Sandfly specimens were collected in June 2019 from sheep, chicken, and cattle pens in two villages in Yangquan County, Shanxi Province. A total of 1535 sandflies were collected from the two villages (1047 and 488 from the first and second villages, respectively). In total, 935 of these sandflies were collected from a sheep pen, 290 from a chicken pen, and 310 from a cattle pen (Table 1).
Collection site Breeding places Total Sheep pen Chicken pen Cattle pen 1 554 183 310 1047 2 381 107 / 488 Total 935 290 310 1535 "/" represents the missing cattle pen.
"1" indicates the first sampling village (east longitude 113°37′12″, north latitude 37°46′48″). There were more than 100 chickens in the chicken pen, 170 sheep in the sheep pen, and 18 cattle in the cattle pen. The distance between the three pens was less than 1000 m. "2" indicates the second sampling village (east longitude 113°38′24″, north latitude 37°45′). There were 70 sheep in the sheep pen and 10 chickens in the chicken pen. Sheep and chicken pens were in the courtyard of the residents, which had two dogs in the house. The two pens were approximately 2 km apart, and the two villages were about 4 km apart.
Table 1. Collection sites of sandfly specimens in Yangquan County, Shanxi Province in 2019.
The collected sandflies were pooled into 23 groups before homogenization, and the supernatant of each pool was inoculated into BHK-21 and C6/36 cells after homogenization. SXYQ1916 sandfly specimens were also inoculated into BHK-21 cells, and CPE was observed after 3 days of inoculation. Notably, cells exhibited significant shrinkage and shedding (Fig. 1). Sandfly specimens were inoculated into BHK-21 cells, and continued culture and passaging of cells, then four virus strains were isolated (Table 2). In contrast, the inoculation of sandfly supernatants into C6/36 cells did not lead to CPE. And we obtained nothing after PCR amplification using C6/36 cell supernatants and WUXV-specific primers.
Figure 1. CPE of the WUXV isolate SXYQ1916 in BHK-21 cells. A BHK-21 cells as negative control were cultured for 3 days. B BHK-21 cells were also cultured for 3 days after the inoculation of WUXV isolate SXYQ1916. Culture in the presence of the SXYQ1916 virus led to a decrease in the number of adherent BHK-21 cells, and profound cell rounding and detachment. Magnification, ×100.
Strain number Collection site Breeding places Number of Phlebotomus chinensiss CPE/ Gene amplification BHK-21 cell WUXV C6/36 cell WUXV L M S M S SXYQ1931 1 Sheep pen 60 + / MW192757 MW192753 – – – SXYQ1916 2 Sheep pen 49 + / MW192754 MW192750 – – – SXYQ1930 2 Sheep pen 74 + / MW192756 MW192752 – – – SXYQ1927-2 2 Chicken pen 54 + MW368897 MW192755 MW192751 – – – Numbers indicate the collection location. Numbers 1 and 2 indicate the first and second sampling villages, respectively; " + " means that the experimental result is positive; "–" means that the experimental result is negative; "/" means that the experiment was not performed.
Table 2. Isolation of Wuxiang virus in Yangquan County, Shanxi Province in 2019.
By dividing all 1535 sandflies into 23 pools and inoculating them into BHK-21 cells, we obtained four virus isolates that caused CPE in BHK-21 cells. The infection rate of these viral pools was 17.39% (4/23), and the MIR was 2.61 (4/1535 × 1000). Among the 14 pools of sandfly specimens collected from the sheep pen, three contained the WUXV. The infection rate of WUXV pools isolated from the sheep pen was 21.43% (3/14), and the MIR was 3.21 (3/935 × 1000). The infection rate of viral pools isolated from the chicken pen was 20.00% (1/5), and the MIR of the WUXV virus was 3.45 (1/290 × 1000). In addition, 310 sandflies were collected from the cattle pen and divided into four pools. We could not isolate a virus from any of these viral pools, and we did not detect any of the WUXV genes. The infection rates of the different WUXV isolates are shown in Table 3.
Breeding places Number of sandflies Pool Infection rate of pools (%) MIR (/1000) Chicken pen 290 5 20.00 (1/5) 3.45 (1/290) Sheep pen 935 14 21.43 (3/14) 3.21 (3/935) Cattle pen 310 4 0.00 (0/4) 0.00 (0/310)
Table 3. Infection rate of Sandfly-borne virus.
The sequences of the coding regions of the virus WUXV S and M genes were determined for all four virus isolates. Interestingly, the two genes were of the same length in all four virus isolates. The coding sequence of the S gene was 1611 base pairs (bp), whereas the coding sequence of the M gene was 4089 bp, similar to the length of the respective genes of the WUXV (SXWX1813-2). The L gene of the SXYQ1927-2 virus isolate was amplified, and the nucleotide sequence was determined and analyzed. The length of the nucleotide and the amino acid sequences of the encoding region of the L gene are 6273 nt and 2090 aa, respectively. BLAST analyses revealed that the sequences of the M gene exhibited similarities of 96.8%-99.5% among the four strains. Similarly, the amino acid sequences of the NS and N proteins encoded by the S gene were 98.2%-99.6% similar among the four strains (Supplementary Tables S2-S4). The results of the homology analysis of the newly isolated virus (SXYQ 1916) and other Phleboviruses are shown in Table 4. We found 96.7%, 97.1%, and 98.0% sequence similarity in genes M, NS, and N between the newly isolated strain SXYQ1916 and a WUXV strain (SXWX1813-2) previously isolated in China (Wang et al. 2020); the respective amino acid sequence similarities were 97.6%, 97.7%, and 99.6%. Moreover, comparisons between SXYQ1916 and a TORV strain (213/Turkey/2012) isolated from Turkey sandfly specimens revealed 72.0% nucleotide (75.1% amino acid) sequence similarity in the M segment, 75.4% (85.1%) similarity of the NS protein in the S segment, and 82.2% (96.4%) similarity of the N protein in the S segment (Table 4). These results suggested that the virus isolated from sandflies in Yangquan County is the same with the WUXV strain previously isolated from sandflies in China.
Virus strains M segment S segment GP NS N nt (%) aa (%) nt (%) aa (%) nt (%) aa (%) SXYQ1916 4089 1362 783 260 741 246 SXWX1813-2 4089(96.7) 1362(97.6) 783(97.1) 260(97.7) 741(98.0) 246(99.6) TORV(213/Turkey/2012) 4080(72.0) 1359(75.1) 783(75.4) 260(85.1) 741(82.2) 246(96.4) TORV(292/Turkey/2012) 4081(72.0) 1359(75.1) 783(75.2) 260(85.1) 741(81.9) 246(96.0) CFUV(Pa Ar 814/Greece/1981) 4080(71.3) 1359(75.7) 783(74.5) 260(84.3) 741(81.9) 246(96.4) SFSV(Ethiopia-2011/Ethiopia / 2011) 4026(62.6) 1341(57.1) / / 741(74.4) 246(84.2) SFSV(Sabin/Italy/1943) / / 783(53.3) 260(63.6) 741(75.3) 246(83.8) DASHV(131/Iran/2011) 4029(65.2) 1342(58.6) 786(58.1) 261(61.7) 741(76.5) 246(85.0) RVFV(ZH-548/Egypt/1977) 3594(36.0) 1197(39.9) 798(15.5) 265(24.1) 738(55.4) 245(52.8) "/" indicates that the sequence was not available in the GenBank database.
Table 4. Homology analysis of the newly isolated virus (SXYQ 1916) and other Phleboviruses.
We performed phylogenetic analysis based on the genetic sequences of the four virus strains isolated from sandfly specimens in Yangquan County in 2019, and those of 60 sandfly viruses reclassified by ICTV in 2020. We found that the four virus strains isolated in this study belonged to the same evolutionary branch as the previously isolated WUXV strain (SXWX1813-2). On the basis of the sequences of the S (including the NS and N coding sequences), M and L gene, these viruses belonged to the same evolutionary population as the TORV isolated from Turkey sandflies in 2012-2013 (Fig. 2).
Figure 2. Phylogenetic analyses of the genetic sequences of the four virus strains isolated from sandflies collected in Yangquan in 2019. A Phylogenetic analysis of isolates SXYQ1916, SXYQ1927-2, SXYQ1930, SXYQ1931 (black dots) based on the M gene sequence. B Phylogenetic analysis of isolates SXYQ1916, SXYQ1927-2, SXYQ1930, SXYQ1931 based on the S gene sequence (black dots). C Phylogenetic analysis of isolates SXYQ1927-2 (black dots) based on the L gene sequence. The phylogenetic analysis was conducted using MEGA 6.0 software and the neighbor-joining method (bootstrap value of 1000). Note For phylogenetic comparison, we used the gene sequences of Phleboviruses from the ICTV 2020 guidelines; "/" indicates that the sequence was not available in the GenBank database.
To identify the species of the collected sandflies, we used PCR to amplify the COI gene. We found that all four pools of sandflies used to isolate the viruses belonged to the species Phlebotomus chinensis (Table 2).
In this study, 46 serum samples of healthy people were collected. The neutralizing antibody level of WUXV in serum samples was detected by plaque neutralization test (when the ratio of WUXV and neutralizing antibody in serum was 1:10, the sample was positive). The results showed that 8.70% (4/46) of the serum samples were positive in local heathy people. The neutralization titers of WX14, WX44 positive specimens were 1:10, WX28 and WX34 positive specimens were 1:20. The serum neutralizing antibodies of four chickens were all positive with titers ranging from 1:10 to 1:160. The results of neutralizing antibody test were shown in Table 5.
Number Gender Age Livestock raised Neutrlization test (Serum dilution) WUXV gene amplification Chicken Sheep Dog WX14 Female 66 10 – 1 + (1:10) – WX28 Male 68 + (1:20) – WX34 Male 61 – – 1 + (1:20) – WX44 Male 59 – – 1 + (1:10) – Table 5 shows the background information of healthy people and chickens with neutralizing antibody positive in serum specimens. The blood donors of WX14 and WX28 came from the same family, which raised 10 chickens and 1 dog. Other positive individual families raised only one dog but not sheep, chickens and other livestock and poultry. "–" means that the experimental result is negative.
Table 5. Detection of neutralizing antibody against WUXV.