Prevalence of PEDV, PDCoV, TGEV, PRoV and SADS-CoV
During a seven-year period, a total of 2,987 samples from five provinces in southern China were tested, and it was found that PEDV was the most dominant virus, with a prevalence varying between 50.21% and 62.1% in 2012-2018 (Table 1). The results were in accordance with the findings presented by other studies that were performed in other areas in China. In addition to PEDV, PDCoV, TGEV, and PRoV have also been found positive in these investigated diarrhea samples. In our study, PDCoV was the second prevalent virus, with detection rates varying between 19.62% to 29.19% from 2012 to 2018. TGEV, and PRoV were both detected at low detection rates (<3%). In the present study, SADS-CoV was not observed in samples from Jiangxi, Zhejiang, Guangdong, and Hunan provinces during 2012 to 2018, however a positive rate of 10.29% (7/68) of this virus was identified in diarrheal piglets from Fujian province. Thus, SADS-CoV was only identified in Guangdong and Fujian provinces.
In the context of the sample sources, the small intestines of suckling piglets showed the highest positive rates of PEDV (63.31%), followed by the feces from diarrheal pigs (52.72%), and milk (22.73%), respectively. Likewise, the detection rates of PDCoV from small intestines, feces, and milk were 29.66%, 25.75%, and 7.92%, respectively (Table 1). As to the growing stage of pigs, PEDV was frequently detected in pigs of all ages, followed by PDCoV. PEDV infection was more common in sows (52.81%) and suckling piglets (62.37%), similar data were recorded for PDCoV infection. These data suggested that suckling piglets were at greater risk for infections of diarrhea viruses, especially PEDV.
Table 1. Categorization of detection results on porcine diarrhea associated viruses of samples collected between 2012 and 2018
Classifications
|
Sample No.
|
Viruses (Number (positive rate,%))
|
PEDV
|
PDCoV
|
TGEV
|
PoRV
|
SADS
-CoV
|
Year
|
2012
|
158
|
91 (57.59)
|
31 (19.62)
|
1 (0.63)
|
0 (0.00)
|
0 (0)
|
2013
|
301
|
187 (62.13)
|
81 (26.91)
|
1 (0.33)
|
3 (1.00)
|
0 (0)
|
2014
|
714
|
413 (57.84)
|
193 (27.03)
|
4 (0.56)
|
5 (0.70)
|
0 (0)
|
2015
|
574
|
343 (59.76)
|
161 (28.05)
|
8 (1.39)
|
9 (1.57)
|
0 (0)
|
2016
|
476
|
239 (50.21)
|
124 (26.05)
|
2 (0.42)
|
3 (0.63)
|
0 (0)
|
2017
|
389
|
218 (56.04)
|
105 (26.99)
|
1 (0.26)
|
3 (0.77)
|
7 (1.80)
|
2018
|
375
|
221 (58.93)
|
118 (31.47)
|
4 (1.07)
|
2 (0.53)
|
0 (0)
|
Total
|
2, 987
|
1, 712 (57.32)
|
813 (27.22)
|
21 (0.70)
|
25 (0.84)
|
7 (0.23)
|
|
Province
|
Jiangxi
|
2, 690
|
1, 524 (56.65)
|
737 (27.40)
|
16 (0.59)
|
23 (0.86)
|
0 (0)
|
Zhejiang
|
81
|
50 (61.73)
|
22 (27.16)
|
1 (1.23)
|
1 (1.23)
|
0 (0)
|
Fujian
|
68
|
45 (66.18)
|
23 (33.82)
|
1 (1.47)
|
0 (0)
|
7 (10.29)
|
Guangdong
|
92
|
58 (63.04)
|
18 (19.57)
|
2 (2.17)
|
1 (1.09)
|
0 (0)
|
Hunan
|
56
|
35 (62.50)
|
13 (23.21)
|
1 (1.79)
|
0 (0)
|
0 (0)
|
|
Sample type
|
Intestine
|
1, 581
|
1, 001 (63.31)
|
469 (29.66)
|
13 (0.82)
|
13 (0.82)
|
5 (0.32)
|
Feces
|
1, 305
|
688 (52.72)
|
336 (25.75)
|
8 (0.61)
|
12 (0.92)
|
2 (0.15)
|
Milk
|
101
|
23 (22.73)
|
8 (7.92)
|
0 (0)
|
0 (0)
|
0 (0)
|
|
Pig stage
|
Sow
|
673
|
355 (52.75)
|
187 (27.79)
|
7 (1.04)
|
2 (0.30)
|
1 (0.15)
|
Suckling piglet
|
1, 697
|
1, 064 (62.70)
|
501 (29.52)
|
12 (0.70)
|
18 (1.05)
|
6 (0.35)
|
Nursery pig
|
357
|
181 (50.70)
|
73 (20.45)
|
1 (0.28)
|
3 (0.84)
|
0 (0)
|
Finishing pig
|
215
|
91 (42.33)
|
36 (16.74)
|
1 (0.47)
|
2 (0.93)
|
0 (0)
|
Abbreviations: PEDV, porcine epidemic diarrhea virus; PDCoV, porcine deltacoronavirus; TGEV, transmissible gastroenteritis virus; PRoV, porcine rotavirus; SADS-CoV, swine acute diarrhea syndrome coronavirus.
Co-infections of diarrhea-associated viruses in pigs in southern China
In this study, co-infection frequency was analyzed. Among the 2, 987 field samples, the positive rates of mono-infection of PEDV, PDCoV, TGEV and PRoV from 2012 to 2018 were 45.53% (1360/2987), 14.23% (425/2987), 0.33% (10/2987), and 0.60% (18/2987), respectively (Fig. 1). The most common co-infection was PEDV with PDCoV, with an average positive rate of 12.72% (380/2987), ranging from 8.26% to 17.33%. The average rates of dual-infections of PEDV and TGEV, PEDV and PRoV, PDCoV and TGEV, were 0.30%, 0.13%, and 0.10%, respectively. In addition, four cases of triple-infections were observed, including 3 cases of a PEDV, PDCoV, and PRoV co-infection, and 1 case of a PEDV, PDCoV, and TGEV co-infection. Four samples collected from the Fujian province in 2017 were determined as a co-infection of SADS-CoV and PEDV. In our study, we didn't observe any other co-infections in the tested samples. It has been reported that mixed infections are common in diarrheal pigs, and might alter pathogenesis and pathogenicity of the agents involved. We also observed severe morbidity and mortality in piglets infected with multiple pathogens, such as PEDV, PDCoV, TGEV, and pathogenic Escherichia coli.
Molecular characterization and phylogenetic analyses of PEDVs circulating in southern China
To elucidate the genetic characteristics of PEDVs circulating in southern China between 2012 and 2018, the the complete S1 genes of 11 PEDV strains were sequenced, and analyzed . Phylogeneticlly, the S1 regions (aa 1~794) of the 11 strains of PEDV identified in this study and other 73 selected reference PEDV strains were divided into two groups (GI and GII). All of the 11 strains determined in this study, along with 3 formerly reported strains were clustered into the group G II, and subgroup G IIa (Fig. 2A). Variations were observed among the 14 strains, which were located into different clades in G IIa, two strains (CH/JX/JJ08/2015 and CH/JX/JGS11/2016) were closely related to CH/JX/01, the strain isolated from the Jiangxi province in 2015 in our lab; six strains were clusered into a independent clade; a strain, CH/JX/ZS03/2014 was in a clade with CH/ZMDZY/11; two strains CH/JX-1/2013 and CH/JX-2/2013, identified in 2013 were closely related to the recombinant Chinese strain, AH2012 that was identifiedin 2012.
The 11 PEDVs identified in this study underwent homology analysis with 3 PEDVs previously reported in our lab and other stains deposited in GenBank. The results showed that all field strains shared 97.8%-100% of nucleotide (nt) and 97.2%-100% of amino acid (aa) identities, as well as 90.7%-92.9% nt and 89.8%-91.6% aa identities with G I strains, and 93.2-99.7% nt and 91.6-100% aa identities with genotype 2 strains. Compared with the CV777 vaccine strain, the strains determined in this study beared same mutation sites of A518S, G521D, L522H/Y, S524G, V528I, T550S, S563F, G594S, A605E, L612F, F617L, K630T, E633V, and I635V (Fig. S1). In addition, regarding the neutralizing epitope SS2 and SS6, mutations were found among differernt genotypes, for the Jiangxi strains determined in this study clustered in G IIa: S764L and T774M were only found in G 1b, and Y764S was observed in genotypes G Ib and GII. Furthermore, three mutations were only observed in the neutralizing epitope SS2 and SS6 between Jiangxi strains and CV777, V747L IN CH/JXJGS11/2016, I751M in CH/JXJA89/2015 and CH/JXWN13/2016, and I77N in CH/JXGZ09/2018 (Fig. 3).
Molecular characterization and phylogenetic analysis of PDCoVs in southern China
Eight complete sequences of the S1 gene of PDCoV strains were obtained from representative positive samples. To analyze the homology and phylogenetic relationships among PDCoV isolates from different countries, the eight PDCoVs determined in this study and 39 PDCoV reference strains from GenBank were used. The eight PDCoV isolates showed 97.7%-99.9% nt and 97.9%-99.8% aa identities with each other; and with 95.2%-99.4% nt identities and 95.5%-99.1% aa identities with the reference strains from China and other countries. Interestingly, sequence alignment analysis indicated that all eight PDCoV strains determined in this study along with all Chinese strains (except HKU15-44 and AN-2004) and a strain identified in Thailand (TT_1115) had the same 3-nt (AAT) deletion in the S gene between nt 19473 and 19477, leading to a deletion of deduced aa Asparagine (Asn or N) when compared with other strains. Phylogenetic analysis based on the aa sequence of the S1 protein demonstrated that PDCoV isolates were divided into two groups, the Chinese- and US-like-groups. The S1 genes of PDCoVs currently circulating in Southern China were more closely related to other Chinese PDCoVs rather than to those isolated previously from the USA, South Korea, and Thailand (Fig. 2B).