Thirty-five groups of newborn mice were inoculated with supernatants clarified from the homogenates of ticks. After the second passage in mice, 11 groups began to demonstrate illness from 4-5 days post inoculation (d p.i.). The signs of illness included slow growth, lethargy, tremors, loss of balance, paralysis of the hind limbs, and progressively diminishing mobility. These mice were sacrificed, and homogenates of brain, heart, liver, spleen, lung, and kidney were prepared. RT-PCR followed by Sanger sequencing showed that the group inoculated with tick homogenates from Yuli County was positive for CCHFV S segment (data not shown). Further, results of high-throughput fluorescence fast assay revealed very high virus load in brain, heart, liver, and kidney (Table 1). BLASTN analysis showed that the sequences of the PCR products shared 99% nucleotide similarity with other CCHFV sequences deposited in GenBank. The supernatants of the homogenates from CCH FV-positive brains were incubated with Vero E6 cells. Green fluorescence was detected in the cytoplasm of Vero E6 cells using IFA (Figure. 2A, white arrowheads), suggesting that Vero E6 cells were infected with CCHFV. Thus, a new strain of CCHFV, designated YL16070, was identified and isolated from tick samples from Xinjiang, and Vero E6 cells were sensitive to the virus after inoculation in mice.
Tissues* MFI Brain* 1698 Heart* 1621.5 Liver* 1613.5 Spleen* 848.5 Lung* 1261.5 Kidney* 1692 Brain from the healthy mouse* 3 CCHFV 1369 Blank 38 Note: *Tissues were collected from newborn mice sacrificed on 5 d p.i. MFI, median fluorescence intensity of each sample.
Table 1. MFI values of the tissues from newborn mice
Figure 2. Immunofluorescence detection of CCHFV NP expression in Vero E6 cells. (A) Vero E6 cells were incubated with supernatants from the homogenates of CCHFV-positive mouse brains. At 6 days post infection, CCHFV NP expression was detected by IFA, as indicated by white arrowheads. (B) Healthy cells were used as a negative control. Bars, 1 mm.
On 5 d p.i., tissues including the brain, heart, spleen, liver, lungs, and kidneys were all collected, and CCHFV RNA was detected in these tissues (Table 1). Notably, higher levels were found in the brain, heart, liver, and kidneys than in the other tissues (Table 1). Additionally, tissue lesions were noted in pathological sections of the liver, kidneys, and brain (Figure 3A). However, no significant changes were observed in the heart, lungs, and spleen (data not shown). The primary lesions in the liver consisted of ballooning degeneration of hepatocytes, congestion, steatosis, and focal necrosis; infiltration of inflammatory cells was also observed in the liver. The kidneys showed edema and cytoplasmic swelling of poorly stained renal tubular epithelial cells in the renal tubular cortex. Hemorrhagic focuses were found in the brain. IHC staining also found CCHFV infection and antigen expression in the liver, kidneys, and brain on 5 d p.i. (Figure 3B). These results suggested that the liver, kidneys, and brain were the primary replication sites of CCHFV in newborn mice.
Figure 3. Detection of CCHFV infection in newborn mice. (A) Tissues from newborn mice were sectioned and stained, and tissue lesions caused by CCHFV infection were detected. Red arrowheads indicate the lesions. (B) Immunohistochemical analysis of CCHFV antigens in different tissues of newborn mice. Red arrowheads indicate the foci of CCHFV antigens.
The genome of the new isolate, designated YL16070, consisted of three segments, the S (1673 nt), M (5379 nt), and L (12156 nt) segments. BLASTN comparisons showed that YL16070 was the most closely related and shared 98% identity with another strain, YL04057, which was isolated from H. asiaticum asiaticum ticks in Yuli County in 2004 (Zhou et al., 2013b). Because the groups of CCHFV were designated according to their geographic distributions, phylogenetic trees built based on 101 complete sequences of S segments showed that eight groups were clustered, i.e., Asia 1 and 2, Africa 1-3, and Europe 1-3, and that YL16070 clustered with all other Chinese strains, belonging to the Asia 2 group (Figure 4A). In the M and L trees, discrepancies were noted, consistent with other studies (Figure 4B and 4C) (Hewson et al., 2004; Anagnostou and Papa, 2009; Chen, 2013; Zhou et al., 2013a). Composed of 90 sequences, the M tree was divided into seven groups, including Asia 1-3, Africa 1-3, and Europe 1 and 2 (Figure 4B); and the L tree (68 sequences included) was divided into six groups, including Asia 1/2 (a group of strains in combination of strains from Asia 1 and 2 in the S and M trees) and 3, Africa 2 and 3, and Europe 1 and 2 (Figure 4C). The Chinese strains did not cluster in just one group in the M and L trees, but dispersed in three Asian groups in the M tree and two Asian groups in the L tree. The new isolate YL16070 belonged to Asia 3 in both the M and L trees. Since there were fewer full-length sequences of M and L segments available for phylogenetic analysis, it is possible that fewer groups were found in the M and L trees than in the S tree. However, the nonexistent group Asia 3 in the S tree appeared in the M and L trees and was composed of at least three Chinese strains. These findings suggested that YL16070 and the closely related Chinese strains (YL04057, 7001, 79121M18, and 79121) may represent a new genotype of CCHFV and that more than one genotype of CCHFV circulated in Xinjiang Province.
Figure 4. Phylogenetic analysis of CCHFV with available complete genome sequences. ML trees were constructed based on the complete sequences of S (A), M (B), and L (C) segments deposited in GenBank. Strains are displayed in the format of "GenBank accession number_strain_country_year". The Chinese strains are shown in red bold characters, and the new isolate in this study, YL16070, is labeled by a black solid circle (●).
Isolation and identification of a new strain of CCHFV from the H. asiaticum asiaticum ticks in Xinjiang
Detection of CCHFV infection in newborn mice
Phylogenetic analysis with the new strain indicated that more than one genotype of CCHFV circulated in Xinjiang Province for years
Primers Sequences (5'-3') Locations of primers S segment S1F* TCTCAAAGAAACACGTGCCGC 1-21 S1R* GACAAATTCCCTGCACCA 653-670 S2F* CCCAGTGAGCCGTGAACAT 628-646 S2R* CAGGGTGCATGTAGATCCTG 1168-1187 S3F* CTTTATGAGCTCTTTGCTGATG 1130-1151 S3R* TCTCAAAGATATCGTTGCCGC 1652-1672 M segment M1F* TCTCAAAGAAATACTTGCGGCAC 1-22 M1R* ATGRTGTGYAYYYTGTGGTGT 504-524 M2F* GTGCATGCAAGCCCATCACC 504-523 M2R* CRTTGATCARCTGMARYCCTG 1201-1221 M3 F TTACTATGCCAAAGGGTCTC 1151-1170 M3 R ATTTGACTGGGATGCTATCT 1901-1920 M4F* TWYAAGCTDTGCGAGAACAGTGC 1833-1855 M4R* CTNACAACCCARGGDATTCTTTC 2481-2503 M5F* CGGARGGARAARGTRGAAGAAACY 2439-2462 M5R* GTWGGTTTGAAGGTTGAYTGRACAT 3307-3331 M6F-2‡* CGTGGGGAGCAATCAATGTTC 3316-3336 M6R-2‡* GTGTGCAGCTCTGTAGCTTGC 4030-4050 M7F* GAAGGTTTYTTTGAYYTRATGCATG 3951-3975 M7R* GCTCTGTGGWTGSTCAAGYTT 4719-4739 M8F* AGCTGCASYRAAGAWGAYACACAA 4680-4703 M8R* TCTCAAAGATATAGTGGCGGCAC 5345-5367 L segment L1 F TCTCAAAGATATCAATCCCC 1-20 L1 R GAGTGCTAATGCAAGGTCTT 652-671 L2F* CTCTCAGAATACTGCCACAG 538-557 L2R* GTTGATAACATGACGCCAAGTG 1123-1144 L3F* CCCTGGAACAGGAATAGAAAG 1052-1072 L3R* ATTGCCCTGCCTGAGGTAC 1666-1684 L4-F1 ACTTCTCGGCTTATCACATT 1500-1519 L4-R1 ATTCAACGGAGTCTCAGGTA 2287-2306 L5 F CAAACCAAGTCTGACCATGT 2048-2067 L5 R TCTGTCCTTATTTTCCCGTC 2571-2590 L6F* GACTATGGAGAGAGGGGAATAG 2463-2484 L6R* TGACTTGATGTCTTCACTTAACC 3130-3152 L7 F CTACCCGTTAGTTTGGAACA 2934-2953 L7 R CTATCTTGGGACACCTCTTG 3311-3330 L8 F GATTGAGCTGCTTGCTTATA 3218-3237 L8 R GACCTTGGCACAGAACACTT 3723-3742 L9 F CAACCTATGCAAAAGGAGTA 3661-3680 L9 R TATGATGAGCCAAGGACAGC 4287-4306 L10+ F AGTAAAGGTAAGGCATTGTG 4154-4173 L10+ R GTTTATTCTGTTGTTGGGAG 4616-4635 L11+ F ACCTAGTTTATGGCTTCCTT 4573-4592 L11+ R ACTGGCAGATGTCTGTTATT 5309-5328 L10F* TGCCCAAATGTGAGAAAAGC 5076-5095 L10R* GAGYYTCTTCTTYAAGCTTCC 5889-5909 L11F* CAATCTCTTCHTCAGTYAAAGG 5869-5890 L11R* CTATCTTTGTYTGATGTAGYAGC 6614-6636 L12F* GAYYTRCACAARACCACTGACG 6588-6609 L12R* CTGACCCATATGGTTGTARCTGTT 7473-7496 L13 F TAGACAGTTGGGAAGGAAAT 7396-7415 L13 R TAAAGGCAGTGTTGGTGATA 7955-7974 L14 F CTTATCACCAACACTGCCTTTA 7953-7974 L14 R AAACTCCCAACCTTTCCAAC 8699-8718 L15 F CATTTGGAGGAGAAGGAACT 8587-8606 L15 R AAGGCTCTTTGACAGTGGTAT 9106-9126 L16+ F GATGGTCCACCAAGCACAGC 8980-8999 L16+ R TGCCCTTTAGCATGTAGAAA 9656-9675 L17 F TACAACAAGCAAAGCCAACC 9628-9647 L17 R GTATGTGAACTTCCCAGACC 10129-10148 L16F* CTGTRAAACGAGATTCTGAACGC 10000-10022 L16R* ACCTCCTGATTTGAGGTACTTGTG 10794-10817 L18+ F AGACTACATACCACGAGAATC 10564-10584 L18+ R CTCTGGAAATAGAAGGCACA 11277-11296 L19 F GTTAATGAACTTAATCAGCCAGTG 11183-11206 L19 R ACCCGTCAACATCAGAATCAAATC 11718-11741 L18F* CAAAGCCTGARAGAGTGGTYATRG 11473-11496 L18R* TCTCAAAGAAATCGTTCCCCCCAC 12133-12156 Note: *Primers were used in reference to a previous study of CCHFV genome announcement (Zhou et al., 2013b).
Table S1. Primers used to sequence the complete genome of the CCHFV isolate