Complete Gene Sequence Analysis of isolated CIAVs
A total of 41 samples were detected to be CIAV positive from 437 tissue samples collected from June 2017 to December 2019 in Henan province. The overall positive rate was 9.4% (95% CI: 6.8%-12.5%). Four CIAV positive vaccines in 120 non-CIAV live attenuated vaccines were detected, and the overall positive rate was 3.33% (95% CI: 0.9%-8.3%) (Table 2). A total of 12 CIAV strains were isolated and identified by PCR from chicken samples and as shown in Fig. 1A, the amplicon sizes were 842 bp, 990 bp and 736 bp, respectively, and this was consistent with prediction.
Table 2. Organize the survey results and the CIAV test form for attenuated vaccine circulating in the market
Sample type
|
Number of samples tested (parts)
|
Number of positive samples (parts)
|
Positive rate/% (95%CI)
|
Tissue disease material
NDV+IB
|
437
53
|
41
3
|
9.4
5.66(1.2-15.7)
|
FPV
|
31
|
1
|
3.23(0.1-16.7)
|
NDV
|
34
|
0
|
0 (0-97.5)
|
MS
|
1
|
0
|
0 (0- 97.5)
|
FPV+ILTV
|
1
|
0
|
0 (0-97.5)
|
The whole genome sequences of 12 CIAV isolates were amplified and aligned to the reference strain. All isolates showed high similarity, ranging from 97.1% to 99.3%. Compared with 32 CIAV reference strains, four strains (HN-3, HN-5, HN-7, HN-10) showed 99% identity with the field strain isolated from Shandong province, China. The similarity between HN-11 strain and the Guangdong strain GD-1-12 was 99.1%, and 96% with Guangdong strain GD-K-12. The three strains HN-2, HN-4 and HN-6 had similarities of 98.4% with the previously isolated HN1405 strain (Fig. S1).
Four strains of CIAV virus (HN-1, HN-8, HN-9, HN-12) had a similarity between 98.1% and 99.7%, of which, HN-1 and HN-9 had the highest similarity. These four isolates are more closely related to the American strain L14767.1, with a similarity of over 98%. Compared to the vaccine strains that have been widely used worldwide, the 12 isolates had high sequence similarity with the Del-Ros vaccine strain, and ranged from 97.8% to 98.6%, and among which, the HN-11 isolate had the highest similarity with the vaccine strain. The similarity between the isolated strains and the Cux-1 vaccine strain worldwide was less than 98% (Fig. S1).
A phylogenetic tree, represented by two large branches, was constructed based on the complete genome sequence of 44 reference strains and 12 CIAV isolates from Henan. All of the Henan isolates were on the same large branch, which was closely related to the CIAV strains derived from Asia. After subdivision, the Henan CIAV isolate became located to three different small branches. The four strains HN-3, HN-5, HN-7, and HN-10 however, were on the same branch as the CIAV strains isolated from Shandong and Liaoning. The HN-11 and the Guangdong isolated strain GD-1-12 were on the same small branch. Both the HN-11 and the Guangdong strain, GD-1-12 evolved from the classical attenuated strain C369, isolated from Japan, and the attenuated strain, CIAVV89-69 isolated from Korea. However, the four strains HN-3, HN-5, HN-7, and HN-10 and the CIAV strains isolated from Shandong and Liaoning were closely related to the classical attenuated strain C369 and CIAVV89-69. The three strains HN-2, HN-4 and HN-6 appear on the same branch as the HN1405 strain isolated from Shandong. HN-1, HN-8, HN-9, and HN-12 are on the same branch as the HN1504 strain. All of the CIAV strains isolated in Henan are far removed from the internationally prevalent vaccine strain Cux-1, and are not on the small branch with the Del-Ros vaccine strain (Fig. 1B).
Amino acid sequence analysis of the VP1 and VP2 proteins
Amino acid sequence analysis based on the VP1 sequence (450 aa) showed that, the overall variation in VP1 protein from all of the isolates was 0-3.4%. The similarity in the VP1 amino acid sequence for the HN-1 and HN-12 strains reached 100%, and the sequence similarity with the American strain L14767.1 was very high (Fig. S2). When comparing the VP1 protein amino acid evolutionary tree (Fig. 2A), and the nucleotide sequence evolutionary tree (Fig. 2B), we found that the VP1 nucleotide tree was roughly the same as the whole genome nucleotide evolutionary tree. When we compared the amino acid trees, we found that the three strains, HN-3, HN-4, and HN-7 were on the same branch as GD-1-12, while the HN-3 and HN-7 isolates were on the same branch as the HN-5, and HN-10 isolates. Finally, the HN-4 isolate was on the same branch as the HN-2, and HN-6 isolates. Therefore, it can be inferred that there may exist site mutations, or sequence recombinations in these strains at the amino acid level. HN-1, HN-8, HN-9, and HN-12 still represent a large branch, which is not different from the phylogenetic tree at the nucleotide level.
VP2 is the most conserved CIAV protein and there are no significant differences at the amino acid level between the Henan isolates and the reference strain, and the co-efficiency of variation was less than 2.3% (Fig. S3). The VP2 nucleotide phylogenetic tree (Fig. 2C) and amino acid phylogenetic tree (Fig. 2D) displayed no obvious phylogenetic pattern. Among them, HN-1, HN-6, HN-7 and HN-8 had an obvious developmental trend within the VP2 amino acid evolutionary tree, which may be related to mutations at their amino acid sites.
From the results seen using similarity analyses, the VP3 protein from the Henan isolates and reference strain, are more conservative at the amino acid level with a difference co-efficient of 0-1.7% (Fig. S4). The nucleotide phylogenetic tree (Fig. 2E) and the amino acid phylogenetic tree (Fig. 2F) of VP3 had no obvious phylogenetic pattern. The HN-1 and HN-2 strains become extended in the amino acid phylogenetic tree, which may be due to the variation in some amino acid sites in these two isolates. Overall, there were no significant differences in apoptotic proteins in CIAV from the Henan isolates and the other isolates.
Analysis of major amino acid sites of VP1, VP2 and VP3 proteins
Previous studies have confirmed that the hypervariable region in the VP1 sequence is at amino acid position 13, in which amino acids 139 and 144 have an effect on the replication rate and infection efficiency of the virus in infected cells. When the 139th and 144th amino acids of the strain were glutamine (Q), the proliferation rate was significantly slower. In this study, the amino acid sites for the VP1 protein in 12 isolates, main vaccine strains, classical attenuated strains, domestic virulent strains, and previously isolated strains from Henan were all analyzed. Among these, the 12 isolates from Henan, and HN-1, HN-8, HN-9, and HN-12) strains all carried Q139 and Q144, and the other eight CIAV isolates (HN-2, HN-3, HN-4, HN-5, HN-6, HN-7, HN-10, and HN-11) carried the amino acid lysine (K) at position 139 and glutamate (E) at position 144. All the viruses isolated from Henan contained Q, at position 394, suggesting that the isolates from Henan had high pathogenicity. The amino acid sites for the four isolates HN-1, HN-8, HN-9, and HN-12 were the same as those of the HN1504 strain on the VP1 protein, which further provides evidence that the four strains (HN-1, HN-8, HN-9, and HN-12) are derived from the HN1504 strain. Except for the four isolates, there were mutations at position 22 histidine (H) 22 asparagine (N)), position 75 (valine (V) 75I) and position 125 leucine (L) (isoleucine (I) 125 L) in the VP1 protein of the remaining eight strains. Among them, HN-4, HN-6, and HN-8 had an alanine (A) 290 to proline (P) mutation at position 290, which has never been previously reported. In addition, there is a serine (S) 287 to N mutation in the HN-4 strain and an S 287 to threonine (T) mutation in the HN-6 strain. However, whether this mutation influences its pathogenicity, needs further investigation (Table 3).
Table 3. Main amino acid positions of VP1 protein
Virus strain
|
VP1 amino acid site
|
22
|
75
|
97
|
125
|
139
|
144
|
157
|
287
|
290
|
370
|
376
|
413
|
446
|
Cux-1
|
H
|
V
|
M
|
I
|
K
|
D
|
V
|
A
|
A
|
S
|
L
|
A
|
T
|
Del-Ros
|
.
|
.
|
.
|
.
|
.
|
E
|
.
|
S
|
.
|
G
|
.
|
S
|
G
|
C369
|
.
|
.
|
.
|
L
|
.
|
E
|
.
|
S
|
.
|
G
|
I
|
S
|
S
|
GD-1-12
|
.
|
.
|
.
|
L
|
.
|
E
|
M
|
S
|
.
|
G
|
I
|
S
|
S
|
HN1405
|
.
|
.
|
.
|
L
|
.
|
E
|
.
|
S
|
.
|
A
|
.
|
.
|
S
|
HN1504
|
N
|
I
|
L
|
.
|
Q
|
Q
|
.
|
.
|
.
|
.
|
.
|
.
|
S
|
SMSC-IP60
|
.
|
.
|
.
|
.
|
.
|
E
|
M
|
S
|
.
|
G
|
.
|
.
|
.
|
SDLY08
|
N
|
.
|
.
|
.
|
.
|
E
|
.
|
S
|
.
|
G
|
.
|
S
|
.
|
HN-1
|
N
|
I
|
L
|
.
|
Q
|
Q
|
.
|
.
|
.
|
.
|
.
|
.
|
S
|
HN-2
|
.
|
.
|
L
|
L
|
.
|
E
|
M
|
S
|
.
|
A
|
.
|
.
|
S
|
HN-3
|
.
|
.
|
.
|
L
|
.
|
E
|
M
|
S
|
.
|
G
|
I
|
.
|
S
|
HN-4
|
.
|
.
|
L
|
L
|
.
|
E
|
M
|
N
|
P
|
G
|
I
|
S
|
S
|
HN-5
|
.
|
.
|
.
|
L
|
.
|
E
|
.
|
S
|
.
|
G
|
I
|
S
|
S
|
HN-6
|
.
|
.
|
L
|
L
|
.
|
E
|
M
|
T
|
P
|
A
|
.
|
.
|
S
|
HN-7
|
.
|
.
|
.
|
L
|
.
|
E
|
M
|
S
|
.
|
G
|
I
|
S
|
S
|
HN-8
|
N
|
I
|
L
|
.
|
Q
|
Q
|
.
|
.
|
P
|
G
|
I
|
.
|
S
|
HN-9
|
N
|
I
|
L
|
.
|
Q
|
Q
|
.
|
.
|
.
|
.
|
.
|
.
|
S
|
HN-10
|
.
|
.
|
.
|
L
|
.
|
E
|
.
|
S
|
.
|
G
|
I
|
S
|
S
|
HN-11
|
.
|
.
|
.
|
L
|
.
|
E
|
.
|
S
|
.
|
G
|
I
|
S
|
S
|
HN-12
|
N
|
I
|
L
|
.
|
Q
|
Q
|
.
|
.
|
.
|
.
|
.
|
.
|
S
|
The amino acid sites of the VP2 and VP3 proteins were relatively conserved with only a few mutated amino acid positions. In VP2, five alternative amino acid mutations were observed: glycine (G) 31 to E mutation at position 31 and alanine (A) 53 to V mutation at position 53 in the HN-1 isolate, a serine (S) 13 to arginine (R) mutation in HN-6 isolate, a lysine (K) 102 to E mutation in HN-7 isolate, and a G 24 to E mutation at position 24 in the HN-8 isolate. In VP3, a P 18 to S mutation was observed in HN-1 isolates, and an A 54 to G mutation was observed in HN-2 isolates (Table 4).
Table 4. Main amino acid positions of VP2 and VP3 proteins
Virus strain
|
VP2 amino acid site
|
VP3 amino acid site
|
13
|
24
|
31
|
53
|
102
|
18
|
54
|
Cux-1
|
S
|
G
|
G
|
A
|
K
|
P
|
A
|
Del-Ros
|
.
|
.
|
.
|
.
|
.
|
.
|
.
|
C369
|
.
|
.
|
.
|
.
|
.
|
.
|
.
|
GD-1-12
|
.
|
.
|
.
|
.
|
.
|
.
|
.
|
HN1405
|
.
|
.
|
.
|
.
|
.
|
.
|
.
|
HN1504
|
.
|
.
|
.
|
.
|
.
|
.
|
.
|
SMSC-IP60
|
.
|
.
|
.
|
.
|
.
|
.
|
.
|
SDLY08
|
.
|
.
|
.
|
.
|
.
|
.
|
.
|
HN-1
|
.
|
.
|
E
|
V
|
.
|
S
|
.
|
HN-2
|
.
|
.
|
.
|
.
|
.
|
.
|
G
|
HN-3
|
.
|
.
|
.
|
.
|
.
|
.
|
.
|
HN-4
|
.
|
.
|
.
|
.
|
.
|
.
|
.
|
HN-5
|
.
|
.
|
.
|
.
|
.
|
.
|
.
|
HN-6
|
R
|
.
|
.
|
.
|
.
|
.
|
.
|
HN-7
|
.
|
.
|
|
.
|
E
|
.
|
.
|
HN-8
|
.
|
E
|
.
|
.
|
.
|
.
|
.
|
HN-9
|
.
|
.
|
.
|
.
|
.
|
.
|
.
|
HN-10
|
.
|
.
|
.
|
.
|
.
|
.
|
.
|
HN-11
|
.
|
.
|
.
|
.
|
.
|
.
|
.
|
HN-12
|
.
|
.
|
.
|
.
|
.
|
.
|
.
|
Recombination sequence analysis
Recombination analysis between all isolates and reference strains by RDP 4 software showed that recombination events occurred between different strains. Among them, HN-4 and HN-8 strains may be two potential recombinant strains. The isolate HN-4 may be a potential recombinant strain between the Korean isolate CIAVV89-69 and the Henan isolate HN-2. Bootscan analysis of the sequence of the HN-4 strain and its major and minor parental strains was conducted. The Korean strain CIAVV89-69 was found to be the major parental strain, while the Henan strain HN-2 represented the minor parental strain, and its recombination breakpoint was mapped to position 787 (initial breakpoint) and 1707 (termination breakpoint) (Fig. 3A).
Isolate HN-8 may represent a potential recombinant strain between the Brazilian isolate RS-BR-15 and the Henan isolate HN-6. Here, we used Bootscan analysis of the sequence of the HN-4 strain and its major and minor parental strains. We found that the Henan isolate HN-6 was the major parental strain, while the Brazilian isolate RS-BR-15 was the minor parental strain, and its recombination breakpoint was mapped to position 36 (initial breakpoint) and 2155 (termination breakpoint) (Fig. 3B).
Pathogenesis of HN-4
To examine the biological characteristics of the CIAV field isolates, we conducted analyses of their pathogenicity by infecting one-day-old SPF chickens with the HN-4 strain. As expected, there were no pathogenic symptoms in the control group. In the experimental group, however, no death was seen from day 1 to day 13, but chickens showed depression, were somnolent, displayed abnormal development and had a significantly lower body weight (P<0.001) (Fig. 4A). Chickens in the experiment group died continuously 14 days post infection, with a total of 10 in 20 deaths seen up until the end of the experiment (21 days) (Fig. 4B). At necropsy, no obvious pathological changes were seen in the liver and kidney, but significant atrophy was seen in the spleen, bursa and thymus, when compared to the control group (Fig. 4C). The tibia of chickens infected with HN-4 was thin and fragile and had yellowish colored bone marrow (Fig. 4D).
To further evaluate the pathogenicity of the HN-4 strain, histological lesions were examined on day 16 in the bursa, thymus, liver, spleen, and kidney. Our findings revealed that there was a significantly reduced number of lymphocytes in the Bursa, Thymus, spleen, and kidney. Whereas liver cells displayed metatropy when compared to the control group (Fig. 4E). These results revealed that HN-4 may be a highly pathogenic strain.