Background
Newcastle disease virus (NDV) and avian influenza virus (AIV) are two of the most important pathogens in poultry worldwide [
1]. Newcastle disease (ND) is usually caused by virulent NDV, which can result in 100% mortality in many species of birds [
2]. The H9N2 low pathogenic avian influenza viruses have been circulating worldwide in multiple avian species, resulting in great economic losses owing to reduced egg production or increased mortality associated with coinfection with other pathogens [
3‐
6]. Thus, severe economic losses in the poultry industry caused by NDV and AIV highlight the importance of vaccine improvement and development.
In China, the implementation of extensive vaccination programs in commercial poultry farms has reduced the number of epizootic outbreaks of ND in recent years. However, genotype VII NDV strains of class II have remained a constant threat to domestic poultry since the 2000s and most recent Chinese isolates belong to a sublineage of genotype VIId [
7‐
9]. The predominance of genotype VIId in China is similarly observed in other countries in Asia and the Middle East, including Iran [
10], Qatar [
11], Japan [
12], Korea [
13], Kazakhstan [
14], Vietnam [
15], Pakistan [
16], Israel [
17], and Malaysia [
18,
19]. In a previous study, we genotypically characterized the NDV isolates recovered from chickens between 2005 and 2012 to establish the nature of the circulating genotype VII strains in China [
20]. Our results indicated that genotype VIId strains were at least 95.1% identical at the nucleotide level across the whole genome. However, they showed only 82–83% nucleotide sequence identity with vaccine strains LaSota and B1. These vaccine strains belong to genotype II in class II and were isolated 60 years ago. The outbreaks of ND in vaccinated poultry flocks in recent years are mainly attributed to the significant differences in the biology, serology, and genetics of the predominant circulating NDV strains compared with the current vaccine strains [
8].
On the basis of clade classification, 69 genotypes were detected among H9N2 chicken viruses isolated in China from 1994 to 2013 [
21]. In contrast, most of the 2010–2014 viruses belonged to a single G57 genotype, which was first detected in chickens in Jiangxi and Jiangsu Provinces in 2007, and its prevalence increased sharply during 2009. Since 2010, it has been the predominant genotype in circulation throughout China. This genotype was generated by continued reassortment that can be traced back to the year 1994. The neuraminidase gene was derived from the A/chicken/Beijing/1/1994-like lineage. Since the late 1990s, vaccination has been carried out in China to prevent and control H9N2 AIV infection in chicken flocks [
21]. However, commercial vaccines did not provide complete protection against endemic H9N2 AIVs [
22].
In this study, we developed an inactivated H9N2 and NDV vaccine based on a H9N2 A/chicken/Hebei/G/2012 (G) virus and a ND Chicken/Shandong/aSG10/2010 (aSG10) attenuated virus that are closely related to the current endemic strains of H9N2 and ND virus in China and evaluated its efficacy in chickens.
Discussion
In China, virulent NDV genotype VII first emerged in the 2000s [
23,
24], and strains recently isolated from chickens have also been shown to belong to this genotype. Recent studies and our present results show that isolates from this prevalent genotype differ from the widely used vaccine strain LaSota and B1 in the immune responses they evoke [
8,
24]. Therefore, antigenic differences between the prevalent circulating genotype and vaccine strains might account for the current ND outbreaks in vaccinated poultry flocks [
25]. To tackle this problem, new vaccines that better match circulating viruses have been developed [
23,
26]. The current study therefore aimed to develop an NDV vaccine strain that is immunogenic and safe for use in chickens.
Vaccination is an effective way to prevent and control the spread of H9N2 AIVs, but the current vaccine used in China is prepared from isolates that were circulating in the early 1990s, and their protective efficacy has been decreasing in recent years. A HI test showed that the commercial vaccine strain SS/94 does not provide robust protection against emerging H9N2 AIVs in China [
22]. It is therefore crucial that novel candidate vaccine strains are employed to combat emerging H9N2 AIVs in China. Previous phylogenetic analysis of the HA genes of H9N2 AIVs isolated in China between 2010 and 2014 demonstrated that group III, as an emerging lineage, has become one of the dominant clades. Increasing evidence suggests that the antigenic drift of field H9N2 AIV in China is a result of the incomplete protection offered by the commercial vaccine, probably caused by the persistent prevalence of H9N2 AIV. Our results also highlight that the currently used vaccine strain should be regularly evaluated and updated to achieve optimal protection against AIV infections [
21].
According to previous phylogenetic analyses, most NDVs isolated in 2010–2014 belonged to genotype VII and most H9N2 AIVs belonged to subgroup III in the BJ/94-like clade, a predominant genotype. These strains showed high homology to each other and a lower level of homology to traditional vaccine strains in China. In this study, a new inactivated vaccine was developed based on the prevalent NDV and H9N2 AIV strains (NDV aSG10 and H9N2 G) and its efficacy was evaluated in chickens [
27,
28].
Evaluation of NDV or AIV vaccine immunogenicity is largely based on HI antibody titers. In this study, the HI antibody titers of all chickens vaccinated with the inactivated vaccine were detected at weekly intervals until 15 wpv by the HI test. The results indicated that the vaccine could induce a fast immunological reaction and the mean HI titers were over 6.9 log2 against both NDV and H9N2 AIV at 2 wpv. At 4 wpv, peak levels of HI antibodies were detected with a mean HI titer of 8.6 log2 against NDV and 9.5 log2 against H9N2. Antibody titers of more than 6.0 log2 were detected up until at least 15 wpv. The results suggested that this new inactivated vaccine was significantly more effective at generating HI antibody responses than the traditional vaccine strain.
We next evaluated whether vaccination of chickens with the inactivated NDV and H9N2 vaccine could reduce the disease rates of both endemic NDV and H9N2 viruses in China by challenge test. We challenged the birds via the injection route in order to the birds receive actual dose of challenge viruses. We also tried a natural route for NDV and H9N2 in our previous studies and the birds demanded higher HI antibodies to obtain good protection [
28,
29]. Reduced mortality rates and decreased viral shedding were detected. Unvaccinated chickens challenged with NDV showed a 100% mortality rate and high levels of virus shedding of 3.01–3.64 log
10 EID
50/0.1 mL, compared with no mortality and a lower rate of viral shedding of 0–1.07 log
10 EID50/0.1 mL in vaccinated groups. All of the unvaccinated chickens challenged with H9N2 shed virus, whereas no virus shedding was detected in vaccinated groups.
Conclusion
Our results showed that the inactivated NDV and H9N2 vaccine (strain NDV aSG10 and H9N2 G) induces a high and prolonged immune response in vaccinated chickens and good protection efficacy against prevalent viruses. This novel vaccine could therefore potentially be used in the poultry industry to prevent and control ND and H9N2 influenza in chickens in China.
Methods
Animals
All SPF chicken eggs and chickens were purchased from Merial Vital Laboratory Animal Technology Co., Ltd (Beijing, China). The chickens were kept in isolators at China Agricultural University throughout the experiment and the animal rearing facilities were approved by the Beijing Administration Committee of Laboratory Animals under the leadership of the Beijing Association for Science and Technology (approval ID SYXK [Jing] 2013–0013).
Vaccine viruses
The selected NDV candidate was strain Chicken/China/Shandong/aSG10/2010 (abbreviated as aSG10) of genotype VIId, which was an attenuated form of the virulent strain SG10 isolated from an outbreak of ND in chickens with an intracerebral pathogenicity index (ICPI) of 1.89 and a mean death time of 45 h [
29]. The selected H9N2 AIV candidate was strain A/chicken/Hebei/G/2012 (abbreviated as G) of BJ/94 (subgroup III), which was isolated from a vaccinated breeder broiler flock in 2012 in northern of China.
Challenge viruses
HA gene segments of 19 H9N2 viruses and F gene segments of 11 NDVs isolated in China from 2012 to 2014 were sequenced as described previously [
21,
27]. Multiple sequence alignment was conducted using the Clustal W program of the MEGA 5.1 software. A phylogenetic tree was constructed using the neighbor-joining method with 1000 bootstrap replicates using the MEGA 5.1 software. According to phylogenetic analyses, three NDV and H9N2 virus isolates were chosen for challenge experiments.
Biological characterization of the viruses
Each virus was amplified in 10-day-old SPF embryonated chicken eggs, and the virus titer (EID
50) was determined and calculated by the Reed–Muench method [
30] based on a hemagglutination assay of the allantoic fluid of eggs inoculated with 10-fold serial dilutions of viruses. The virulence of the viruses was evaluated using standard pathogenicity tests, the ICPI [
31] for NDVs, and the IVPI [
6] for H9N2 AIVs.
Vaccine development
NDV strains aSG10 and H9N2 G were propagated in the allantoic cavities of 10-day-old SPF embryonated chicken eggs. Allantoic fluid was harvested 72–96 h after inoculation, and the virus titer in the allantoic fluid of 108.50 EID50/0.1 mL for two virus was used for antigen production. The egg-derived virus was inactivated with formalin at 37 °C at a final concentration of 0.1% for 18 h for NDV aSG10 or 0.2% for 24 h for H9N2 G. Complete inactivation of the virus was confirmed through three passages in 10-day-old embryonated SPF chicken eggs. To produce the vaccine, the inactivated viral antigens were blended at a ratio of 1:1 (V/V), then mixed with oil emulsion adjuvants Marcol 52 (Seppic, Paris, France) at a ratio of 1:2 (V/V).
Efficacy experiment
Dynamics of HI antibody
Forty 3-week-old SPF While Leghorn chickens were divided into two groups and 20 for each. The birds in Group 1 were vaccinated with one single dose (0.25 mL/bird) of the inactivated vaccine via subcutaneous injection. Group 2 was the control. Blood was collected from each group (n = 10) at weekly intervals until 15 weeks post-vaccination (wpv). Serum was separated and stored at −20 °C until use. The antibody titers were determined by HI tests using 1% chicken red blood cells according to the OIE recommended protocol [
32]. The HI titers were expressed as the log2 of the reciprocal of the highest serum dilution, resulting in complete inhibition of red blood cells.
Challenge of vaccinated chickens with LPAI H9N2 and virulent NDV
SPF chickens were challenged with one of three LPAI H9N2 avian influenza viruses or one of three virulent NDV challenge strains three weeks after vaccination and observed for clinical disease and shedding for two weeks after challenge. A total of 95, 3-week-old, SPF chickens were randomly divided into 13 groups (A–M) and treated as shown in Table
4. In brief, six groups (A–C and G–I, n = 10) were injected subcutaneously with one single dose (0.25 mL/bird) of the inactivated vaccine, another seven unimmunized groups served as challenged controls (D–F and J–L, n = 5) and unchallenged controls (M, n = 5). Chickens of six immunized groups and six control groups were infected with 10
5.0 EID
50 for NDV (Intramuscular injection) and 10
6.0 EID
50 for H9N2 (Intravenous injection) at 3 wpv. Chickens were monitored daily for morbidity and mortality for 14 days.
Table 4
Assignment of group and the treatment of chickens
A |
n = 10 | Yes | NDV-HB12 | 105.0EID50
| Intramuscular injection |
B |
n = 10 | Yes | NDV-SD13 |
C |
n = 10 | Yes | NDV-HB14 |
D |
n = 5 | No | NDV-HB12 |
E |
n = 5 | No | NDV-SD13 |
F |
n = 5 | No | NDV-HB14 |
G |
n = 10 | Yes | H9-JS12 | 106.0EID50
| Intravenous injection |
H |
n = 10 | Yes | H9-HB13 |
I |
n = 10 | Yes | H9-SD14 |
J |
n = 5 | No | H9-JS12 |
K |
n = 5 | No | H9-HB13 |
L |
n = 5 | No | H9-SD14 |
M |
n = 5 | No | No | No | No |
Virus isolation and titration
To determine viral shedding from individual chickens, cloacal or throat swabs were obtained at 5 days post-challenge for virus isolation in eggs. Swab samples were suspended in 1.0 mL of PBS supplemented with penicillin-streptomycin (10,000 IU/mL) and then inoculated into 10-day-old embryonated chicken eggs (n = 3 eggs/sample) through the intra-allantoic route. The allantoic fluid was harvested at 5 days post-inoculation and checked for NDV and H9 AIV growth by a HA test using 1% chicken red blood cells. Positive samples were further titrated by EID50 determination in 10-day-old embryonated chicken eggs.
Acknowledgments
Grammar assistance was provided by Dr. Jia Xue of College of Veterinary Medicine,China Agricultural University, Beijing, P.R. China.
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