Tick collection
In this study, we collected 170 engorged ticks from 22 sheep in Taian and Yantai regions of Shandong Province from May through September 2019, of which 89 ticks from 12 sheep in Taian city, and 81 ticks from 10 sheep in Yantai city (Table 1). All ticks collected from sheep were engorged Haemaphysalis longicornis and store in a cool and ventilated place to digest the blood in body. Then, each tick and serum sample was grouped into individual pool in preparation for viral RNA detection assays.
Table 1
Detection of DTV viral RNA in sheep and ticks collected from Shandong, China.
|
|
Sheep
|
Attached ticks
|
|
Sample ID
|
qRT-PCR
|
Nested-PCR
|
NO.
|
qRT-PCR
|
Nested-PCR
|
|
|
TADL-001
|
-
|
∕
|
12
|
-
|
∕
|
|
TADL-002
|
-
|
∕
|
9
|
-
|
∕
|
|
TADL-003
|
+
|
+
|
5
|
+(4 of 5)
|
+(2 of 4)
|
|
TADL-004
|
-
|
∕
|
3
|
-
|
∕
|
|
TADL-005
|
-
|
∕
|
5
|
-
|
∕
|
|
TADL-006
|
-
|
∕
|
7
|
-
|
∕
|
Taian
|
TADL-007
|
-
|
∕
|
7
|
-
|
∕
|
|
TADL-008
|
-
|
∕
|
6
|
-
|
∕
|
|
TADL-009
|
-
|
∕
|
9
|
+(2 of 9)
|
+(1 of 2)
|
|
TADL-010
|
-
|
∕
|
15
|
+(2 of 15)
|
+(1 of 2)
|
|
TADL-011
|
+
|
-
|
3
|
+(1 of 3)
|
-
|
|
TADL-012
|
-
|
∕
|
8
|
-
|
∕
|
|
total
|
|
2
|
1
|
89
|
9
|
4
|
|
|
LZSS-001
|
-
|
∕
|
11
|
-
|
∕
|
|
LZSS-002
|
-
|
∕
|
14
|
-
|
∕
|
|
LZSS-003
|
-
|
∕
|
7
|
+(1 of 7)
|
+(1 of 1)
|
|
LZSS-004
|
-
|
∕
|
8
|
-
|
∕
|
Yantai
|
LZSS-005
|
-
|
∕
|
8
|
-
|
∕
|
|
LZSS-006
|
+
|
+
|
4
|
+(3 of 4)
|
+(1 of 3)
|
|
LZSS-007
|
-
|
∕
|
7
|
-
|
-
|
|
LZSS-008
|
-
|
∕
|
9
|
+(1 of 9)
|
-
|
|
LZSS-009
|
-
|
∕
|
7
|
-
|
∕
|
|
LZSS-010
|
-
|
∕
|
6
|
-
|
∕
|
|
total
|
|
1
|
1
|
81
|
5
|
2
|
“∕”indicates that no experimental operation has been carried out. |
Detection of DTV RNA in ticks and sheep
According to the result of qRT-PCR, DTV RNA were detected in 14 tick pools (out of 170, 8.2%), three sera pools (out of 22, 13.6%) in this study, including nine tick pools (out of 89, 10.1%), two sera pools (out of 12, 16.7%) from Taian, and five tick pools (out of 81, 6.2%), one sera pool (out of 10, 10%) from Yantai, repectively(Table 1). This result indicated a high prevalence of DTV in engorged ticks and domestic animals, compared to that of unfed ticks (0.67%) in the same region[7]. It seemed probable that domestic animals could enhance virus replication or play a positive role in virus spread, which also verified the role of domestic animals in the life cycle of DTV. Besides, we also found that the DTV RNA was detected in sheep from different areas, and the prevalences were high, maintaining at about 10%. It perhaps because the sheep in this study were grazed on pastures or hills during the day and kept in household backyards at night, leading to a heavy infestation with ticks carrying DTV.
In addition to transmission by ingesting the blood of infected hosts, arboviruses have been proved to spread from infected ticks to other ticks that infect the same host by co-feeding, even in seropositive hosts[9, 10]. Under experimental conditions, transmission of SFTSV from infected to uninfected Haemaphysalis longicornis occurred by co-feeding in BALB/c mice and the author speculated that this transmission pathway is of great significance to the survival of TBEV in nature[11]. In the present study, 22 groups of corresponding RNA samples were obtained from ticks and sheep serum samples and, DTV was detected simultaneously in sheep serum and corresponding ticks among three groups, which suggested that the potential viremia might promote the flow and transmission of virus in ticks and animal hosts. Nevertheless, not all ticks from sheep with viremia were detected carrying DTV,perhaps because of the insufficient blood intake of some ticks, resulting in too few copies of the virus to reach the detection threshold of qRT-PCR. Otherwise, the viral RNA of DTV was positive in some ticks of four groups of samples, but negative in corresponding sheep blood and the discordant results suggest that ticks carrying DTV failed to cause high level of viremia in host animals owing to some factors, such as high level of serum antibody and insufficient blood exchange.
Infectious virus, a competent insert vector, and susceptible reverse hosts are the three basic elements for the stable existence of virus cycle[12]. It has been proved that threshold viremia levels of 102.0–4.7 50% lethal dose/mL is sufficient for some TBVs infections, like Colorado tick fever virus, Russian spring-summer encephalitis virus, and louping ill virus[13–15]. In our study, the DTV RNA in the domestic animal were detected, although the copy numbers of viral RNA were low (approximately 6.0×104 copies/mL, 95% CI = 5.6–7×104 copies/mL). Successful amplification of the partial S segment of DTV confirmed the occurrence of DTV viremia in this kind of domestic animal. However, only 2 isolates were obtained from all serum samples, which indicated that most infected sheep might have either a short period of viremia, a low level of viremia, or that few infectious virions might be present under the neutralization pressure of high level of serum antibodies.
Phylogenetic analysis
According to the latest taxonomic information on viruses from the International Committee on Taxonomy of Viruses (ICTV) in 2019, DTV is classified as a new member of Uukuvirus genus, Phenuiviridae[16]. Recent studies have demonstrated that DTV was closely related with Yongjia tick virus, Uukuniemi virus and Okutama tick virus in phylogeny and its pathogenicity to animals and human remains unclear due to the lack of further research on this novel virus[7, 8]. However, DTV deserves more attention as an emerging threat to public health. In this study, the partial S segment of DTV was amplified and sequenced from 8 of 17 qRT-PCR-positive samples and all the sequences obtained from sheep and ticks have a 95.1–99.8% nucleotide identity with each other. Pairwise distances analysis demonstrated a close evolutionary relationship among those DTV isolates from animal and ticks in these epidemic regions. Interestingly, the isolate called 19YTLZ-13-P from tick shared nearly the same identity (98.2%) with 19YTLZ-17-P from attached sheep (LZSS-006). Meanwhile, similar results occurred in another group of specimens from Taian, that is, the isolates 19TADL-12-P and 19TADL-16-P from ticks grouped more closely with19TADL-8-P from attached sheep(TADL-003). These suggested a potential link of DTV infections between sheep and ticks, and DTV might circulate in the endemic areas among ticks and many kinds of domestic animals, such as cattle, pigs, dogs, etc. Besides, these eight S sequences of DTV strains obtained in this study were closely related to that found in Hubei Province, indicating that the sequences of DTV from different regions had high similarity and DTV was relatively conservative in evolution, which was also consistent with our previous research results.