PCR confirmation of Guangxi ChPV and TuPV strains
Non-structural (NS) and VP genes of positive samples were PCR amplified using primers targeting the conserved 561-bp NS1 region and 249-bp VP1/VP2 region, respectively. Epidemiological research results are shown in Table 1. Table 1 shows that the total positive rate is 69.72%, the positive rate of RSS-like cases is up to 91.86%, and the positive rate of healthy chickens is 66.91%. Positive samples were further confirmed by sequencing of the NS1 and VP genes. NCBI BLAST results showed that the samples had 98-100% homology with the ChPV ABU-P1 strain isolated from Hungary and the TuPV 260 strain isolated from the United States. The whole genome was successfully sequenced from 32 PCR-positive chicken throat and cloacal swab samples and 3 PCR-positive turkey throat and cloacal swab samples using Sanger sequencing.
General characteristics of genomes
The genomes of the Guangxi ChPV and TuPV strains were 4612 to 4642 bp long. The genomes had an approximate GC content of 42.88% and contained 3 segments, each encoding 4 viral proteins. Genomic segments ranged in length from 305 bp (NP1) to 2085 bp (NS1), and ORF analysis of nucleotide (nt) sequences showed that 2 out of 3 genome segments encode a single ORF; ChPV/TuPV reference strains. The first ORF was predicted to encode 2 putative proteins (NS1 on NS1 and NP1 on NP1) ranging in size from 101 to 695 amino acids (aa). The 2028-bp VP segment was found to contain two partially overlapping genes encoding VP1 (2028 bp, 676 aa) and VP2 (1611 bp, 537 aa).
Comparison of the similarities between the nt sequences of Guangxi ChPV/TuPV strains and the nt sequences of 17 ChPV/TuPV reference strains revealed that all 3 segments identified in Guangxi ChPV/TuPV strains showed varying degrees of homology with the reference ChPV/TuPV strains. 35 Guangxi isolates with 79.4–99.7 nt identity to each other and 78.7–99.7 nt with 11 classical ChPV reference strains including ChPV ABU-P1, ChPV ADL120686, ChPV ADL120019, ChPV ADL120035, ChPV 367, ChPV 736 showed identity. , CHPV 798, CHPV 841, CHPV parvod62/2013, CHPV parvod11/2007, and CHPV IPV strains and 6 classic TUPV reference strains, including Tupv 260, Tupv 1078, Tupv 1030, Tupv 1085, Tupv 1090, and Tupv JO11 strains.
Nucleotide and amino acid comparisons
Comparison of the nt and aa sequences of the NS1 gene revealed high sequence identities between 35 Guangxi ChPV and TuPV strains and 11 ChPV reference strains and 6 TuPV reference strains. GPV (accession no. NC_001701 from the USA) and DPV (accession no. U22967 from Hungary) were used as the outgroup. Accession numbers of reference sequences of ChPVs/TuPVs are listed in Supplementary Tables S1 and S2. Homology analysis of the NS1 gene showed that the homology of the nt and derived aa sequences of 35 Guangxi isolates was 88.1–99.9% and 89.1–100.0%, respectively. The nt sequence alignment between ChPV and TuPV strains from Guangxi and other countries showed 85.2-99.9% identity, and aa sequences showed 87.8-100% identity. The sequence identity of the NP1-encoding genes was highest (>95%); however, the role of this putative protein is unknown.3.
Compared with the genome fragments encoded by NS1 and NP1, the segments encoding VP showed higher genetic diversity. For the VP1 protein, the Guangxi ChPV and TuPV strains showed similar identities as the ChPV/TuPV reference strains (nt, 73-98%; aa, 77.1-100%). Conversely, the VP2 protein shared the lowest identity with the ChPV 367 strain and the highest identity with the TuPV JO11 strain (nt, 72-98%; aa, 76.9-100%). For the VP1 gene, the homologies of the nt and derived aa sequences of 32 Guangxi isolates were 72.6-99.9% and 78.0-99.7%, respectively, and the homologies between ChPV and TuPV isolates from Guangxi and other countries were 72.7. –99.9% and 77.2–99.6%, respectively. For the VP2 gene, the homologies of the nt and derived aa sequences of 32 Guangxi isolates were 70.7–99.9% and 77.3–99.6%, respectively, and the homologies between Guangxi ChPV and TuPV isolates and those from other countries were 71.1–. 99.9% and 77.1–99.4%, respectively.
Interestingly, an 8-nt (TTATTTTG) deletion (corresponding to nts 2778 to 2785 in the NP1 gene of strain ABU-P1) was observed in all ChPV and TuPV strains except GX-CH-PV-1 and ChPV 841. strains TuPV 1078, 1085, 1090, GX-Tu-PV-1, GX-Tu-PV-2 and GX-Tu-PV-3 (see Supplementary Figure S1). In addition, a 4-nt (CTAA) deletion (corresponding to nt 2789 to 2792 in the NP1 gene of strain ABU-P1) was found in all strains of ChPV and TuPV except GX-CH-PV-1 and ChPV 841. strains TuPV 1078, 1085, 1090, GX-Tu-PV-1, GX-Tu-PV-2 and GX-Tu-PV-3. Also, a 9-nt (TCCATAATG) deletion (corresponding to nt 3275 to 3283 in the VP1 gene of strain ABU-P1) was found in all strains of ChPV and TuPV except GX-CH-PV-1 and ChPV 841. strains TuPV 1078, 1085, 1090, GX-Tu-PV-1, GX-Tu-PV-2 and GX-Tu-PV-3 (see Supplementary Figure S2). Finally, a 3-nt (GAA) deletion (corresponding to nt 3570 to 3572 in the VP2 gene of strain ABU-P1) was observed in all strains of ChPV and TuPV except GX-CH-PV-1 and ChPV 841. strains TuPV 1078, 1085, 1090, GX-Tu-PV-1, GX-Tu-PV-2 and GX-Tu-PV-3 (see Supplementary Figure S3).
Sixteen of the Guangxi ChPV/TuPV strains (ie, GX-CH-PV-4, GX-CH-PV-6, GX-CH-PV-7, GX-CH-PV-13, GX-CH-PV-14) , GX-CH-PV-15, GX-CH-PV-17, GX-CH-PV-18, GX-CH-PV-20, GX-CH-PV-21, GX-CH-PV-22, GX -CH-PV-25, GX-CH-PV-27, GX-CH-PV-28, GX-CH-PV-29, and GX-CH-PV-30) sequenced in this study contained the putative VP3 start codon ChPV ABU-P1 has also been identified in strains ChPV 367, ChPV 736, ChPV 798 and TuPV 260 (see Supplementary Figure S4). Therefore, the VP3 protein of ChPV is not produced by alternative splicing of ORF2.
A highly conserved phosphate binding ring (P-loop) motif (aa 392 to 399, GPANTGKT) and NTP binding motif (aa 436 to 437, EE) corresponding to the NS1 gene of strain ABU-P1 were present in all Guangxi ChPV/. TuPV strains. Start codons for VP1 (nts 2998 – 3000; ABU-P1) and VP2 (nts 3415 – 3417; ABU-P1), leucine residue (L; aa 293 of ABU-P1, VP1; ABU- aa 152, VP2) P1) and the five-fold cylinder region (LQVIQKTVTDSGTQYSND; aa 275 to 292, VP1 of ABU-P1; aa 134 to 151, VP2 of ABU-P1) were defined (see Supplementary Fig. S5), but a glycine-rich sequence (GGGGGGGGGG; aa 164 – 172, VP1 of ABU-P1; aa 23 – 31, VP2 of ABU-P1) GX-TU-PV-1, GX-TU-PV-2, GX-TU-PV- 3 was identified in GX-CH-PV-1 and GX-CH-PV-24, TVGGGGGGG was identified in other Guangxi ChPV isolates.
Evolutionary relationships between Guangxi ChPV and TuPV strains and their different members aveparvovirus The genus containing DPV and GPV used as outgroup controls was determined by phylogenetic analysis. Based on the nt sequences of the NS1, VP1 and VP2 genome segments and the entire ChPV/TuPV genomes, the neighbor-joining method with 1000 bootstrap copies was used to construct phylogenetic trees (Fig. All generated phylogenetic trees showed marked differences between Guangxi ChPV and TuPV strains and other reference ChPV and TuPV strains. For the 3 genome segments, the majority of ChPV strains formed a host-associated group (excluding strains TuPV 260, ChPV 841, GX-Tu-PV-2 and GX-CH-PV-1). turkey strains, FM duck strain and lethal B goose strain. Moreover, segments encoding VP1/VP2 proteins showed significantly higher divergence from NS1 in ChPV and TuPV strains, as demonstrated by sequence comparisons. A phylogenetic tree based on the VP gene revealed that 35 Guangxi ChPV and TuPV isolates sequenced in our research clustered into 5 ChPV/TuPV groups, designated Groups A, B, C, D, E, and F (Figure 1d). Genotyping cluster A of 12 Guangxi ChPV field strains, 2 prototype ChPV and TuPV strains (strains ABU-P1 and 260), 1 a rooster a rooster enteric parvovirus isolate (strain 736) from the USA and 1 prototype ChPV strain from South Korea (strains ParvoD11-2007); Genotyping cluster B of 4 Guangxi ChPV field strains included 7 prototype ChPV strains from the USA, South Korea, and Brazil; and genotyping cluster F of 3 Guangxi TuPV and 3 ChPV field strains included 6 prototype ChPV and TuPV strains, all from the USA. Of the 35 ChPV/TuPV isolates, 18 were identified as Groups A and Group F, while 3 Guangxi ChPV and 3 Guangxi TuPV field strains of Group F were field variants from the prototype ChPV (strain 841) and TuPV (strains 1085, 1078, 1090, 1030 and JO11) strains, all from the USA. More importantly, 3 new ChPV/TuPV groups (Groups C, D and E) were identified for the first time, all from Guangxi. Interestingly, ChPV/TuPV whole genome sequences from chickens with RSS-like symptoms were more concentrated in Groups C, D and E (Fig. 1d).
nt alignment of the complete genome of Guangxi ChPV/TuPV strains and 17 reference ChPV/TuPV strains (Figure 2a,b) revealed conserved and divergent regions across genomes. This visualization of genomes supported the results of the phylogenetic study described above.
As shown in Table 2, fifteen recombination events were detected in the NS1, VP1 and VP2 genes of 13 Guangxi strains. To further validate the recombination events identified by RDP 5.0, Simplot 3.5.1 software was used to analyze the homology of recombinant strains. . These recombination sequence signals were confirmed by SimPlot analysis (see Supplementary Figures S6, S7).
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