In the present study, 19 (13.4%) of the 141 rectal swab samples tested positive for PAstV by RT-PCR of partial ORF1b/ORF2 region. Of the positive samples, 16/111 (14.4%) were from diarrheic pigs and 3/30 (10%) were from non-diarrheic pigs. Since PAstV was detected in both healthy and diarrheic pigs, no clear association of PAstV infection with diarrhea could be ruled out. Our results concur with earlier reports showing 17.6% occurrence of PAstV from ten different states of India (Kattoor et al., 2019).Whereas in an earlier study from Haryana using partial ORF1bgene primers, 31.8% of samples tested positive for PAstV. The difference in the results could be due to the difference in the efficiency of primers used and the high genetic variability in the ORF2 region (Kour et al., 2021).
Among the positive samples, the percent positivity was morein weaning pigletsfollowed by suckling piglets (Table 1). The weaning piglets are more susceptible to viral diarrhea possibly due to a decline in maternal antibody titer and inefficient immune response making them more prone to infections (Wang et al., 2006; Amimo et al., 2014).
Table 1
Age wise distribution of PAstV in the samples collected from both diarrheic and non-diarrheic pigs of Haryana, India
Age categories
|
Diarrheic pigs
|
Non-diarrheic pigs
|
Total
|
Suckling piglets
(0-3 weeks)
|
4/ 22 (18.1%)
|
0/4 (0%)
|
4/26 (15.3%)
|
Weaning piglets
(3-9 weeks)
|
11/ 45 (24.4%)
|
3/16 (18.7%)
|
14/61 (22.9%)
|
Fattening pigs
(9-24 weeks)
|
1/28 (3.5%)
|
0/7 (0%)
|
1/35 (2.8%)
|
Adult pigs
(>24 weeks)
|
0/16 (0%)
|
0/3 (0%)
|
0/19 (0%)
|
Total
|
16/111 (14.4%)
|
3/30 (10%)
|
19/141 (13.4%)
|
Out of the 19 positive samples, 9 were commercially sequenced by the Sanger sequencing method. The obtained sequences were submitted to NCBI GenBank and accession numbers MW357980 to MW357988 were assigned.
On phylogenetic analysis of the partial ORF1b/ORF2 nucleotide region, two lineages viz; PAstV4 (5/9) and PAstV2 (4/9) were found circulating in Haryana with PAstV4 (55.5%) being predominant (Fig. 1). These findings concur with the earlier study from India, where PAstV4 (21/22) and PAstV2 (1/22) were reported in India with PAstV4 being predominant (Kattoor et al., 2019).Similarly, the lineage PAstV4 and PAstV2 were also detected in Thailand with PAstV4 being predominant (Kumthip et al., 2018) using the same primer set.
Five nucleotide sequences from the present study (MW357981, MW357982, MW357983, MW357984 and MW357988) clustered within PAstV4 group and were closely related to PAstV strain; KX431949 from China and LC201614 from Japan sharing 77.5–81% and 71.9–75.5% nucleotide identities, respectively. In addition to these one of the nucleotide sequences; MW357984 clustered more closely with the Indian strain, KJ650565 with 87.5% nucleotide identity. However, the nucleotide identities of all the PAstV4 sequences from the present study with previously reported PAstV4 Indian strains ranged from 70.3–78.8%. The remaining four PAstV strains (MW357980, MW357985, MW357987 and MW357986) from this study clustered with PAstV2 group and were related to PAstV strains; KP982872 from Belgium sharing 83.6–85.3% nucleotide identity, MG930777 from Italysharing79.3–84.8% nucleotide identity and KP759770 from South Korea sharing 80.5–83.5% nucleotide identity. Moreover, the nucleotide identities with previously reported PAstV2 strain, KJ650651 from India ranged from 69.4–71.0%.
Phylogenetic analysis based on the amino acid sequence of the partial ORF2 gene segment also revealed the presence of two lineages; PAstV4 and PAstV2 circulating in Haryana (Fig. 2). In the PAstV4 clade, same five sequences were clustered sharing amino acid identities of 50.3–81.8% and 43.0–68.5% with PAstV strain; KX431949 from China and LC201614 from Japan, respectively. Furthermore, the amino acid identities with previously reported PAstV4 Indian strains ranged from 41.3–74.1%. The remaining four sequences in the PAstV2 clade shared amino acid identities of 82.6–91.7% with KP982872 from Belgium, 79.7–83.3% with MG930777 from Italy and 79–83.9% with KP759770 from South Korea. The amino acid identities with the India isolate, KJ650561 ranged from 46.55 to 50%.
The sequences of rodent AstV clustered along with PAstV4 group whereas the sequences of bovine AstV and the deer AstV clustered along with PAstV2 group. The sequences of the human AstV clustered within PAstV1 group. This could possibly be due to the multiple recombination events or interspecies transmissions in the past (Lukashov and Goudsmit 2002; Xiao et al., 2013)
The nucleotide sequences of the partial ORF1b/ORF2 region of samples processed in the present study have sequence identities ranging from 22.7–100% at the nucleotide level and 79.9–100% at the amino acid level, respectively. Similar results have been reported from India earlier showing nucleotide identities ranging between 16.9%-99.2% (Kattoor et al., 2019) and 31.5% - 100% (Kour et al., 2020)The percent identity within PAstV4 and PAstV2 lineages was found to range between 79.1%-100% and 81.1%-100% respectively based on nucleotide sequences. The amino acid identity within PAstV4 and PAstV2 sequences ranged between 60.1–100% and 79.9–100% respectively. Hence from the present analysis, it can be concluded that a wider range of genetic variation is present in PAstVs which is a common feature of the family Astroviridae (Wohlgemuth et al., 2019). However, it was observed that the range of divergence is more when comparing the sequences based on partial ORF2 amino acid region, and this might be due to high variability in the ORF2 region of the PAstV genome(Strain et al., 2008).
Besides this the sequence analysis by MEGA X software revealed the presence of this conserved nucleotide sequence; “UUUGGAGGGG (A/C) GGACCAAA N(11) AUG GC” (where N stands for any of 4 nucleotides) at the overlapping region of ORF1b/ORF2 gene just before the start codon of ORF2 gene (Fig. 3) in all the 9 sequences. This conserved region is hypothesized to act as a regulatory element, serving as a promoter for sub-genomic RNA transcription (Lv et al., 2019; Qin et al., 2019). The sequence comparison indicated that there is a presence of N (11) in the samples sequenced in the present study instead of N(4-8), as reported earlier (Xiao et al., 2013).