of 3 weeks at 21 to 27 ºC (Echeonwu et

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al., 2008a), and still able to spread between chickens by direct contact (Bancroft &

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Spradbrow, 1978). A total of 1.0 ml of I2 strain reconstituted from lyophilisate could

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vaccinate several thousands of chickens after stored at 26 to 32 ºC for 6 days (Tu et al.,

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1998). The I2 vaccine diluted with 1% gelatin could still produce an antibody response

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after being stored for 12 weeks at 22 ºC (Bensink & Spradbrow, 1999). Furthermore,

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these thermstable NDV vaccines can be administered through several immunization

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routes, such as eye drop, drinking water, spray, and food (Bell et al., 1995; Echeonwu

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et al., 2008a; Mazija et al., 2010). Therefore, these thermostable NDV strains are

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suitable vaccines for village chickens, especially in developing and least-developed

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countries.

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During the past decade, NDV has been confirmed to be a suitable vector to express

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foreign genes using reverse genetics technology for vaccine and gene therapy

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purposes. A number of foreign antigens derived from avian viral pathogens such as

5

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the hemagglutinin (HA) gene of avian influenza virus, the VP2 gene of infectious

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bursal disease virus, the S2 gene of infectious bronchitis virus, the glycoprotein B (gB)

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and D (gD) of infectious laryngotracheitis virus, and the glycoprotein (G) gene of

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avian metapneumovirus (DiNapoli et al., 2010; Hu et al., 2011; Huang et al., 2004;

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Toro et al., 2014; Zhao et al., 2014), have been expressed by NDV as bivalent

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vaccines against NDV and the target avian pathogen challenges. However, most of the

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NDV vaccine vector strains are thermolabile, and may not be suitable for use as

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vaccine vectors for the backyard chickens.

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Previously, we developed a new thermostable NDV strain TS09-C by serial passage

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of the V4 strain in BHK-21 cells. The TS09-C strain retained the thermostability and

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lentogenic pathotype as its parental virus, but grew to a higher titer in BHK-21 cells

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than the V4 strain (Wen et al., 2013). In the present study, we utilized the TS09-C

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strain as a backbone to develop a thermostable NDV vaccine vector using the reverse

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genetics technology. The green fluorescence protein (GFP) gene as a reporter, along

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with the self-cleaving 2A gene of foot-and-mouth disease virus and Ubiquitin

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monomer (2AUbi) (Tscherne et al., 2006), were inserted immediately upstream of the

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NP, M, or L gene translation start codon in the TS09-C vector. Evaluation of the

104

thermostability, pathogenicity, growth dynamics, GFP expression of these rescued

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recombinant viruses and their protective efficacy against virulent NDV challenge

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demonstrated that the TS09-C virus can be used as a thermostable vaccine vector.

107

RESULTS

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Generation of rTS09-C and rTS-GFP/M expressing GFP

6

109

A full-length cDNA clone of the thermostable NDV TS09-C strain was constructed

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and used as a backbone for generation of recombinant cNDA clones containing the

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GFP reporter gene. A fusion sequence encoding GFP and 2AUbi was inserted

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immediately upstream of the M gene translation start codon to generate the

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recombinant plasmid pTS-GFP/M (Fig. 1a). The C- and N-termini of GFP were fused

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with the 2AUbi and the first 20 amino acids of M gene, respectively. The 2AUbi was

115

a 94 amino acids fragment containing self-cleavage FMDV 2A peptide and Ubiquitin

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coding sequences (Tscherne et al., 2006). The inserted GFP protein could be

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self-cleaved from the NDV M protein by the unique feature of 2AUbi. The total

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length of cDNA clone in the pTS-GFP/M plasmid is 16,332 nucleotides and divisible

119

by 6, abiding by the “Rule of Six” (Kolakofsky et al., 2005). The recombinant NDV

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rTS09-C and rTS-GFP/M were rescued successfully in BHK-21 cells.

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To detect the recombinant virus replication and GFP expression, infected cells were

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immunostained with anti-NDV polyclonal antibody and examined by fluorescence

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microscopy. Fig. 1b illustrated that rTS-GFP/M infected cells showed both red and

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green fluorescence whereas rTS09-C infected cells displaced only red fluorescence,

125

demonstrating that rTS-GFP/M expressed GFP in the infected cells. To investigate

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whether GFP was incorporated into rTS-GFP/M particles, the virions of rTS-GFP/M

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and rTS09-C were purified by sedimentation through sucrose gradients, and examined

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by electron microscopy and Western-blot analysis. Both rTS-GFP/M and rTS09-C

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virions showed typical NDV morphology, with densely arrayed spikes on their

130

envelopes (Fig. 1c). The purified virions of rTS09-C and rTS-GFP/M contained the

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NDV structural proteins, including HN, NP, F1, P, and M (Fig. 1d). The GFP proteins

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were recognized by anti-GFP serum in the purified virions of rTS-GFP/M, but not in

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those of rTS09-C (Fig. 1e), suggesting that the GFP might be incorporated into the

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viral particles.

135

Optimization of GFP expression in rTS09-C

136

In order to determine the optimal insertion site of GFP into the NDV vector, three

137

more rTS-GFPs with GFP inserted at different locations in the backbone of NDV

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strain rTS09-C were generated as illustrated in Fig. 2a. The possible effect of GFP

139

insertion on the growth of the rTS-GFPs was examined by virus titration. As shown in

140

Fig. 2b, most of the recombinant viruses displayed similar growth dynamics as the

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parental rTS09-C virus, except the rTS-GFP/L had a slow growth curve with a

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proximately 2 log10 titer lower than other recombinant viruses at 72 h post-infection.

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The result indicated that the effect of GFP insertion on the recombinant virus growth

144

was depended on the locations of insert. The levels of GFP expression from the

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rTS-GFPs infected BHK-21 cells were examined by fluorescence microscopy and

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Western-blot analysis. As shown in Fig. 2c, all the rTS-GFPs were able to express

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GFP, but with different levels of fluorescence intensity in the order of rTS-GFP/M >

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rTS-GFP/NP > rTS-GFP/M2 > rTS-GFP/L. As the infection progresses, the increase

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in GFP expression was observed in the infected cells, and also detected by

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Western-blot analysis (Fig. 2d). Clearly, the insertion of GFP-2AUbi into the M gene

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of NDV vector resulted in the highest level of GFP expression in BHK-21 cells.

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Biological characterization of rTS09-C expressing GFP

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To determine whether the insertion of GFP affects the viral thermostability,

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pathogenicity and replication, the recombinant viruses, rTS-GFP/M and rTS09-C,

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were examined in vitro and in vivo by performing the thermostability, ICPI, MDT and

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titration assays. As shown in Fig. 3a, the average times of 2 log2 decrease in HA

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activity of rTS09-C, rTS-GFP/M, and LaSota were 87 min, 47 min, and 1 min,

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respectively, demonstrating that the HA thermostability of NDV rTS09-C and

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rTS-GFP/M strains was much higher than that of the control LaSota strain. The

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average times of 2 log10 decrease in infectivity of rTS09-C, rTS-GFP/M, and LaSota

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were 20, 15, and 3 min, respectively (Fig. 3b). The infectivity inactivation rate of

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rTS-GFP/M was 5-fold slower than that of the LaSota strain at 56 ºC. According to

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the criteria for the thermostability of NDV strains (Lomniczi, 1975), the NDV strains

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rTS09-C and rTS-GFP/M belong to the thermostable virus (the time of 2 log10

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decrease in infectivity >10 min at 56 ºC). Both rTS09-C and rTS-GFP/M viruses

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retained their lentogenic pathotype with the MDT values greater than 168 h and the

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ICPI values being 0. There were no significant differences between the rTS09-C and

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rTS-GFP/M in growth in either BHK-21cells or eggs (Table 1). These data

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demonstrated that the NDV rTS09-C vector was thermostable and avirulent, and the

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insertion of GFP did not apparently change the biological property of this vector.

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Efficient replication of rTS09-C in BHK-21 cells in the absence of trypsin

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The fusion protein of most NDV avirulent strains could not be cleaved by cellular

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proteases in infected cells. An extracellular protease, such as trypsin, was required for

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the cleavage of the fusion protein to allow virus multiple replicating. Therefore, most

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avirulent NDV strains could not replicate in cells in the absence of trypsin. Here, the

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ability of trypsin-dependence of NDV avirulent strain rTS09-C was evaluated. As

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shown in Fig. 4a, in the absence of trypsin, the virus titers of rTS09-C increased from

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104.0 TCID50/ml at the 1st passage to 106.5 TCID50/ml at the 3rd passage, with no

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further significant difference in virus titers between the 3rd and the 4th passage of

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rTS09-C. In the presence of trypsin, the virus titers from different passages changed

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little (~ 106.6 - 107.0 TCID50/ml). In the absence of trypsin, the rTS09-C virus

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replicated slightly slower and reached to a peak titer one day later than in the presence

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of trypsin (Fig. 4b). Similar results were also observed by fluorescence microscopy on

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the BHK-21 cells infected with the NDV rTS-GFP/M in the presence or absence of

185

trypsin (Fig. 4c). Nevertheless, these results proved that the NDV avirulent strains

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rTS09-C and rTS-GFP/M replicated efficiently in BHK-21 cells in the absence of

187

trypsin.

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Protective efficacy and immunogenicity of rTS09-C expressing GFP

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To evaluate whether the thermostable avirulent virus rTS-GFP/M could protect the

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immunized chickens against virulent NDV challenge, chickens were vaccinated by

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IN/IO routes with rTS-GFP/M, V4, or PBS and challenged with the virulent strain

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CA02. As expected, the chickens in the PBS control group developed conjunctivitis

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and severe depression from 3 day post-challenge (DPC) and all birds died at 5 PDC.

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Whereas the birds in rTS-GFP/M and V4 vaccinated groups survived the challenge

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and did not show any clinical signs (Table 2). The immunogenicity of the NDV

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recombinant viruses determined by the HI test showed that rTS-GFP/M induced a

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slightly lower NDV-specific HI titer than V4 strain. The data confirmed that the

198

rTS-GFP/M virus maintained its immunogenicity and conferred completely protection

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of chickens against the virulent NDV challenge.

200

DISCUSSION

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The thermostable avirulent NDV strain V4 was isolated from proventriculus of a

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chicken in Australia in 1966 (Simmons, 1967). After that, several thermostable NDV

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strains, such as V4-UPM, and I2, were isolated and characterized (Bensink &

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Spradbrow, 1999; Ideris et al., 1990). These NDV isolates have several advantages,

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including thermostable, avirulent, spread between chickens, and easy to be

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administered through drinking water, spray, and food. Thus, they have been widely

207

used as vaccines to control ND for village flocks (Aini et al., 1990). Since the

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establishment of reverse genetics systems for NDV, many thermolabile NDV strains

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have been developed as vaccine vectors for generation of bivalent vaccines against

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avian diseases. However, the potential use of thermostable NDV as a vaccine vector

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has been seldom reported. In the present study, a reverse genetics system for NDV

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thermostable avirulent strain TS09-C was developed. The GFP gene was inserted into

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the genome of the TS09-C strain at various locations as a reporter. Evaluation of the

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optimal GFP expression, thermostability, pathogenicity, and immunogenicity of the

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rTS09-C based recombinant viruses demonstrated that the rTS09-C virus could be

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used as a thermostable vaccine vector.

217 218

For most of the NDV vaccine vectors, the foreign genes were expressed from an additional transcriptional unit in the NDV genome (Engel-Herbert et al., 2003;

11

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Nakaya et al., 2001), which may affect transcription efficiency of the downstream

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viral genes and virus replication dynamics (Zhao et al., 2015). In this study, we

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developed a novel approach for expression of a foreign gene by inserting the reporter

222

gene, GFP, into the M gene transcriptional unit of the thermostable rTS09-C strain to

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produce a polyprotein GFP-2AUbi-M. The self-cleavage of the polyprotein

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GFP-2AUbi-M resulted in generation of the GFP and NDV M proteins. Thus, the

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expression level of the foreign gene would be the same as the NDV M protein. Our

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results confirmed that the GFP was expressed as a fusion protein with NDV M protein,

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and then separated from the M protein by self-cleaving of 2AUbi in embryonated eggs

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and DF1 cells. The Western blot analysis of the purified virions indicated that the GFP

229

might be incorporated into the viral particles of rTS-GFP/M. However, we cannot rule

230

out the possibility that a small amount of GFP was co-purified with NDV particles

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which could not be detected by Coomassie staining but Western blot.

232

The level of foreign gene expression is an important criterion for evaluation of a

233

vaccine vector. Usually, a higher level of foreign protein expression by a vaccine

234

vector would induce a stronger immune response against the foreign protein.

235

Therefore, it is necessary to maximize the foreign gene expression from the NDV

236

vaccine vector by identifying the optimal insertion site. For expression of a foreign

237

gene from an independent transcriptional unit, the gene junction region between the P

238

and M gene has been approved to be the optimal site (Carnero et al., 2009; Zhao &

239

Peeters, 2003; Zhao et al., 2015). But for expression of a foreign gene from an

240

integrating transcriptional unit, the optimal insertion site has not been studied until

12

241

now. Our results showed that the GFP expression level was higher when the GFP gene

242

was integrated into the M gene transcriptional unit than it was integrated into the NP

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or L gene, and also higher than that expressed from an independent transcriptional

244

unit inserted between the P and M gene. The reason for the higher level of the GFP

245

expression by the M fusion protein approach than the traditional extra transcription

246

unit method may be due to the 5’ untranslated region (UTR) of the M gene in

247

rTS-GFP/M but not in rTS-GFP/M2. It has been reported that the 5’ UTR of NDV M

248

gene can enhance levels of GFP expression at the junction of the P and M genes

249

without altering replication of NDV (Kim & Samal, 2010).

250

Thermostable NDV vaccines have been widely used to control ND for village

251

chickens, especially in developing and least-developed countries. These vaccines

252

could be prepared simply by inoculating chicken embryos, harvesting and diluting

253

allantoic fluid, and storing at 4 ºC without freeze-drying (Bensink & Spradbrow,

254

1999). Cold chain and refrigeration were not essential for transport and short-period

255

storage of these vaccines in the village (Bensink & Spradbrow, 1999). Our

256

thermostability assays of the rTS09-C and rTS-GFP showed that rescued NDV

257

rTS09-C virus retained the thermostability as its parental V4 strain (Lomniczi, 1975),

258

and the insertion of the GFP gene into the rTS09-C vector did not apparently effect on

259

its thermostability. When diluted the rTS-GFP/M virus with 3% gelatin, the virus titer

260

decreased less than of 2 log10 after being stored at 21 ºC for 2 months (our

261

unpublished data). These results suggested that this novel thermosatble rTS09-C virus

262

could be used as a vector for the development of bivalent thermostable avian vaccines

13

263

that could be transported and short-period stored in the village without the cold chains

264

and refrigeration.

265

The importance of the activation process of NDV F protein in virus pathogenicity

266

has been well characterized. The F proteins of most virulent NDV strains have two

267

pairs of basic amino acids in the F-cleavage site, and can be cleaved by host-cell

268

proteases, as existed in a variety of tissues and cells. Consequently, the rapid spread of

269

infectious viral particles throughout the organism results in fatal systematic disease

270

(Garten et al., 1980; Nagai et al., 1976). Whereas the F proteins of most avirulent

271

strains possess two single basic amino acids in the F-cleavage site, and can be cleaved

272

only by trypsin-like protease found in the respiratory and intestinal tracts. Therefore,

273

the avirulent NDV cannot multiple-cycle replicate in cells in the absence of

274

exogenous trypsin, such as LaSota, V4 and Clone-30 strains (Ge et al., 2011; King,

275

1993; Wu et al., 1999). However, we found that the avirulent NDV stain rTS09-C, as

276

well as rTS-GFP/M, could replicate efficiently in BHK-21 cells without trypsin.

277

Sequence analysis revealed that the F-cleavage site of TS09-C strain had an

278

uncommon connecting peptide sequence (112G-K-Q-R-R-L117) with a single and a

279

paired basic amino acids. The same sequence motif (112G-K-Q-R-R-L117) at the

280

cleavage site was also observed in an Ulster 2C mutant, which can multi-cycle

281

replicate in MDBK cells in the absence of trypsin (Pritzer et al., 1990). These

282

observations indicated that the first pair of basic amino acids at position 112-113 was

283

not necessary for efficient cleavage by some host-cell proteases, and this is in

284

agreement with the protease furin, detected in mammalian cells which required a

14

285 286

R-X-R/K-R motif rather than two pairs of basic amino acids (Hosaka et al., 1991). In addition to the conservation of its parental virus thermostability, avirulent

287

pathogenicity, and growth ability, the rTS-GFP/M virus retained the immunogenicity

288

of the V4 strain. Our animal experiment showed that vaccination of SPF chickens

289

with the rTS-GFP/M conferred complete protection against virulent NDV challenge

290

although a low mean HI antibody titer was induced. This was consistent with the

291

finding that chickens vaccinated orally with V4 strain induced low levels of NDV HI

292

titer, but still survived NDV challenge (Spradbrow, 1993/94). Besides the humoral

293

immune response, both the mucosal and cell-mediated immune responses were also

294

induced in chickens by vaccination with V4 strain, which would contribute to the

295

protection against NDV challenge (Jayawardane & Spradbrow, 1995a; b).

296

In summary, this study involved the development of a novel thermostable NDV

297

vector, rTS09-C, by utilizing the reverse genetic approach. This vector has several

298

advantages including thermostable, avirulent, immunogenic, high level of foreign

299

gene expression, and efficient replication in BHK-21 cells without trypsin. Thus, the

300

rTS09-C vaccine vector can be used to develop thermostable bivalent vaccines for the

301

control of ND and a targeted avian disease in village chickens, especially in the

302

developing and least-developed countries.

303

METHODS

304

Animals and ethics statement

305 306

All animal experiments that were carried out in the present study were approved by the Institutional Animal Care and Use Committee of Southeast Poultry Research

15

307

Laboratory (SEPRL, USDA-ARS, Athens, GA). Specific pathogen free (SPF)

308

Leghorn chickens and embryonated eggs were obtained from the Southeast Poultry

309

Research Laboratory. Chickens were raised in Horsfal isolation units (Federal Designs,

310

Inc., Comer, GA) with feed and water administered ad libitum. Vaccinations of chickens and

311

challenge against virulent NDV were conducted in the BSL-3E animal facility at SEPRL. At

312

the termination of the experiments all birds were humanely euthanized in accordance to

313

an SEPRL’s Institutional Animal Care and Use Committee approved animal use

314

protocol.

315

Cells and viruses

316

BHK-21 cells (C-13; ATCC) were cultured in Dulbecco’s Modified Eagle Medium

317

(DMEM, Life Technologies, Carlsbad, CA) with 10% Fetal Bovine Serum (FBS, Life

318

Technologies, Carlsbad, CA). The NDV lentogenic strains TS09-C, V4 and LaSota

319

were maintained in the Hubei Academy of Agricultural Sciences (HBAAS, Wuhan,

320

China). The velogenic NDV strain gamefowl/USA(California)/212676/2002 (CA/02)

321

was obtained from the repository bank of pathogens in the SEPRL, USDA-ARS,

322

Athens, GA, USA.

323

Plasmid construction and virus rescue

324

For the construction of full-length cDNA clone of TS09-C strain, the genomic RNA

325

was extracted from allantoic fluid of SPF chicken embryos infected with the NDV

326

TS09-C virus by using TRIZOL (Invitrogen). Five overlapping cDNA fragments

327

covering the whole viral genome were amplified by RT-PCR using the genomic RNA

328

as template. Subsequently, the five cDNA fragments were assembled into a modified

16

329

pBR322 vector under the control of the T7 promoter and followed by a partial HDV

330

ribozyme and T7 terminator, resulting in a full length cDNA clone, designated as

331

pTS09-C. For construction of the supporting plasmids, the NP, P and L genes were

332

amplified by PCR using pTS09-C as a template with gene-specific primers, and

333

inserted into pVAX1 vector, respectively.

334

The plasmid pTS09-C was utilized as a backbone for the construction of the

335

recombinant cDNA clones with GFP inserted into different regions of the TS09-C

336

genome. The GFP gene was PCR amplified using the pEGFP-C1 vector as a template.

337

A cDNA cassette encoding the self-cleaving foot-and-mouth disease virus (FMDV)

338

2A peptide and ubiquitin monomer in tandem (2AUbi) (Tscherne et al., 2006) was

339

synthesized by Invitrogen, Carlsbad, CA, USA. The GFP fusing with 2AUbi

340

(GFP-2AUbi) was amplified by PCR using the mixture of overlapped GFP and 2AUbi

341

fragment as templates. To construct the pTS-GFP/M, two fragments of pTS09-C, the

342

short fragment (S-TS) and long fragment (L-TS), were amplified by PCR using the

343

primers S-M-F and S-M-R, and L-M-F and L-M-R, respectively. The PCR

344

amplification was performed in a final volume of 50 μl containing 30 ng of template

345

pTS09-C, 5 μl of 10×PCR buffer, 0.5 μl of dNTPs (25 mM each), 1.0 μl of each

346

primer (10 μM), and 1 μl of PfuUltra II Fusion HS DNA polymerase (Agilent). The

347

PCR was carried out at 92 ºC for 2min for denaturation, 30 cycles of 92 ºC for 10 sec,

348

55 ºC for 20sec, and 68 ºC for 15 min, and final extension at 68 ºC for 10 min. The

349

pTS-GFP/M was generated by ligation of the three fragments, GFP-2AUbi, S-TS and

350

L-TS, using the In-fusion® PCR clone kit (Clontech). The same cloning approach was

17

351

used to construct the recombinant clones, pTS-GFP/NP and pTS-GFP/L, with

352

corresponding primers. To construct the pTS-GFP/M2, the GFP transcription cassette

353

was ligated to the long fragment of pTS09-C (L2-TS) that was amplified by PCR

354

using the primers M2-F and M2-R, so that the GFP was inserted into the intergenic

355

region between the P and M genes as an additional transcription unit. The sequences

356

of all primers used in this study will be available upon request.

357

Rescue of recombinant NDV rTS09-C and rTS-GFPs viruses were performed by

358

co-transfection of MVA-T7 infected BHK-21 cells in a 6-well plate with the

359

full-length cDNA clone, NP, P and L expression plasmids using LipofectamineTM

360

2000 (Invitrogen) according to the manufacture’s instruction. The cells were washed

361

with PBS and cultured in DMEM with 2% FBS, antibiotics and 0.2 μg/ml of

362

TPCK-trypsin (Sigma-aldrich) at 6 h post-transfection. The cell lysates were collected

363

by freeze-thawing for three times at 72 h post-transfection, and inoculated into

364

10-day-old SPF chicken embryos. After 96 h of inoculation, the allantoic fluids were

365

collected and the viruses were identified by the HA assay using 0.5% chicken red

366

blood cells.

367

Virus titration and growth kinetics

368

The virus stocks were titrated by using the standard HA assay in 96-well

369

micro-plate, the 50% egg infectious dose (EID50) assay in 10-day-old SPF chicken

370

embryos, the 50% tissue culture infectious dose (TCID50) assay on the BHK-21 cells

371

in the presence of 0.2 μg/ml of TPCK-trypsin (Sigma) (Alexander, 1998). The EID50

372

and TCID50 of virus were calculated by the Reed and Muench method (Reed, 1938).

18

373

To examine virus growth dynamics, BHK-21 cells grown to 90% confluence were

374

infected with 0.1 multiplicity of infection (MOI) of virus for 1.5 h. Then the cells

375

were washed twice, and the medium was added to cells. The media from infected cells

376

were collected at 24, 48, 72, and 96 h post-infection, and the TCID50 of virus in

377

collected media was determined in BHK-21 cells.

378

Virus pathogenicity assays

379

The pathogenicity of NDV for chickens was tested by performing the intracerebral

380

pathogenicity index (ICPI) assay in 1-day-old SPF chickens and the mean death time

381

(MDT) assay in 10-day-old SPF chicken embryos (Alexander, 1998).

382

Thermostability test

383

Aliquots of undiluted allantoic fluid infected with NDV at 1.0 ml/vial were sealed

384

airtightly in vials, submerged into a water bath and incubated at 56 ºC for different

385

time intervals. After heat treatment, the vials were transferred to an ice-cold water

386

bath to stop heat treatment. The HA activity and infectivity of virus in the vials were

387

titrated by the standard HA assay and TCID50 assay in BHK-21 cells, respectively.

388

The decreased HA activity and infectivity of viruses were showed in a logarithmic

389

scale as a function of heat treat time. Regression lines were plotted from 4 time points.

390

The thermostability of NDV was shown as the average time required for 2 log2 and 2

391

log10 decrease in HA activity and infectivity, respectively.

392

Indirect immuno-fluorescence assay (IFA)

393 394

BHK-21 cells grown in a 24-well plate were infected with 0.1 MOI of virus. At 24 h post-infection, the cells were fixed with 4% paraformaldehyde and washed with

19

395

PBS for three times. The fixed cells were blocked in PBS containing 1% Bovine

396

Serum Albumin (BSA) at 4 ºC for 1 h, and incubated with primary antibody,

397

anti-NDV polyclonal antibody for 1 h at 37 ºC. Subsequently, the cells were washed

398

three times with PBS and incubated with secondary antibody, goat-anti-chicken IgG

399

antibody conjugated with Alexa (Invitrogen) for 1 h at 37 ºC. The cells were

400

counterstained with DAPI, and examined with a con-focal laser microscopy after

401

further washed with PBS.

402

SDS-PAGE and Western blot

403

For analysis of virion-associated proteins, the allantoic fluid of embryonating eggs

404

infected with rNDV was clarified by the low-speed centrifugation. Viral particles were

405

purified from clarified allantoic fluid by ultracentrifugation in a 40 to 60% (wt/wt)

406

sucrose gradient (100,000 x g for 2 h). The sucrose gradient purified viral particles

407

were pelleted by centrifugation at 100,000 x g for 2 h, and resuspended in PBS. For

408

analysis of GFP expressed by rTS-GFPs, BHK-21 cells were infected with rNDV at

409

0.1 MOI, and lysed at the different time points. Proteins from the purified viral

410

particles or from the lysates of infected cells were separated by 12% SDS-PAGE,

411

followed by transferred to nitrocellulose membrane. The membrane was then blocked

412

in PBS containing 5% skim milk and incubated with anti-GFP Mab (Clontech). The

413

secondary antibody was Horseradish Peroxidase (HRP)-conjugated goat-anti-mouse

414

IgG antibody (Invitrogen). The membrane-bound antibodies were detected with 3,

415

3´-Diaminobenzidine (DAB).

416

Immunization and challenge experiments

20

417

To evaluate the immunogenicity of the thermostable recombinant virus and

418

protection against lethal NDV challenge, thirty 1-day-old SPF chickens were divided

419

randomly into 3 groups of 10 chickens. Birds were inoculated with 0.1 ml of

420

rTS-GFP/M at 107.0 EID50/ml (group 1), 0.1 ml of V4 at 107.0 EID50/ml (group 2), or

421

0.1 ml of PBS (group 3), respectively, via the routes of intranasal (IN) and intraocular

422

(IO). At 14 day post-vaccination, the immunized chickens were challenged with 0.1

423

ml of 106.0 EID50/ml of the virulent NDV/CA02 virus by IN/IO routes. Serum samples

424

were collected from the immunized chickens immediately before challenge. Chickens

425

challenged with the NDV/CA02 were observed daily for two weeks, and the mortality

426

was calculated.

427

Serological analysis

428

To examine the NDV-specific serum antibody response of immunized chickens, the

429

hemagglutination inhibition (HI) titers of serum samples were measured by

430

performing the HI assay (Alexander, 1998), the NDV strain V4 was used as the

431

antigen. The HI titer was presented as the mean value of log2 plus the standard

432

deviation in each group.

433

ACKNOWLEDGEMENTS

434

The authors thank Xiuqin Xia for her excellent technical assistance, and Patti Miller

435

for critical reading of the manuscript. This research was supported by China

436

Agriculture Research System (CARS-42-G11), Special Fund for Agro-scientific

437

Research in the Public Interest (201303033), the National Natural Science Foundation

438

of China (31000082), and USDA, ARS CRIS project 6612-32000-067-00D.

21

439

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440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481

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metapneumovirus subgroup C as a bivalent vaccine in turkeys. Vaccine 29, 8624-8633. Huang, Z., Elankumaran, S., Yunus, A. S. & Samal, S. K. (2004). A recombinant Newcastle disease virus (NDV) expressing VP2 protein of infectious bursal disease virus (IBDV) protects against NDV and IBDV. Journal of virology 78, 10054-10063. Ideris, A., Ibrahim, A. L. & Spradbrow, P. B. (1990). Vaccination of chickens against Newcastle disease with a food pellet vaccine. Avian Pathol 19, 371-384. Jayawardane, G. W. & Spradbrow, P. B. (1995a). Cell-mediated immunity in chickens vaccinated with the V4 strain of Newcastle disease virus. Veterinary microbiology 46, 37-41. Jayawardane, G. W. & Spradbrow, P. B. (1995b). Mucosal immunity in chickens vaccinated with the V4 strain of Newcastle disease virus. Veterinary microbiology 46, 69-77. Kim, S. H. & Samal, S. K. (2010). Role of untranslated regions in regulation of gene expression, replication, and pathogenicity of Newcastle disease virus expressing green fluorescent protein. J Virol 84, 2629-2634. King, D. J. (1993). Newcastle disease virus passage in MDBK cells as an aid in detection of a virulent subpopulation. Avian Dis 37, 961-969. Kolakofsky, D., Roux, L., Garcin, D. & Ruigrok, R. W. (2005). Paramyxovirus mRNA editing, the "rule of six" and error catastrophe: a hypothesis. J Gen Virol 86, 1869-1877. Lamb, R. A., Collins, P. L., Kolakofsky, D., Melero, J. A., Nagai, Y., Oldstone, M. B. A., Pringle, C. R. & Rima, B. K. (2005). Family Paramyxoviridae. In Virus Taxonomy, Eighth Report of the International Committee on Taxonomy of Viruses, pp. 655-668. Edited by C. M. Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger & L. A. Ball. San Diego: Elsevier Academic Press. Lomniczi, B. (1975). Thermostability of Newcastle disease virus strains of different virulence. Arch Virol 47, 249-255. Mazija, H., Cajavec, S., Ergotic, N., Ciglar-Grozdanic, I., Gottstein, Z. & Ragland, W. L. (2010). Immunogenicity and safety of Queensland V4 and Ulster 2C strains of Newcastle disease virus given to maternally immune, newly hatched chickens by nebulization. Avian Dis 54, 99-103. Miller, P. J. & Guus, K. (2013). Newcastle Disease. In Diseases of Poultry, 13th edn, pp. 98-107. Edited by D. E. Swayne, J. R. Glisson, L. R. McDougald, L. K. Nolan, D. L. Suarez & V. Nair. Ames, Iowa, USA: Wiley-Blackwell Publishing. Nagai, Y., Klenk, H. D. & Rott, R. (1976). Proteolytic cleavage of the viral glycoproteins and its significance for the virulence of Newcastle disease virus. Virology 72, 494-508. Nakaya, T., Cros, J., Park, M. S., Nakaya, Y., Zheng, H., Sagrera, A., Villar, E., Garcia-Sastre, A. & Palese, P. (2001). Recombinant Newcastle disease virus as a vaccine vector. J Virol 75, 11868-11873. Pedersen, J. C., Senne, D. A., Woolcock, P. R., Kinde, H., King, D. J., Wise, M. G., Panigrahy, B. & Seal, B. S. (2004). Phylogenetic relationships among virulent Newcastle disease virus isolates from the 2002-2003 outbreak in California and other recent outbreaks in North America. J Clin Microbiol 42, 2329-2334. Peeters, B. P., Gruijthuijsen, Y. K., de Leeuw, O. S. & Gielkens, A. L. (2000). Genome replication of Newcastle disease virus: involvement of the rule-of-six. Arch Virol 145, 1829-1845. Pritzer, E., Kuroda, K., Garten, W., Nagai, Y. & Klenk, H. D. (1990). A host range mutant of Newcastle disease virus with an altered cleavage site for proteolytic activation of the F protein. Virus Res 15, 237-242.

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Reed, L. J. (1938). A simple method for estimating fifty percent endpoint. Am J Hyg 27, 493-497. Simmons, G. C. (1967). The isolation of Newcastle disease virus in Queensland. Aust Vet J 43, 29-30. Spradbrow, P. B. (1993/94). Newcastle disease in village chickens. Poultry Science Review 5, 57-96. Steward, M., Vipond, I. B., Millar, N. S. & Emmerson, P. T. (1993). RNA editing in Newcastle disease virus. J Gen Virol 74 ( Pt 12), 2539-2547. Toro, H., Zhao, W., Breedlove, C., Zhang, Z. & Yu, Q. (2014). Infectious bronchitis virus S2 expressed from recombinant virus confers broad protection against challenge. Avian Dis 58, 83-89. Tscherne, D. M., Jones, C. T., Evans, M. J., Lindenbach, B. D., McKeating, J. A. & Rice, C. M. (2006). Time- and temperature-dependent activation of hepatitis C virus for low-pH-triggered entry. J Virol 80, 1734-1741. Tu, T. D., Phuc, K. V., Dinh, N. T., Quoc, D. N. & Spradbrow, P. B. (1998). Vietnamese trials with a thermostable Newcastle disease vaccine (strain I2) in experimental and village chickens. Prev Vet Med 34, 205-214. Wen, G., Shang, Y., Guo, J., Chen, C., Shao, H., Luo, Q., Yang, J., Wang, H. & Cheng, G. (2013). Complete genome sequence and molecular characterization of thermostable Newcastle disease virus strain TS09-C. Virus Genes 46, 542-545. Wu, C., Zhan, A., Liu, H. & Zhang, R. (1999). Studies on effects of trypsin on Newcastle disease virus LaSota strain. Journal of Yangzhou University (natural science edition) 2, 33-36. Zhao, H. & Peeters, B. P. (2003). Recombinant Newcastle disease virus as a viral vector: effect of genomic location of foreign gene on gene expression and virus replication. J Gen Virol 84, 781-788. Zhao, W., Spatz, S., Zhang, Z., Wen, G., Garcia, M., Zsak, L. & Yu, Q. (2014). Newcastle Disease Virus (NDV) Recombinants Expressing Infectious Laryngotracheitis Virus (ILTV) Glycoproteins gB and gD Protect Chickens against ILTV and NDV Challenges. Journal of virology 88, 8397-8406. Zhao, W., Zhang, Z., Zsak, L. & Yu, Q. (2015). P and M gene junction is the optimal insertion site in Newcastle disease virus vaccine vector for foreign gene expression. The Journal of general virology 96, 40-45.

556 557

24

558

Fig. 1. Generation and detection of recombinant NDV strain rTS09-C and

559

rTS-GFP/M. (a) Schematic presentation of the genomes of NDV strain rTS09-C and

560

rTS-GFP/M. (b) BHK-21 cells infected with either rTS09-C or rTS-GFP/M at an MOI

561

of 0.1, were fixed and labeled for the presence of NDV (red); GFP was detected by its

562

natural fluorescence; Cell nuclei were counterstained with DAPI. Cells were analyzed

563

by using confocal laser microscopy. (c-e) rTS09-C or rTS-GFP/M were propagated in

564

eggs and purified by differential centrifugation and sedimentation through sucrose

565

gradients. Viral particles were analyzed by using electron microscopy (c). Viral

566

proteins were analyzed by using SDS-PAGE (d), and were subjected to Western-blot

567

analyses with the rabbit serum against GFP (e).

568 569 570

Fig. 2. GFP expression levels of different recombinant NDVs. (a) Schematic

571

presentation of the genomes of different rNDVs expressing GFP. (b) BHK-21 cells

572

were infected with the different rNDVs at an MOI of 0.1. Media from infected cells

573

were harvested at the indicated time points, and virus titers were measured by TCID50

574

titration in BHK-21 cells. Mean values ± standard deviations were shown from three

575

tests. Significant differences (p < 0.01) were seen between the rTS-GFP/L and other

576

viruses at 72hpi. (c) BHK-21 cells infected with the indicated rNDVs were analyzed

577

by using fluorescence microscopy at 24, 48, and 72 hpi. (d) Lysates from BHK-21

578

cells infected with the indicated rNDVs were harvested at 24, 48, and 72 hpi, and GFP

579

expression were analyzed by Western-blot with rabbit serum against GFP.

25

580

Fig. 3. In vitro test of NDV thermostability. Heat-inactivation kinetics of HA activity

581

(a) and infectivity (b) of the indicated NDV strains were determined at 56 ºC. The

582

remaining percentage of HA activity (a) and infectivity (b) were represented in a

583

logarithmic scale as a function of heat treat time.

584 585 586

Fig. 4. Propagation of NDV in BHK-21 cells without trypsin. (a) NDV strain rTS09-C

587

was serially passaged in BHK-21 cells with or without trypsin. Cell cultures of

588

passage 1, 2, 3, and 4 (P1, P2, P3 and P4) were harvested at 72 hpi, and virus titers

589

were determined by TCID50 titration in BHK-21 cells. (b) Growth curves of the 4th

590

passage of rTS09-C with or without trypsin. Virus titers were tested by TCID50

591

titration in BHK-21 cells. (c) BHK-21 cells infected with the 4th passage of

592

rTS-GFP/M in the presence or absence of trypsin, were analyzed by using

593

fluorescence microscopy at the indicated time points. More than 50% of BHK21 cells

594

infected with rTS-GFP/M with trypsin died at 72hpi.

595 596 597 598 599 600 601 602 26

603

Table 1. Biological characterization of the NDV recombinant viruses Virus titer Virus

MDT*

ICPI†

HA titer In allantoic fluid‡

In BHK-21 cells§

rTS09-C

>168h

0.00

29

109.50

106.94

rTS-GFP/M

>168h

0.00

29

109.25

107.05

604 605

* Mean death time

606 607

‡ Virus titer in allantoic fluid was presented as EID50/ml § Virus titer in BHK-21 cells was presented as TCID50/ml

† Intracerebral pathogenicity index

608 609 610

Table 2. Protective efficacy of the recombinant viruses in 1-day-old chickens against the highly pathogenic NDV challenge* Virus Immunized

No. of birds

HI titer†

Protection Rate‡ (%)

rTS-GFP/M

10

2.6 ± 0.52

10/10 (100%)

V4

10

3.2 ± 0.63

10/10 (100%)

PBS

10

0.0

0/10 (0%)

611 612 613

* All day-old chickens in each group were immunized with 106.0EID50 of rTS-GFP/M or V4 in a 0.1-ml

614 615 616

† Hemagglutination inhibition (HI) titer was presented as log2 of the mean ± standard deviation

volume by oculonasal administration and challenged with 10 5.0EID50 of NDV strain CA02 at 2 weeks post vaccination. ‡ Protection rate was evaluated by counting numbers of survived birds without showing Newcastle disease clinical signs after challenge for 2 weeks.

617

27

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