The TW I-type IBV strain, CK/CH/GD/GZ14 was isolated in 2014 by our lab (Feng et al. 2017) and purified using the limiting dilution method (Zhang et al. 2018). CK/CH/GD/GZ14 p5 strain was purified with following procedure: Nine-day-old SPF chicken embryos were purchased from the SPF Experimental Animal Center of Guangdong Dahuanong Animal Health Co., Ltd. The virus stock were diluted into five gradients (from 10−5 to 10−9), and five SPF chicken embryos were inoculated for each gradient. After inoculation, the embryos were cultured in an incubator at 37 ℃ for seven days, the dead chicken embryos were discarded within 24 h, after 7 days, the allantoic fluid of chicken embryo was collected, and the chicken embryo was dissected, and the allantoic fluid which produced the maximum dilution multiple of dwarf embryo was selected to continue dilution and passage.
The passage procedure was: The purified strain, CK/CH/GD/GZ14 p5 strain, was diluted 100-fold with sterile physiological saline. Then, 0.1 mL of the virus dilution was inoculated into the allantoic fluid of ten day-old SPF chicken embryo and cultured in a 37 ℃ incubator for 72 h, after which the allantoic fluid was collected. During the later stages (40 generation), the time of death of the chicken embryos advanced due to the adaptation of the strain to the chicken embryo. The embryo was harvested 48 h after it was inoculated, and a continuous passage of 145 generations was performed. Viral purification was performed once every 30 passages [p35 (35th generation), p65 (65th generation), p95 (95th generation), p105 (105th generation), p120 (120th generation), and p135 (135th generation)].
A total of twenty 14 day-old chickens were purchased from the SPF Experimental Animal Center of Guangdong Dahuanong Animal Health Co., Ltd and were divided into a control group and an experimental group (n = 10 in each group). The chickens were kept in a positive pressure isolator and were free to eat and drink. The experimental group was challenged with CK/CH/GD/GZ14 (104.5 EID50/0.1 mL) per chicken by the nose and eye. The control group was inoculated with the same dose of PBS. The chickens were observed for morbidity characteristics daily for 15 days after challenge.
Hematoxylin and eosin (H&E) staining was used to evaluate the pathological characteristics of tissues of chickens infected with the CK/CH/GD/GZ14 strain. A histopathological section experiment was performed as previously described (Zhang et al. 2015).
A total of 90 ten-day-old SPF chickens were divided into nine groups, including a control group (10 chickens), p5 group (10 chickens), p70 group (10 chickens), p80 group (20 chickens), p95 group (20 chickens), and p105 group (20 chickens). The chickens in the experimental group were inoculated with allantoic fluid containing different doses of virus. For the p5 and p70 groups, the inoculation dose was 104.5 EID50/0.1 mL. Ten chickens in the p80, p95, and p105 groups were inoculated with 104.5 EID50/0.1 mL, whereas 10 chickens in the p80, p95, and p105 groups were inoculated with 103.5 EID50/0.1 mL. The virulence of different generations was evaluated by assessing the clinical symptoms and pathological changes of the tissues. The clinical symptoms and death of the chickens were recorded daily. Five days after the inoculation, three chickens were randomly selected from each group to evaluate the pathological changes in the trachea, lung, and kidney tissues.
The effectiveness of the attenuated virus was evaluated in the 95th and 105th generations. A total of 132 one-day-old SPF chickens were randomly divided into six groups (22 chickens per group): groups 1–4 were vaccinated with the 95th and 105th generations at the dose of 103.5 or 104.5 EID50 at day 10; group 5 was non-immunized (positive control group); and group 6 was the negative control group. At the age of 25 days, the chickens in the vaccinated and positive control groups were challenged with the 5th generation of CK/CH/GD/GZ14 at the dose of 104.5 EID50. The detailed grouping is shown in Table 1.
Group Generation of vaccinated strain Vaccination dose Generation of the challenge strain Challenge day Challenge dose 1 p95 103.5 EID50 p5 25 104.5 EID50 2 p95 104.5 EID50 p5 25 104.5 EID50 3 p105 103.5 EID50 p5 25 104.5 EID50 4 p105 104.5 EID50 p5 25 104.5 EID50 5 – – p5 25 104.5 EID50 6 – – – – –
Table 1. Specific grouping for evaluation of vaccine effectiveness.
At 5 and 10 days after vaccination or 5 days and 10 days after challenge, three chickens from each group were sacrificed, and the tracheal, lung, and kidney tissues of important IBV-infected organs were collected. Detection of the viral copy number in tissues was assessed by qPCR after reverse transcription. Real-time PCR quantification of the viral load of each organ was conducted using standard curves as described previously (Chen et al. 2011). Briefly, two primers F: 5′-TTTGCTGGAACTTGTCTTGCAAGTATTAATG-3′ and R: 5′-CCTTCGTCTTTACTCTTGCTGATTGAAACAG-3′ were used to amplify a 600-bp IBV PCR product. Then the reaction product was analyzed on 1% agarose gels and ligated into a PMD-18-T vector (TaKaRa, Biotechnology, Dalian, China) to transform TOP10 competent cells. The positive clones were sequenced, and plasmids were extracted. The DNA concentration was calculated by measuring the absorbance at 260 nm. Using the DNA concentration, the plasmid copy number was calculated using the following formula: copy/μL = 6.02 × 1023 (copy/mol) × DNA concentration (g/μL)/MW (g/mol). Serial tenfold dilutions from 103 to 1010 copies of the purified plasmid were prepared in duplicate to produce a standard curve.
Three chickens from each group were sacrificed at day 5 after vaccination and day 5 after challenge and the trachea of each chicken was collected. The upper, middle, and lower sections of the tracheas were separated to observe the level of ciliary movement and shedding. The tracheal cilia activity was scored using a 0–4 scoring system. If the cilia in the whole trachea section display activity, the score is 0; if the cilia in the whole section of the trachea have approximately 75%–100% activity, the score is 1; if the cilia in the whole section of the trachea have approximately 50%–75% activity, the score is 2; if, in the whole trachea, 25%–50% of the cilia have activity, the score is 3; if less than 25% of the cilia in the whole trachea have an activity or the cilia have no activity at all, the score is 4. A significant difference analysis was performed between the experimental groups and the negative control group.
Throat and cloaca swabs of each group were collected after vaccination and after challenge at day 5, 10, and 15. The throat and cloacal swabs from the same chicken were mixed and shaken in 0.5 mL physiological saline (containing penicillin 8000 U and streptomycin 8000 U), repeatedly frozen, and thawed three times. The samples were centrifuged at 9300 ×g for 5 min. The supernatant was collected and 0.1 mL of the supernatant was inoculated into SPF chicken embryo. After six days, the chicken embryos were dissected to observe whether there were lesions characteristic of IBV, and the morbidity of each group was counted.
The clinical symptoms and death of the chickens were recorded daily following the challenge. The behaviour and clinical symptoms of the chickens were recorded daily. All of the surviving chickens in each group were subjected to the post-mortem examination, and pathological changes in the trachea, lungs and kidneys were examined by hematoxylin and eosin (H&E) staining. The morbidity and mortality of each group of chickens were assessed.
A total of 24 pairs of primers used to amplify the complete genome sequence of IBV were designed and synthesized by AuGCT DNA-SYN Biotechnology Co., Ltd. (Beijing, China) (Supplementary Table S1). Viral RNA extracted from the allantoic fluid of different generations was amplified using 24 primer pairs by reverse-transcription polymerase chain reaction (RT-PCR). The products of each RT-PCR were ligated to the cloning vector, pMD18-T (Takara, Japan), and transformed into competent cells. Positive clones were screened by PCR and sequenced by Shanghai Sang-gong Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). The nucleotide sequences of each generation were sequenced and assembled into a complete genome sequence respectively. The genome sequences were compared with that of the CK/CH/GD/GZ14 strain using DNAstar software.
Statistical analyses were performed with GraphPad Prism software and expressed as means and standard deviation. The statistical significance of data was calculated with a one-way analysis of variance (ANOVA) between the experimental groups. Significant differences are indicated as * P < 0.05 and ** P < 0.01.
The Serial Passage of the TW I-Type IBV CK/CH/GD/GZ14 Strain
Pathogenicity Study of the TW I-Type IBV Strain, CK/CH/GD/GZ14
Evaluation of the Attenuated Effects of CK/CH/GD/GZ14 with Different Generations
Evaluation of Vaccine Efficacy Based on the Viral Load in the Organs
Evaluation of Vaccine Efficacy Using the Tracheal Cilia Stagnation Experiment
Evaluation of Vaccine Efficacy Based on the Virus Shedding
Evaluation of Vaccine Efficacy Based on Clinical Morbidity and Histopathological Sections
Complete Gene Sequencing Analysis of Different Generations of CK/CH/GD/GZ14 Viruses
The 14 day-old chickens in the experimental group were infected with the CK/CH/GD/GZ14 strain (n = 10). On the sixth day post-infection, one chicken exhibited sneezing, listlessness, and huddling. Chicken death began on the seventh day post-infection. The post-mortem examination revealed tracheal congestion, massive urate deposition in the heart and ureter, and renal enlargement with variegated appearance, whereas the trachea, heart, ureter and kidney in the negative control group were unaffected (Fig. 1). The follow-up record was continued until 15 days post-challenge. A total of eight chickens died, thus the mortality rate of CK/CH/GD/GZ14 strain was 80%. At the end of the experiment, all the chickens in experimental group displayed obvious renal swelling. However, the chickens in the negative control group had no sneezing, listlessness, and huddling, and the kidneys were normal after dissection. The results showed that the TW I-type IBV CK/CH/GD/GZ14 strain had strong pathogenicity to chickens, especially causing kidney enlargement, with the morbidity rate of 100%.
Figure 1. Representative images of severe kidney enlargement and congestion induced by CK/CH/GD/GZ14 to SPF chickens. Fourteen-day-old SPF chickens were infected with CK/CH/GD/GZ14 (104.5 EID50/0.1 mL per chicken) or PBS via the nose and eye. The kidney was macroscopically examined at 7 days post-challenge. Representative images from the uninfected group and infected SPF chickens are shown in A and B respectively. A Normal kidney of SPF chickens inoculated with PBS in the negative control group. B The enlarged and mottled kidney of SPF chickens infected with CK/CH/GD/GZ14 (black arrow).
Our results showed that CK/CH/GD/GZ14 strain could cause 100% morbidity and 70% mortality in infected chicken embryos. With the increase in passage generation on SPF embryos, the virulence of the virus against 10 day-old SPF chickens gradually weakened, and the morbidity rate of the 95th generation (p95) and the 105th generation (p105) was 20% after infection, which was milder than the virulence associated with the 5th generation.
As shown in Table 2, compared with the p80/103.5 EID50 group, the p80/104.5 EID50 group had high morbidity and mortality in SPF chickens, whereas none of the p80/103.5 EID50 group died. The p95 and p105 groups had fewer diseased chickens than the p80, p70, and p5 groups. The p95 and p105 groups did not have any dead chickens and the symptoms were milder than those in the p5 group were.
Group Generation/Vaccination dose Morbidity Mortality 1 p5/104.5 EID50 10/10 7/10 2 p70/104.5 EID50 5/10 3/10 3 p80/104.5 EID50 3/10 3/10 4 p80/103.5 EID50 3/10 0/10 5 p95/103.5 EID50 2/10 0/10 6 p95/104.5 EID50 2/10 0/10 7 p105/103.5 EID50 2/10 0/10 8 p105/104.5 EID50 2/10 0/10 Negative control – 0/10 0/10
Table 2. The morbidity and mortality of chickens after vaccination of CK/CH/GD/GZ14 strain of different generation.
As shown in Fig. 2, in the p5/104.5 EID50 infected group, the tracheal lamina propria displayed edema, the mucosal upper layer had blood cell shedding; the pulmonary bronchial interstitium showed a large number of inflammatory cell hyperplasia; the alveolar cavity exhibited inflammation and was blocked, and there was a large amount of inflammatory cell infiltration in the kidney. However, the lung and kidney tissues of the p95/103.5 EID50-, p95/104.5 EID50-, p105/103.5 EID50-, and p105/104.5 EID50-infected groups were normal; only the trachea in the p95/103.5 EID50 and p95/104.5 EID50 groups had edema of the lamina propria. According to the histopathological sectioning results, the pathogenicity of the CK/CH/GD/GZ14 strain in the trachea, lungs, and kidneys gradually weakened with the increasing number of passages, and pathogenicity was significantly reduced when passaged to p105.
Figure 2. Histopathological changes in the lungs, trachea, and kidneys of SPF chickens infected with CK/CH/GD/GZ14 of different generations. Ten-day-old SPF chickens were infected with 104.5 EID50/0.1 mL 5th (p5), 103.5 and 104.5 EID50/0.1 mL 95th (p95) and 105th (p105) generations of CK/CH/GD/GZ14 passaged in SPF chicken embryos. After infection for 5 days, the lungs, trachea, and kidneys were examined by H&E staining (200 ×). The chickens in the negative control group were inoculated with 0.1 mL PBS. The histopathological changes were denoted by arrows. In the p5/104.5 EID50 infected group: a large number of inflammatory cells proliferated and infiltrated in the bronchiolar interstitium and a large number of inflammatory substances were blocked in the alveolar cavity; Edema of lamina propria and exfoliation of blood cells in the upper layer of mucosa; Congestion and infiltration of a large number of inflammatory cells. In the p5/103.5 EID50 infected group, the trachea showed the edema of lamina propria and infiltration of inflammatory cells in mucosal layer. In the p5/103.5 EID50 infected group, the structure of trachea is normal while the lamina propria is edema for the trachea.
In the non-immunized group (positive control group), the chickens exhibited sneezing, listlessness, and huddling on the fourth day after challenge with the CK/CH/GD/GZ14 p5 strain. However, the chickens vaccinated with the CK/CH/GD/GZ14 p95 and p105 strains appeared normal following the challenge. All chickens were sacrificed on day 15 post-challenge, and two chickens in the p95 group displayed serious renal swelling, while two chickens in the group vaccinated with the CK/CH/GD/GZ14 p105 strain exhibited moderate renal enlargement. Only four chickens in the non-immunized group (n = 22) remained at 15 days post-challenge, with kidney enlargement (Fig. 3). Furthermore, the lung, trachea, and kidneys of the remaining chickens in the vaccinated groups were normal, while lungs of chickens in the positive control group showed hemorrhaging and extensive shedding of bloody tissue. Large amounts of bloody and inflammatory substances were observed in the upper layer of the trachea, and the kidney showed a large degree of inflammatory cell proliferation and infiltration (Fig. 4).
Figure 3. Representative images of kidneys in p95-, p105-vaccinated chickens following challenge. A dose of 104.5 EID50/0.1 mL of the 95th-generation strain (p95) and the 105th-generation strain (p105) was used for vaccination in 10 day-old SPF chickens, and a dose of 104.5 EID50/0.1 mL of the 5th-generation strain (p5) was used for viral challenge. Representative images of the kidneys from unvaccinated (positive control), p95-vaccinated, p105-vaccinated chickens after challenge were shown. The enlarged and mottled kidney in unvaccinated group were indicated by blue arrows, while the kidneys in vaccinated (p95 or p105) and challenged chickens were normal.
Figure 4. Histopathological changes in the lungs, trachea, and kidneys of 10 day-old chickens in different vaccinated groups at 15 days post-challenge with CK/CH/GD/GZ14 (p5). SPF chickens were vaccinated with 103.5 and 104.5 EID50/0.1 mL CK/CH/GD/GZ14 p95 or p105 strains and then challenged with the 5th-generation strain (p5) at the dose of 104.5 EID50/0.1 mL at 15 days post-vaccination. The lungs, trachea, and kidneys were examined by hematoxylin and eosin (H&E) staining (200 ×) at 15 days post-challenge. The lung, trachea, and kidneys of the remaining chickens in the vaccinated groups were normal. The histopathological changes in the positive control group were denoted by arrows: Congestion and bleeding, with a large amount of bloody material shedding in lung. Edema of lamina propria of mucous membrane and exfoliation of a lot of bloody and inflammatory substances in the upper layer of trachea. Proliferation and infiltration of a large number of inflammatory cells in kidney.
The challenge experiment results showed that the CK/CH/GD/GZ14 strain of the 95th and 105th generations could provide better protection against the CK/CH/GD/GZ14 strain, with a relative protection rate of 80% (Table 3). There was no significant difference in the tracheal ciliary motility between the p105/p95 (103.5 EID50)-vaccinated group and negative control group at five days post-vaccination (P > 0.05) (Fig. 5A). However, the p95 (104.5 EID50) group showed ciliary movement stagnation and ciliary shedding, and the difference was extremely significant compared with the negative control group (P < 0.01), while there was no significant difference in the tracheal ciliary motility between the p105 (104.5 EID50)-vaccinated group and the non-vaccinated control group (P > 0.05), illustrating that p105 generation is more suitable for vaccine development. Furthermore, the difference of tracheal cilia activity between the vaccinated/challenged groups and the unvaccinated/challenged positive control group was extremely significant at 5 days post-challenge (P < 0.01) (Fig. 5B).
Group Generation of vaccinated strain Vaccinated dose Generation of the challenge strain Anatomy results Morbidity (n = 10 in each group) Protection rate 1 p105 103.5 EID50 p5 1 chicken showed mild kidney enlargement and 1 chicken showed kidney enlargement 2/10 80% 2 p105 104.5 EID50 p5 1 chicken showed mild kidney enlargement, 1 chicken showed kidney enlargement and yolk malabsorption 2/10 80% 3 p95 103.5 EID50 p5 2 chickens showed kidney enlargement 2/10 80% 4 p95 104.5 EID50 p5 2 chickens showed kidney enlargement 2/10 80% 5 – – p5 10 chickens showed kidney enlargement and urate deposition 10/10 0 6 – – – The kidneys of all chickens were normal 0 –
Table 3. Protective efficacy of the attenuated virus CK/CH/GD/GZ14 of different generation to 10-day-old SPF chickens against challenge with the CK/CH/GD/GZ14 virus of the 5th generation.
Figure 5. The cilia stagnation of chickens in the different vaccinated and vaccinated/challenged groups. A Ten-day-old SPF chickens were vaccinated with CK/CH/GD/GZ14 strains of different generations using different inoculation doses. At five days post-vaccination, the ciliary movement stagnation in different groups was analyzed. The p105-vaccinated group was better than the p95-vaccinated group according to the score of tracheal cilia stagnation. B Ten-day-old SPF chickens were vaccinated with CK/CH/GD/GZ14 strains of different generations using different inoculation doses. After 15 days post-vaccination, the chickens were challenged with CK/CH/GD/GZ14 of the 5th generation for 5 days, and then, the ciliary movement stagnation in different groups was further analyzed. A dose of 104.5 EID50/0.1 mL was used for viral challenge. There was no significant difference for the score of tracheal cilia stagnation between the p95-vaccinated/challenged and p105-vaccinated/challenged groups. ** denotes P < 0.01, n.s. denotes not significant, compared with the control group.
As shown in Supplementary Table S2, after vaccination with the same dose of virus, the rate of virus shedding in the p105-vaccinated group was lower than that of the p95 group. At 10 days post-challenge, the virus shedding rate of the throat swab in the p105-vaccinated group was significantly lower than that in the p95-vaccinated group, suggesting that with the increasing number of passages, the ability of the attenuated strain to block virus shedding gradually enhanced.
The CK/CH/GD/GZ14 virus was detected in the trachea, lungs, and kidneys in the p95 and p105 vaccinated groups at 5 and 10 days post-vaccination, of which the highest number of viral RNA copies was observed in the trachea (Fig. 6A, 6B). The viral RNA copies in the lungs, trachea, and kidneys of the p105-vaccinated group were significantly lower than those in the same dose p95-vaccinated group post-challenge (Fig. 6C, 6D). Collectively, these data suggest that p105 providing efficient protection from infection with the TW I-type CK/CH/GD/GZ14 virus.
Figure 6. Viral RNA copies of the major organs (kidney, lung and trachea) at 5 and 10 days after p95 and p105 vaccination or at 5 and 10 days post-challenge with CK/CH/GD/GZ14 of the 5th generation in different vaccinated groups. A, B The viral RNA copies were detected in the trachea, lungs and kidneys at 5 (A) or 10 (B) days post-vaccination. C, D The viral RNA copies were tested in the trachea, lungs and kidneys of p95- and p105vaccinated chickens followed by challenging with CK/CH/GD/GZ14 of the 5th generation at day 5 post-challenge. *denotes P < 0.05, ** denotes P < 0.01, compared with the control group.
The complete gene sequences of the p50, p80, p95, p105 and p120 strains were found to have nucleotide point mutations at multiple positions compared with p5. There were 11 nucleotide point mutations in p50 which were highly stable and continued to p120. There were six mutations generated during passaging from the p50 generation to the p80 generation, as well as one nucleotide insertion and one nucleotide deletion in the 5′ UTR region. Furthermore, three mutations occurred from p80 to p95 passaging, and one mutation generated from p95 to p105 passaging. Nucleotide mutations, insertions, and deletions ultimately resulted in the insertion and replacement of 19 amino acids in the entire CK/CH/GD/GZ14 genome. Among the 19 amino acid mutations, seven amino acid mutations occurred in the 1a gene (V957F, A1209S, W2249R, Y2491H, Q2707R, Q3500K and A3929V); four amino acid mutations occurred in the S1 gene (P118L, T179A, V387F, and R411L); three amino acid mutations occurred in the S2 gene (A222V, S345F, and S468F); two amino acid mutations occurred in the N gene (P145L and A403P); and one amino acid mutation occurred in each of the 1b (A423S), E (R70I), and 5a (E32K) genes (Table 4), indicating that low pathogenicity and good antigenicity may have a relationship with the stable amino acid point mutation of 1a, S1, S2, N, 1b, E, and 5a genes, as well as nucleotide insertions and deletions in the 5′ UTR region.
Gene Nucleotide position p5–p50 p50–p80 p80–p95 p95–p105 p105–p120 Amino acid position Amino acid insertion and replacement 5'-UTR 16 – insert A – – – – – 5'-UTR 18 – A → T – – – – – 5'-UTR 21 – deletion T – – – – – 1a 2854 G → T – – – – 957 V → F 1a 3625 G → T – – – – 1209 A → S 1a 6844 – T → C – – – 2249 W → R 1a 7471 T → C – – – – 2491 Y → H 1a 8280 A → G – – – – 2707 Q → R 1a 10, 498 C → A – – – – 3500 Q → K 1a 11, 457 T → A – – – – 3820 Silent 1a 11, 786 C → T – – – – 3929 A → V 1b 1414 – G → T – – – 423 A → S E 209 – – G → T – – 70 R → I S1 353 C → T – – – – 118 P → L S1 535 – A → G – – – 179 T → A S1 1159 – G → T – – – 387 V → F S1 1232 – G → T – – – 411 R → L S2 665 – – – C → T – 222 A → V S2 1034 C → T – – – – 345 S → F S2 1403 C → T – – – – 468 S → F 5a 94 – – G → A – – 32 E → K N 434 – – C → T – – 145 P → L N 1207 G → C – – – – 403 A → P
Table 4. Summary of nucleotide and amino acid mutations during CK/CH/GD/GZ14 passage.