Clinical symptoms of infection
The rectal temperatures of the SVV-inoculation groups showed transient increases but did not exceed 40 ℃ in the 5-8 dpi period (Fig. 1A). Pigs infected with SVV showed symptoms of mild depression 5-10 dpi, whoever, feed intake and weights showed no significant differences in comparison to the control PBS group (Fig. 1B). From day 5 post-infection, the SVV group showed erythema near the pig hooves which was vesicular (Fig. 2B). Thereafter vesicular lesions were observed on the snout (day 6, Fig. 2A), and fluid-filled vesicles burst in the hooves and blood-like lesions were observed (7-14 dpi, Fig. 2C). In order to further describe the SVV infected clinical symptoms more accurately, the incidence of SVV infection in the pigs was also analyzed (Table 1).
Table 1
Statistics of clinical symptoms of the pigs infected with the SVV
Groups
|
Lesion on the snout
|
Lesion on the hoof
|
Blood-like lesions on the hoof
|
PBS control group
|
0/5
|
0/5
|
0/5
|
SVV-infection group
|
1/5
|
2/5
|
4/5
|
Viremia analysis and viral load detection in tissues
In order to analyze the duration of viremia caused by SVV in pigs, blood samples were collected at post-infection days 3, 7, 10, 14, 21, and 28. qRT-PCR was established for viral load detection (Fig. S1). The results showed that SVV could be detected in the blood on the 3rd day after the challenge, and the viral load peaked at 7 dpi, at about 104.5 Copies/μl. Thereafter, the viral load in blood decreased rapidly to about 101.5 Copies/μl at 14 dpi, until elimination at 21 dpi. Therefore, in this study, viremia persisted for more than 14 days following infection with SVV in pigs (Fig. 3A).
Two pigs were euthanized on the 14th day after being challenged with SVV. The tissues the including heart, liver, spleen, lung, kidney, submaxillary LN, inguinal LN, intestine, tongue, tonsil and hooves with blisters were collected and the cDNA was obtained from them for qRT-PCR analysis. The results showed that SVV was detected in all of these tissues, with the viral loads highest in the hooves with blisters (at approximately 105 copies/μl). The virus copies within the submaxillary LN, inguinal LN and tonsil were higher than 104 copies/μl (Fig. 3B).
Neutralization antibody levels
A neutralization antibody assay was set up during this research to test serum samples collected on 0, 7, 10,14, 21, and 28 dpi. All of the SVV-infected pigs seroconverted and generated neutralizing antibodies on the 7th dpi, and the neutralizing antibodies titers ranged from 1:64 to 1:128. The neutralizing antibody titers reached their highest levels around 10 days post-infection with SVV, and the neutralizing antibody titers reached from 1:128 to 1:512 at that point. The neutralizing antibodies persisted for at least 28 days in the pigs infected with the CH-GX-01-2019 SVV strain, and were largely unabated at around 28 days (Fig. 4 and Table 2).
Table 2
Neutralizing antibody titers of pigs to infection with CH-GX-01-2019 SVV strain.
Groups
|
Pig No.
|
Days-post-infection (dpi)
|
0
|
7
|
10
|
14
|
21
|
28
|
PBS control group
|
1
|
<1:4
|
<1:4
|
<1:4
|
<1:4
|
<1:4
|
<1:4
|
2
|
<1:4
|
<1:4
|
<1:4
|
<1:4
|
<1:4
|
<1:4
|
3
|
<1:4
|
<1:4
|
<1:4
|
<1:4
|
<1:4
|
<1:4
|
4
|
<1:4
|
<1:4
|
<1:4
|
<1:4
|
|
|
5
|
<1:4
|
<1:4
|
<1:4
|
<1:4
|
|
|
SVV-infection group
|
6
|
<1:4
|
1:128
|
1:512
|
1:128
|
1:128
|
1:256
|
7
|
<1:4
|
1:64
|
1:128
|
1:128
|
1:256
|
1:256
|
8
|
<1:4
|
1:128
|
1:256
|
1:128
|
1:128
|
1:128
|
9
|
<1:4
|
1:64
|
1:128
|
1:128
|
|
|
10
|
<1:4
|
1:128
|
1:128
|
1:128
|
|
|
Detection of SVV in mosquitoes and Culicoides
About 1000 mosquitoes were collected and SVV detection was carried out using RT-PCR. The results showed that SVV was not detected in any of the samples (Table S3).
About 54000 Culicoides were collected and again SVV infection was detected using RT-PCR. The results showed that four out of ten samples were SVV positive (Table S3, Fig. S2). When samples 11-30 underwent RT-PCR, 13 out of the 20 samples were SVV positive (Table S3, Fig. S2). The complete gene sequences of the positive samples were sequenced and spliced successfully, the sequence detected was completely consistent with that shown by the CH-GX-01-2019 strain. Positive sample grinding fluid was to undertake virus isolation from the BHK-21 cells. Blind passages were carried out on the BHK-21 cells for 5 passages, and RT-PCR and indirect immunofluorescence were used for identification purposes for each generation of cells. Cell cultures in passages 3-5 were all SVV negative according to the RT-PCR results. None had visible green fluorescence under indirect immunofluorescence microscopy analysis in any passage. SVV was therefore not isolated from positive Culicoides.
As previously reported, SVV nucleic acids have been detected in mice and houseflies [17]; and although SVV nucleic acids were not detected in cattle, neutralizing antibodies were present [12], therefore we inferred that cattle may be infected with SVV. So far, the pig is the only host of SVV, therefore we infer that possible transmission routes of SVV, including houseflies, mice, Culicoides and cattle may serve as vehicles for SVV transmission and promote the spread of SVV (Fig. 5).