Two reassortant viruses (RG-HB29578 and RG-FJ21099) were obtained using the (6 + 2) RG method in vero cells. To confirm the reassortant viruses, we sequenced the first-generation rescued viruses RG-HB29578 V1E1 and RG-FJ21099 V1E1 (initial growth in Vero cells followed by one passage in eggs). Sequence analysis indicated that the cleavage site of the HA sequence was consistent with the desired cleavage motif (Figure 1A and 1B). No other mutations were detected when comparing the HA and NA sequences with those of the parental strains HB29578 and FJ21099. Six internal gene segments were derived from the high-yield PR8 strain. The sequencing results for RG-HB29578 V1E1 and RG-FJ21099 V1E1 viruses confirmed that the gene sequence was identical to that carried by plasmids used in their construction.
Rescued V1E1 virus was propagated in embryonated eggs for an additional nine passages, and the resulting passages were designated as V1E2–V1E10 to determine the genetic stability of the HA and NA genes. No mortality among the eggs was observed for any of the virus passages. The HA titers of RG-HB28578 for passages V1E2 to V1E10 were 256–512 HA units, with the exception of V1E1 (HA 128) and RG-FJ21099, between 256–512 for passages V1E1 to V1E10 (Table 1). The two CVVs from each passage were sequenced and compared with their respective V1E1 sequences. The HA sequence of the two CVVs retained the modified motif at the cleavage site, and no mutations had occurred from V1E2 to V1E10. Sequence analysis of the NA gene of the two CVVs from V1E2 to V1E10 revealed no mutations compared with the respective NA gene sequences of V1E1. In the internal gene, sequencing results revealed a mixture of amino acids (A/T) at position 648 in basic protein 1 (PB1) of RG-HB29578 V1E10 compared with a homozygous A at the same position of V1E1 to V1E9. At position 347 in nucleoprotein (NP), a mutation from E to K first appeared in RG-HB29578 V1E7, and at position 384 in NP, a mixture of amino acids (R/K) was found in RG-FJ21099 V1E10 (Table 1).
Viruses Passage historya HA titer LogTCID50/mLb
(mean ± SD)
IVPI PB1 648 NP 372 384 RG-HB29578 V1E1 128 − − A E R V1E2 256 7.34 ± 0.13 0 A E R V1E3 256 − − A E R V1E4 256 − − A E R V1E5 256 − − A E R V1E6 256 − − A E R V1E7 256 7.54 ± 0.12 0 A K R V1E8 256 − − A K R V1E9 512 − − A K R V1E10 512 7.81 ± 0.21 0 A/T K R RG-FJ21099 V1E1 256 − − A E R V1E2 256 8.28 ± 0.18 0 A E R V1E3 256 − − A E R V1E4 512 − − A E R V1E5 256 − − A E R V1E6 512 − − A E R V1E7 512 − − A E R V1E8 256 − − A E R V1E9 512 − − A E R V1E10 512 8.44 ± 0.08 0 A E R/K PR8 C1E2 1,024 8.48 ± 0.16 − A E R Note. a: V. Vero cells; E. eggs; C. MDCK cells; bMean ± standard deviations represent three replicates; TCID50 was determined by hemagglutination assay and calculated by the Reed-Muench method. The 1% Turkey red blood cells were used; IVPI: intravenous pathogenicity index; −: excluded from analysis. Changed amino acids are in bold.
Table 1. Virus titers and amino acid changes of CVVs during passage in eggs
The viral growth characteristics were assessed in MDCK cells and in eggs as per previous reports to analyze the replication efficiency of different passage viruses [26,27]. No significant difference was observed among RG-HB29578 V1E2, V1E7, and V1E10 at any of the indicated time points, although a single amino acid mutation was recorded at position 372 from E to K in NP of V1E7 and one mixture of amino acids A/T at position 648 in PB1 of V1E10 (Table 1, Figure 2A and 2B). No significant differences were detected between RG-HB21099 V1E2 and V1E10 at any of the indicated time points, although one mixture of amino acids R/K was detected at position 384 in NP of V1E10 (Table 1, Figure 2C and 2D). All detected viruses had higher titers in embryonated eggs than in cells at each time point, and these viral titers were all above 28. We observed that the detected viruses had similar growth characteristics to that of PR8 at most p.i. time points in MDCK cells, except for RG-HB29578 at 24 h p.i. (*P < 0.05).
For CVVs derived from highly pathogenic H5 strains by RG, the HA was modified from possessing multiple basic amino acids at the HA cleavage site to a single basic amino acid; as a result, such viruses were unable to form plaques in the absence of added trypsin . The CVVs should maintain a HA cleavage site consistent with a low pathogenic phenotype upon multiple passages in embryonated eggs. We selected V1E1 and V1E10 of the two CVVs to determine whether they maintained a low pathogenic phenotype using a TPCK-treated trypsin-dependent plaque formation assay in MDCK cells. The results showed that none of the tested viruses can form plaques in the absence of TPCK-treated trypsin, whereas in the presence of TPCK-treated trypsin, plaques can be identified, indicating the low pathogenicity of these viruses (Figure 3).
Figure 3. Replication of two CVVs in MDCK cells with or without TPCK-treated trypsin. V1Ex: V1Ex with initial growth in Vero cells followed by x passage in eggs; the original viruses were diluted from 10−1 to 10−6, and 10−2 to 10−6 diluted viruses were inoculated in MDCK cells. Mock cells were inoculated with PBS.
To confirm that the antigenicity of the two CVVs, which were passaged ten times, was consistent with the homologous wild-type viruses, we tested the reactivity of ferret antisera to RG-HB29578 (V1E2 and V1E10) and RG-FJ21099 (V1E2 and V1E10) viruses (Table 2). RG-HB29578 (V1E2 and V1E10) were immunogenic and induced HI antibody titers (160 and 640, respectively) against homologous viruses. RG-FJ21099 (V1E2 and V1E10) were immunogenic and induced HI antibody titers (160) against homologous viruses. Antisera to RG-HB29578 (V1E2 and V1E10) can inhibit the wild-type HB29578 virus well. Antisera to RG-FJ21099 (V1E2 and V1E10) reacted well with wild-type FJ21099 virus. Antisera to RG-HB29578 inhibited neither wild-type FJ21099 nor reassortant RG-FJ21099, and similarly, antisera to RG-FJ21099 inhibited neither wild-type HB29578 nor reassortant RG-HB29578. The above results indicate that both CVVs maintained consistent antigenicity to wild-type viruses after 10 passages, with no cross-reactivity between them.
Antigens Passage history Antisera RG-HB29578 V1E2 RG-HB29578 V1E10 RG-FJ21099 V1E2 RG-FJ21099 V1E10 HB-29578 wt E2 80 320 < 20 < 20 FJ-21099 wt E2 < 20 < 20 80 80 RG-HB29578 V1E2 160 640 < 20 < 20 RG-HB29578 V1E10 160 640 < 20 < 20 RG-FJ21099 V1E2 < 20 < 20 160 160 RG-FJ21099 V1E10 < 20 < 20 160 160 Note. wt: Wild-type. Boldface denotes titers of ferret sera with homologous antigens.
Table 2. Antigenicity analysis of two CVVs
To verify the low pathogenicity of the two CVVs, we tested viral pathogenicity in chickens. SPF chickens (n = 10) were inoculated with 1:10 diluted HA titers of RG-HB29578 (V1E2 and V1E10) and RG-FJ21099 (V1E2 and V1E10) via the intravenous route. All chickens remained healthy throughout the 10-day observation period with no mortalities (Table 1). The IVPI was calculated in accordance with the standards of OIE . The IVPI for both viruses was zero. These results demonstrate that both CVVs exhibited similar pathogenic characteristics that are indicative of low pathogenic avian influenza virus after 10 passages. Pathogenicity was unaffected by the substitutions in PB1 and/or NP.
Ferrets have been used extensively as an ideal indicator of influenza virus virulence in humans . The attenuation of CVVs in ferrets must be demonstrated in accordance with WHO guidelines. Thus, two RG viruses were tested in ferrets, with PR8 as the control virus. All ferrets survived the two-week observation period with slight weight loss, and the most weight loss was observed in the PR8-infected group (Figure 4). Nasal discharge was observed in all infected animals on 1–3 dpi. In the nasal wash samples, viral titers were detected at 1, 3, and 5 dpi. in the three groups but were not detected at 7 dpi (Table 3). In the nasal turbinate samples, viral titers were detected in all the infected animals on day 3, and CVVs titers were similar to those of PR8 (Table 3). In the lung samples, viral titers were detected in both PR8-infected animals and one of the animals for CVVs (Table 3). Viruses were not detected in any other organs (i.e., the spleen, intestine, brain, or olfactory bulb of the brain). Viruses were only detected in the respiratory system, including the lungs, nasal turbinate, and nasal wash fluid.
Virus Animal No. Nasal washesa Nasal turbinatesb Lungb Other organsb,c Day 1 Day 3 Day 5 Day 7 Day 3 Day 3 Day 3 PR8 1 4.00 2.23 3.83 / 2 4.17 2.00 2.00 / 3 2.50 2.00 / 4 2.17 1.67 / RG-HB29578 1 2.5 2.25 2.00 / 2 2.83 2.77 2.00 / 3 2.50 / / 4 1.67 1.33 / RG-FJ21099 1 2.00 2.50 1.73 / 2 2.67 2.23 2.00 / 3 2.33 1.50 / 4 1.67 / / Note. aLog10TCID50/mL. bLog10TCID50/g. cOther organs include brain, spleen, intestine, and olfactory bulb of the brain. /, Denotes that no virus titer was detected.
Table 3. Virus titer in different organs
Figure 4. Body weight change (%) post-infection. The change in weight is shown as an average with the standard deviation. Ferrets (n = 2 for each group) were lightly anesthetized via inhalation of isoflurane and inoculated intranasally with 1.0 mL containing 106 50% TCID50 RG-HB29578 or RG-FJ21099 or PR8 influenza virus.
Development and Assessment of Two Highly Pathogenic Avian Influenza (HPAI) H5N6 Candidate Vaccine Viruses for Pandemic Preparedness
- Received Date: 2020-02-05
- Accepted Date: 2020-07-22
- Highly pathogenic avian influenza H5N6 virus /
- Genetic stability /
- Candidate vaccine virus /
- Reverse genetic technology
|Citation:||LIU Li Qi, LI Zi, JIAO Ming, LU Jian, ZHOU Jian Fang, LI Xi Yan, LIU Jia, GUO Jun Feng, XIAO Ning, ZHAO Xiang, WANG Da Yan. Development and Assessment of Two Highly Pathogenic Avian Influenza (HPAI) H5N6 Candidate Vaccine Viruses for Pandemic Preparedness[J]. Biomedical and Environmental Sciences, 2020, 33(9): 670-679. doi: 10.3967/bes2020.088|