Development of a highly immunogenic Newcastle disease virus chicken vaccine strain of duck origin

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1 Development of a highly immunogenic Newcastle disease virus chicken vaccine strain of duck origin J. Y. Kim, S. J. Kye, H. J. Lee, S. Gaikwad, H. S. Lee, S. C. Jung, and K. S. Choi,1 Avian Disease Division, Animal and Plant Quarantine Agency, Anyang, Republic of Korea; Foot and Mouth Disease Diagnosis Division, Animal and Plant Quarantine Agency, Anyang, Republic of Korea; Department of Microbiology, College of Veterinary and Animal Sciences, Maharashtra Animal and Fishery Sciences University, Parbhani, India; and Bacterial Disease Division, Animal and Plant Quarantine Agency, Anyang, Republic of Korea ABSTRACT Newcastle disease virus (NDV) strain quences and avian sequences, and passage of the duck NDRL0901 was developed as a live vaccine candidate for control of Newcastle disease. NDV isolate KR/duck/13/07 (DK1307) of duck origin was used as the selected vaccine strain. DK1307 was passaged 6 times in chickens. Then a single clone from the chickenadapted virus (DK1307C) was finally selected, and the vaccine strain was named NDRL0901. DK1307C and the clone NDRL0901 viruses showed enhanced immunogenicity compared to the DK1307 virus. Principal component analysis based on fusion and hemagglutininneuraminidase genes revealed the codon usage pattern in the dataset is distinct separating duck viral se- origin virus into the chicken host causes deviation in the codon usage pattern. The NDRL0901 virus was avirulent and did not acquire viral virulence even after 7 back passages in chickens. When day-old chicks were vaccinated with the NDRL0901 virus via spray, eye drops, and drinking water, the vaccinated birds showed no clinical signs and had significant protection efficacy (>80%) against very virulent NDV (Kr005 strain) infection regardless of the administration route employed. The results indicate that the NDRL0901 strain is safe in chickens and can offer protective immunity. Key words: Newcastle disease virus, live vaccine, immunogenicity, protective efficacy 2016 Poultry Science 95: INTRODUCTION Newcastle disease virus (NDV), the causative agent of the disease, is avian paramyxovirus type 1 that belongs to the genus Avulavirus in the family Paramyxoviridae (Mayo, 2002). NDV has a negative sense, single-stranded RNA genome encoding at least 6 proteins, i.e., nucleoprotein, phosphoprotein, matrix protein, fusion (F) protein, hemagglutinin-neuraminidase (HN) and large polymerase (Yusoff and Tan, 2001; Cattoli et al., 2011). In general, NDV strains vary in virulence and have been classified into at least 3 major pathotypes based on pathogenicity in chickens such as the intracerebral pathogenicity index (ICPI) of inoculated day-old chickens and the mean death time (MDT) of inoculated embryonated chicken eggs: velogenic (very virulent), mesogenic (moderately virulent) and lentogenic (avirulent), in order of virulence (Cattoli et al., 2011;OIE2012). Alternatively, virulence C 2016 Poultry Science Association Inc. Received August 24, Accepted October 26, Corresponding author: kchoi0608@korea.kr can also be evaluated based on the amino acid sequence of a precursor F (F0) protein cleavage site (OIE, 2012). NDV isolates exhibit a broad range of genetic diversity. The most widely used genotype classification system proposed by Diel et al., (2012) divides NDV isolates into 2 distinct classes, class I and class II In particular, class II NDVs are important for the poultry industry. Velogenic class II NDVs are associated with ND outbreaks in poultry all over the world (Miller et al., 2010; Diel et al., 2012; Dortmans et al., 2012). Class II NDVs under genotype VII are the main genotype responsible for ND outbreaks in Asia (Berhanu et al., 2010; Keet al. 2010; Rui et al., 2010; Tan et al., 2010; Choi et al., 2014). Some lentogenic strains of class II NDVs (genotype I or II) have been widely used as NDV vaccines (Senne et al., 2004; Miller et al., 2010). In commercial poultry flocks, a massive intensive vaccination program is commonly practiced to keep ND under control (Dortmans et al., 2012). Nevertheless, ND outbreaks still occur despite intensive vaccination programs against NDV. These outbreaks are attributed to poor vaccination practices and poor vaccine quality and may be responsible for poor immunity levels. This also gives the way for the spread of viruses especially in young birds (Jeon et al., 2008; van Boven et al., 2008). 790

2 DEVELOPMENT OF NEWCASTLE DISEASE VIRUS VACCINE 791 Thus, a highly immunogenic and safe live vaccine can provide an opportunity to enhance herd immunity through a massive vaccination program. In the present study, a lentogenic strain of NDV of duck origin was selected for the development of an NDV vaccine candidate. The NDV strain was adapted to chickens through serial passage. A single clone from the chicken-adapted NDV was chosen as the vaccine strain and was evaluated for immunogenicity, safety, and protection efficacy against velogenic NDV infection. Viruses MATERIALS AND METHODS Avirulent NDV isolate KR/duck/13/07 (DK1307) of domestic duck origin, under genotype I in class II (Lee et al., 2009), was used in the study as the source to generate an NDV vaccine candidate. Velogenic NDV (vndv) strain NDV/KR/chicken/005/2000 (Kr005), under genotype VII in class II (Lee et al., 2004), was used as the challenge NDV. All viruses were propagated with the egg inoculation method in specific pathogen free (SPF) chicken embryonated eggs (ECEs; Valo Biomedia, Adel, IA), and the aliquots were then stored at 70 C until use. The infective titer was expressed as 50% egg infective dose (EID 50 ) according to the Reed and Muench (1938) formula. Chickens White leghorn chickens hatched from SPF ECEs or commercial broiler chickens were used in the study. The experimental chickens were maintained in positivepressure high-efficiency particulate air-filtered stainless steel isolation cabinets (Three Shine Inc., Daejeon, Korea) and received food and water ad libitum. All experimental procedures and animal care activities were conducted according to the institutional guidelines to ensure that the animal experiments conformed to national animal welfare legislation. The animal experiments were approved by the institutional Animal Experiment and Ethics Committee. Chicken-to-chicken Passage The DK1307 virus was serially passaged in SPF chickens. Briefly, the DK1307 virus was inoculated into 3 SPF chicks (3 to 5 d old) via the eye drop and intranasal routes. On d 3 post-inoculation (pi), 3 chicks were humanely sacrificed according to the standard procedures described by Bermudez and Stewart-Brown (2008), trachea samples were homogenized, and the virus recovered was propagated by the egg inoculation method. The procedure was repeated 6 times. After the final passage, the chicken-adapted virus (DK1307C) was recovered from the inoculated chickens and was propagated with the egg inoculation method. The aliquots were then stored at 70 C until use. Plaque Assay A plaque assay was conducted in primary chicken embryo kidney (CEK) cell monolayers in 6-well plates with some modifications as described previously (Choi et al., 2013). Briefly, the CEK cells in the 6-well plates were infected with 10-fold dilutions of DK1307C and then overlaid with 1% (wt/vol) agar gel. Following incubation for 3 d at 37 C, the cells were overlaid again with 1% (wt/vol) agar gel containing 0.01% neutral red. Several plaques formed in CEK cells were picked at 96 h pi, and each plaque clone was tested for hemagglutination (HA) using 1% (vol/vol) chicken red blood cells on the plate. The HA-positive clones were propagated in ECEs as described. Selection of Vaccine Virus Clone Several representative NDV clones in the study were tested for immunogenicity in week-old SPF chickens. DK1307 (duck virus) and DK1307C (chicken-adapted virus) were included for comparison. Each experimental group was inoculated via the eye drop route with each of the NDVs tested (10 6 EID 50 /bird) while the control group was inoculated with phosphate buffered saline (PBS), ph 7.2. All birds were observed twice daily for 14 d. Sera were taken from the chickens at 0, 14,and21dpi,andtheNDV-specificantibodiesinthe serum samples were measured with the hemagglutination inhibition (HI) test in V-bottom microtiter plates according to the OIE (2012) manual. The HI titer was determined as the last dilution that showed complete inhibition of HA activity. All tests were repeated twice. As a result of the animal experiment in week-old SPF chickens, the most immunogenic and safest virus clone was determined as the NDV vaccine strain candidate. Reversion to Virulence The selected NDV vaccine strain candidate was examined with an in vivo test of reversion of virulence using SPF chickens. NDRL0901 was serially passaged in SPF chicks (3 to 5 d old) by the eye drop route and sacrificed on 3 d pi. The trachea samples from the chicks were pooled and homogenized for egg inoculation. This procedure was repeated 7 times, and the final passaged virus (NDRL0901bp) was propagated using SPF ECEs and stored at 70 C. Intracerebral Pathogenicity Index (ICPI) Test The pathogenicity of the NDVs in the study was determined with the ICPI test in day-old SPF chicks, according to standard procedures (Alexander, 1988; OIE 2012). The ICPI was assessed by inoculating day-old

3 792 KIM ET AL. SPF chicks (10 birds per test virus) via the intracerebral route with 1:10 dilution of fresh infective allantoic fluid from the NDVs. The birds were observed daily for 8 d and scored as normal, sick, or dead. Calculation of the ICPI was performed as described (Alexander, 1988). Molecular Changes in Surface Glyocprotein Genes during Adaptation We sequenced fusion and hemagglutininneuraminidase (HN) genes of DK1307 and NDRL0901 to examine whether the substitutions in the F and HN genes during adaptation occurred. A reversetranscription polymerase chain reaction (PCR) assay was performed for amplification of the F and HN genes using 2 primer sets, HNf/HNr and Ff/Fr, as described previously (Choi et al., 2013). The PCR products were purified using a Qiagen gel extraction kit (Qiagen, Valencia, CA), and then the resulting products were sequenced directly (Macrogen, Seoul, Korea) with an ABI 3730XL Analyzer (Applied Biosystems, Foster, CA). Two separate datasets of the 2 gene sequences for duck origin NDVs and various NDV genotypes were retrieved from NCBI. In order to analyze the pattern of codon usage among the F and HN sequences of duck origin and chicken adapted viruses, the set of Relative Synonymous Codon Usage (RSCU) values are computed for each gene. The RSCU value for a codon is defined as the ratio of the observed frequency to the expected frequency for the same codon. Here we calculated the RSCU values of the 59 relevant codons for all sequences in DAMBE version software. The RSCU reflects the codon usage bias in a single codon family (Sharp et al., 1986). Principal component analysis of all codons was performed in the R statistical environment. Accession Numbers The nucleotide sequence data reported in this study have been submitted to GenBank and have been assigned the following accession numbers: KT (isolate DK1307, F and HN) and KT and KT (strain NDRL0901, F and HN respectively). The accession numbers and genes of interest in the NDV reference strains included in this study are as follows: JX (CBU2249, F and HN), JX (CBU2179, F and HN), JX (CBU2374, F and HN), JX193077(duck/China/Guangxi16/2008, F and HN), JX (duck/china/guangxi20/2010, F and HN), JX193083(duck/China/Guangxi22/2010, F), HM (R8, HN), KF (237/Jilin/2011, HN), KF (94/Jilin/2011, HN), KF (12/Jilin/2011, HN), KF (119/Jilin/2011, HN), KF (NEFU1301, HN), HM (D3, HN), JX (duck/china/guangxi17/2009, HN), KM (Du/CH/LAH/224/2011, HN), KF (87/Jilin/2011, HN), for duck NDVs; AY (HB92, F and HN), AY (SP13, F), KM (B1/1961, F and HN), KJ (BD12, F), HM (NDV- 4/chicken/Namakkal/Tamil Nadu/India, F), JX (H37, F), JN (New Jersey-Roakin/1946, F), KJ (VRDC/Ventri/LaSota/WS, F and HN), JF (Lasota, F and HN), GQ (YC Ch, HN), EU (D58, HN), for class II genotypes I and II NDVs; KF (chicken/israel/ 2011/ , F), KF (752, F), KF (755, F), KF (759, F), KF (761, F), KF (763, F), KF (764, F), GQ (QG/Hebei/07, HN), KC (chicken/china/ Hebei/01/2006, HN), GQ (XD/Shandong/08, HN), DQ (JS02, HN), DQ (lchen-1-99, HN) for class II genotype VII NDVs; GU (chicken/spvc/karachi/ndv/33/2007, F), JN (chicken/byp/lahore/2010, F), JN (chicken/ CP/Islamabad1/2010, F), JN (chicken/byp/ Rawalpindi/2010, F), JN (chicken/cp/attock/ 2010, F), JN (chicken/cp/islamabad3/2010, F), JQ (chicken/pakistan/ndv/udl8/2011, F), KF (751, F) for class II genotype XII NDVs. Protection Efficacy Test Day-old SPF chicks were used. The birds were vaccinated with EID 50 of the NDRL0901 strain (vaccinated) via the spray (SP) (Desvac R Kit 2, pore size μm, Ceva, France), eye drop (ED) or drinking water (DW) route. Control birds were mock infected with PBS solution via the eye drop route (unvaccinated). At 14 d post-vaccination (dpv), all birds were challenged with EID 50 of vndv strain Kr005 via the intramuscular route. The birds were monitored daily for overt clinical signs of disease during the 14-d period. Field Trials on Commercial Broiler Farms Pilot live NDV vaccines containing the NDRL0901 virus ( to EID 50 per dose) were manufactured and applied on 3 farms where commercial broilers with maternal antibodies were raised. Before the birds were administered the vaccination, maternal antibodies to NDV were measured with the HI test. Broilers on the farm were vaccinated with 1 dose of the NDRL0901 pilot vaccine and the VG/GA vaccine each by spray (at 1 d of age) and/or drinking water (at 14 d of age) on the farm, respectively. For each experimental farm, unvaccinated birds were included as the control group. On d 14 after the last vaccination, 36 birds on each farm, including the vaccinated (n = 26) and control (n = 10) animals, were randomly selected, bled for the HI test, and then moved to the animal experimentation facility. Five d later, all birds were challenged by the intramuscular route with a dose of EID 50 /bird of

4 DEVELOPMENT OF NEWCASTLE DISEASE VIRUS VACCINE 793 Table 1. Immunogenicity of NDV DK1307C clones in SPF chickens and comparison with parent NDVs. HI titer (log 2 ) 3 Vaccine virus No. 1 Clinical sign 2 0 dpv 14 dpv 21 dpv DK /10 <1 1.5 ± 1.9 p 2.9 ± 1.7 p DK1307C 8 0/8 <1 4.1 ± 1.6 q 5.5 ± 1.3 q DK1307C-6 8 0/8 <1 3.8 ± 1.4 q 5.1 ± 1.1 q DK1307C-7 (NDRL0901) 8 0/8 <1 4.6 ± 1.2 q 6.3 ± 1.1 q DK1307C-8 8 0/8 <1 3.3 ± 1.0 q 4.1 ± 1.0 q Control 5 0/8 <1 <1 p <1 r 1 Week-old SPF birds were inoculated with NDV (10 6 EID 50 per dose) via the eye drop route. 2 Number of birds showing clinical signs/number of birds inoculated. 3 HI titers are expressed as log 2 geometric mean HI antibody titers ± standard deviations. p,q,r Values with different superscripts within column differ significantly (Student s t-test, P < 0.05). vndv Kr005 strain. The birds were monitored daily for overt clinical signs of disease during the 14-d period. Statistical Analysis Comparison of serological data was performed using the statistical software program Statistica. Variables were compared using the Student s t-test, and variation within groups was considered to be significant at P < RESULTS Chicken Adaptation of Duck NDV Isolate DK1307 In order to facilitate adaptation to chickens, the DK1307 virus of duck origin was serially passaged in SPF chicks. After the 6th passage, a chicken-adapted virus (DK1307C) was finally recovered from the inoculated birds. The plaque assay using CEK cells was performed to clone plaque from DK1307C. Twelve wellisolated plaque clones formed on the CEK cells were randomly selected and propagated in ECEs. When mixed with chicken red blood cells (RBCs) on a glass plate, the allantoic fluids of the plaque clones showed various patterns of HA activity such as strong, medium, and weak HA (data not shown). Of the plaque clones, the number 6, 7, and 8 clones were selected as NDV vaccine candidates based on the HA pattern. Selection of Highly Immunogenic NDV Clone Table 1 shows the immunogenicity results of NDV strains of the study in week-old SPF chickens. No overt clinical signs of ND were observed in any birds (including the control birds) before they were administered the vaccination. In addition to the unvaccinated birds in the control group, the vaccinated birds also showed no clinical signs of ND during the experiment. The mockinfected control birds had no detectable HI antibodies during the experiment. Furthermore, no birds in the 5 vaccinated groups had detectable HI antibodies before receiving the vaccination. Seroconversion to NDV occurred on 14 dpv in all 5 groups. The HI titers of the antibodies to the NDV-vaccinated birds increased up to 21 dpv. Of the 5 viruses tested, the duck virus DK1307 showed the weakest immunogenicity (log 2 HI titers of 2.9 ± 1.7, 21 dpv); on d 21 dpv, sera of 6 out of 10 had HI titers of 2. After the chicken passages, the chicken-adapted virus DK1307C (log 2 HI titers of 5.5 ± 1.3, 21 dpv) showed significantly enhanced immunogenicity in the chickens compared to the wild-type duck virus DK1307 (P < 0.05). Clone No. 7 virus (DK1307C-7) induced better humoral immune response (log 2 HI titer of 6.3 ± 1.1, 21 dpv) in vaccinated chickens than DK1307C while the other two clones (DK1307C-6 and DK1307C-8) did not, although the difference in the HI titers between groups was not significant (P > 0.05). In the study, the DK1307C- 7 virus was selected as the vaccine strain and named NDRL0901. The NDRL0901 strain propagated in ECEs had virus titers of EID 50 /ml, and the allantoic fluid containing the NDRL0901 strain had 2 11 HA titers per 25 μl. Pathogenicity of NDV Strain NDRL0901 in Chickens The pathogenicity of the NDRL0901 strain was assessed with the ICPI test, the prescribed OIE method for pathogenicity and molecular pathotyping assay based on the amino acid sequence of F0 protein cleavage site (OIE 2012). The predicted amino acid sequence at the F0 protein cleavage site (positions 112 to 119) was determined. All NDVs examined including DK1307, NDRL0901 and NDRL0901bp had the same cleavage site motif ( 112 GKQGRL 117 ) containing monobasic amino acids and leucine (L) at position 117, which are commonly found in avirulent chicken NDV strains (OIE 2012). The ICPI value of duck virus DK1307 was 0.2, which belongs to category of the lentogenic viruses (ICPI of < 0.6). The NDRL0901 strain had an ICPI of 0.05, indicating that the virus was more attenuated after adaptation in chickens. The ICPI of the NDRL0901 strain (ICPI of 0.18) increased slightly after 7 back

5 794 KIM ET AL. Figure 1. A plot of principal component analysis of fusion (F) and hemagglutinin-neuraminidase (HN) protein genes in duck origin virus (, DK1307) and chicken adapted virus (, NDRL0901). Principal component 1 (PC1) and 2 (PC2) represent the values of the first and the second axis of each gene in the study as coefficients associated with the first two extracted principal components. The two axes, PC1 and PC2, in the plot contributed to the codon usage bias., duck origin NDVs;, class II genotype I and II NDVs; +, class II genotype VII NDVs;, classii genotype XII NDVs. Each symbol represents 1 NDV strain (isolate). passages in chickens, but the virus (NDRL0901bp) was still lentogenic. confines of codon usage values of other duck virus sequences considered in graph. Protective Efficacy of the NDRL0901 Strain Molecular Changes in Fusion and HN Proteins during Adaptation In the F gene from NDRL0901, two nucleotide substitutions at position 1159 (A to G) and 1609 (T to C) were observed. Of both, single nucleotide substitution from A to G recorded at position 1159 induced a lysine to glutamic acid change. In the HN gene, a single synonymous mutation (from A to G) at nucleotide position 645 was observed in NDRL0901 (data not shown). For the sequences of F and H genes, the RSCU values of the 59 relevant codons for all sequences were calculated. NDV strains of duck origin and chicken origin (class II genotypes I, II, VII and XII) are included. As a result, principal component analysis revealed that chicken adapted virus sequence have distinct codon usage than that of duck virus sequence from which it was derived, as represented by its distinct location away from its parent on graph (Figure 1). Nevertheless it still is in The protective efficacy of the NDRL0901 strain following vaccination via spray (SP), ED, or DW routes was assessed by challenging several day-old SPF chicks with vndv strain Kr005 via the intramuscular route (Table 2). All unvaccinated challenged birds had no detectable antibody to NDV before the challenge as measured with the HI test. Following the challenge with the Kr005 strain, all unvaccinated birds died within 5 d after the challenge. In the vaccination groups, the SPF chicks vaccinated with the NDRL0901 strain via SP, ED or DW showed HI titers of antibodies to NDV measured 14 dpv (before the challenge). ED vaccination had the highest HI titers (log 2 mean HI titers of 4.6 ± 1.2), followed by SP vaccination (log 2 HI titers of 4.1 ± 0.8) and DW vaccination (HI titers of 2.9 ± 1.5) in the order. The NDRL0901 strain gave significant protection (>80% protection rate) from mortality regardless the administration route when challenged with vndv Kr005. The protection rates for the SP, ED, and DW Table 2. Protection efficacy in day-old chicks of the NDRL0901 strain vaccination following challenge with the NDV Kr005 strain. Group Vaccine administration No. 1 HI titer (log 2 ) 2 at 14 dpv Protection rate (n/n) 3 ED Eye drop ± 1.2 p 90.0% (26/30) SP Spray ± 0.8 p 100% (40/40) DW Drinking water ± 1.5 q 83.3% (25/30) Control 10 <1 r 0% (0/10) 1 SPF birds were vaccinated with the NDRL0901 strain ( EID 50 per dose) via 1 of 3 vaccination routes (eye drop, spray, and drinking water) and challenged 2 wk later with the NDV Kr005 strain ( EID 50 per dose) via the intramuscular route. 2 HI titers are expressed as log 2 geometric mean HI antibody titers ± standard deviations. 3 Number of birds survived (n) /number of birds challenged (N). p,q,r Values with different superscripts within columndiffer significantly (Student s t-test, P < 0.05).

6 DEVELOPMENT OF NEWCASTLE DISEASE VIRUS VACCINE 795 groups were 100% (40/40), 90.0% (27/30) and 83.3% (25/30), respectively. Field Trials with the NDRL0901 Strain Field trials with the NDRL0901 pilot vaccines were performed on 3 commercial farms (J1, J2 and J3) where commercial broilers with maternally derived antibodies (MDAs) were raised. The NDRL0901 strain was compared with the VG/GA commercial vaccine (Table 3). Single spray vaccination at 1 d of age was performed on 1 broiler farm (farm J3). When the birds were 14 d old, the log 2 HI titers for the NDRL0901 pilot vaccine, VG/GA commercial vaccine, and control groups were 2.1 ± 1.3, 2.0 ± 1.2 and 3.7 ± 1.2, respectively. The difference in HI titers was not significant (P > 0.05). However, when challenged with the vndv Kr005 strain at 19 d of age, the vaccination groups (NDRL0901 and VG/GA) gave high protection of >80% while the protection rate for the unvaccinated control group was 10% (1/10). Twovaccinations(SPond1andDWond14)were performed on 3 farms (farms J1, J2, and J3). When the birds were 28 d old, the log 2 HI titers for the NDRL0901 pilot vaccine and the VG/GA commercial vaccine were 2.8 ± 1.4to3.1± 1.5 and 2.4 ± 1.3to2.5± 1.5, respectively. The difference between the 2 vaccine groups was not significant (P > 0.05). Unvaccinated birds had no detectable maternal antibody as measured with the HI test. In both vaccines, 2 vaccinations provoked a high humoral immune response compared to a single vaccination, although the difference was not significant (P > 0.05). When the birds were challenged with the vndv Kr005 strain at 33 d of age, the protection rate for the NDRL0901 pilot vaccine and the VG/GA commercial vaccine was 83.3% to 88.9% and 72.2% to 88.9%, respectively, while the unvaccinated challenged birds (n = 10) died within 1 wk after the challenge. DISCUSSION Currently, throughout the world, lentogenic strains of NDV are recommended worldwide for use as live vaccine strains. NDVs with low virulence have the biological property of either respiratory tract tropism (respirotropic) or digestive tract tropism (enterotropic) (Choi, 2014). In general, the more immunogenic live vaccines are more virulent in chickens (OIE 2012). Respirotropic viruses (e.g., La Sota) have a tendency to more immunogenic than enterotropic viruses (e.g., VG/GA) in chickens (Choi, 2014). However, live respirotropic NDVs often cause mild respiratory illness. In addition, complications with other respiratory pathogens (e.g., Escherichia coli) might lead to more severe respiratory illness that causes significant production losses (OIE 2012). In the field, vaccination practices are therefore directed at avoiding side effects rather than at obtaining optimal immunity (Dortmans et al., 2012). Thus, this study aimed at the development of an NDV vaccine strain that was immunogenic and safe for chickens. In the study, we developed the new NDV vaccine strain NDRL0901. The NDRL0901 strain originated from the DK1307 virus, an NDV isolated from domestic duck feces on a farm in Korea. As expected, the DK1307 virus isolate induced poor humoral immunity in a different host (chickens); some birds even had no detectable HI titers (Table 1). Such poor humoral immunity may be explained by poor virus replication in chickens. To overcome this limitation and acquire increased immunogenicity in chickens, we passaged the duck virus in chickens 6 times. As a result, the chicken-adapted virus (DK1307C) induced significantly higher HI titers in chickens than the duck virus DK1307 (Table 1). This suggests that DK1307C may replicate more efficiently in chickens than the duck virus. We sequenced two surface glycoprotein (F and HN) genes to study molecular changes during chicken adaptation. Two and single nucleotide mutations occurred at both F and HN genes, respectively during the adaption, but a single amino acid change was observed in the F protein only. Here we applied principal component analysis of RSCU to understand the pattern of codon usage among the sequences of duck and chicken NDVs and to examine the change in the codon usage pattern during adaption of duck NDV (DK1307 strain) to chickens. The principal component analysis revealed that with a few mutations in sequenced genes there is deviation in the codon usage pattern away from duck NDVs during adaptation of the DK1307 strain to chickens (Figure 1). Thus, we could not exclude the possibility that a few point mutations in these genes may impart cellular attachment fitness to virus in different hosts such as chickens. In the study, the principal component analysis revealed that duck NDVs formed a cluster distinct from chicken NDVs. However, the NDRL0901 strain in the study still clustered with other duck viruses rather than chicken NDVs following passages in chickens. Other proteins of NDV such as phosphoprotein and polymerase protein may also contribute to the replicative fitness of the virus since they are known to be involved in the efficiency of viral replication (Dortmans et al., 2011). Thus, further sequencing of genes encoding other proteins of NDV in the study would be helpful to investigate whether the chicken passages might result in genetic mutations involved in the efficiency of viral replication. To select a highly immunogenic virus clone, 10 plaque clones were randomly selected from the DK1307C virus. When tested for the plate HA test using chicken RBCs, the clones showed several distinct patterns of HA, indicating viral quasi-species in the DK1307C virus. NDRL0901, a single clone from the DK1307C virus, was highly immunogenic in chickens. The NDRL0901 virus had very low virulence (ICPI value of 0.05) and did not acquire virulence after serial back passages in chicks. In addition, the NDRL0901 virus caused no clinical signs

7 796 KIM ET AL. Table 3. Results of field trials for the NDV vaccine the NDRL0901 strain in commercial broilers with maternal antibodies. Vaccination HI titer (log 2 ) 2 Farm Vaccine strain Vaccine program 1 1 d.o. 14 d.o. 28 d.o. Protection rate (n/n) 3 J3 NDRL0901 SP (1 d.o.) 4.4 ± ± 1.3 NT 83.3% (15/18) VG/GA SP (1 d.o.) 2.0 ± 1.2 NT 83.3% (15/18) Control 3.7 ± 1.2 NT 10.0% (1/10) J1 NDRL0901 SP (1 d.o.)+dw (14 d.o.) 4.9 ± 1.0 NT 2.8 ± % (15/18) VG/GA SP (1 d.o.)+dw (14 d.o.) NT 2.5 ± % (13/18) Control NT <1 0% (0/10) J2 NDRL0901 SP (1 d.o.)+dw (14 d.o.) 4.5 ± 0.9 NT 3.1 ± % (15/18) VG/GA SP (1 d.o.)+dw (14 d.o.) NT 2.4 ± % (14/18) Control NT <1 0% (0/10) J3 NDRL0901 SP (1 d.o.)+dw (14 d.o.) 4.4 ± 1.3 NT 3.1 ± % (16/18) VG/GA SP (1 d.o.)+dw (14 d.o.) NT 2.5 ± % (16/18) Control NT <1 0% (0/10) 1 Day-old SPF birds were vaccinated with the NDRL0901 strain ( EID 50 per dose) at 1 d old (spray; SP) and/or 14 d old (drinking water; DW). 2 HI titers are expressed as log 2 mean HI antibody titers ± standard deviations. 3 At 2 wk after the last vaccination, the birds were challenged with the NDV Kr005 strain ( EID 50 per dose) via the intramuscular route. The protection rate is expressed as number of birds that survived (n)/number of birds challenged (N). (especially respiratory signs) in day-old chicks. This indicates that the NDRL0901 strain can be safely used as a live NDV vaccine in the field. In the field conditions, ED, SP, and DW are the major routes of NDV vaccine administration on farms (OIE 2012). In particular, the DW route in young birds is the most widely used method of administration of live NDV vaccine on the farm in many countries including Korea. This route has the advantage of ease of administration and reduction of labor on intensive commercial farms where many birds are raised. However, in this study the method resulted in a lower level of immunity and thus increased mortality when compared with those that were vaccinated through then ED and SP routes, as also reported in previous studies (Okwor et al., 2013; Rehmani, 1996). Nevertheless, DW vaccination of the NDRL0901 pilot vaccine gave good protection of >80% 14 dpv in the SPF chickens. To prevent an NDV outbreak in the field, the most widely used live NDV vaccination program employed is drinking water vaccination on the farm (especially when the chickens are 2 wk old) combined with spray vaccination at the hatchery. In field trials on broiler farms, the SP administration of the NDRL0901 pilot vaccine at hatcheries gave good protection of >80% when the chickens were challenged at 19 d of age. Unvaccinated birds on the same farm had little protection efficacy although the level of humoral immunity at 14 d old was comparable with that in the vaccinated birds. This may be because active immunity by vaccination is on the increase and the titers of MDAs in unvaccinated broilers decreased over time. In addition, booster vaccination via DW on d 14 also gave good protection when the chickens were challenged at 33 d of age. Our results indicate that the NDRL0901 strain might be a suitable NDV live vaccine candidate for use at the hatchery as well as on a farm that is safe while maintaining solid immunity under a combined SP and DW vaccination program. ACKNOWLEDGMENTS This work was fully supported by the Animal and Plant Quarantine Agency, Ministry of Agriculture, Food and Rural Affairs (MAFRA), Republic of Korea (Project M-AD ). REFERENCES Alexander, D. J Newcastle disease diagnosis. Pages in Newcastle Disease. D. J. Alexander, ed. Kluwer Academic Publishers, Boston, MA. 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