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Novel subgenotypes of bovine viral diarrhea virus based on 5’ UTR molecular epidemiology in cattle from huhhot of Inner mongolia autonomous region, China

*Yong Jun Wen
College Of Veterinary Medicine, Inner Mongolia Agricultural University, China

*Corresponding Author:
Yong Jun Wen
College Of Veterinary Medicine, Inner Mongolia Agricultural University, China

Published on: 2020-04-05

Abstract

Bovine viral diarrhea (BVD) causes high economic losses in the cattle population worldwide. Here, we present the results of an epidemiological survey for Bovine viral diarrhea virus (BVDV) in Northwest of China. In this study, a total of 167 samples were collected from the bovine farms and cattle slaughters for epidemiology nearby Huhhot in Inner Mongolia Autonomous Region during 2017 and 2018. Positive BVDV isolates were genotyped based on a comparison of gene sequences from their 5’untranslated regions (5’UTR). Results indicated that, out of 167 samples, 68 (40.72%) were BVDV-RNA-positive. 28 of them were sequenced and analyzed with 5’UTR and used for constructing phylogenetic tree. Phylogenetic analysis based on 5’UTR revealed that13 of the BVDV isolates belong to BVDV-1 and 15 belong to BVDV-2 genotype. Interesting, two novel BVDV-1 subgenotypes and one novel BVDV-2 subgenotype were found in present study. Therefore, the result of this study will be useful to understand epidemiology in Inner Mongolia Autonomous Region and allow producers to better protect their livestock.

Keywords

Bovine viral diarrhea viruses; Epidemiology; 5’UTR; Novel Genotype

Importance

An epidemiological survey indicated two novel BVDV-1 subgenotypes and one novel BVDV-2 subgenotypewere found based on 5’UTR phylogenetic tree in Northwest of China.

Introduction

BVDV can cause bovine respiratory, gastroenteric and reproductive clinical consequences, and still lead to the birth of immunotolerant. It is the main viral infectious disease of cattle, which is a worldwide epidemic disease. Bovine viral diarrhea virus (BVDV) is a member of the genus Pestivirus. According to the genetic characteristics of the genome, BVDV is divided into two genotypes, BVDV-1 and BVDV-2. At present, BVDV-1 strains have further been divided into 21 subtypes (1a-1u) (Tajima et al., 2001; Vilcek et al., 2001; Yamamoto et al., 2008; Gong et al., 2013; Deng M. et al., 2015). To date, the BVDV circulating in the Chinese cattle population is mainly BVDV-1b, -1c, -1m, -1p (Zhong et al., 2011). According to the difference of secondary structure of 5’ -UTR sequence, BVDV-2 is divided into 4 subtypes, BVDV-2 a, 2 b, 2 c and 2 d (Giangaspero et al., 2008).
Bovine viral diarrhea virus infection in pregnant animals can also result in the birth of a persistently infected (PI) calf. PI animals are the main source of virus transmission to susceptible animals. It is an obstacle for the elimination of the virus in a susceptible population. The combined economic impact of BVDV has been estimated at a 20 to 57 million dollar loss per million calving’s in the USA. It also caused a huge economic loss from BVDV in China. 5’-UTR, Npro, and E2 genes were also used for genetic typing of the pestiviruses (Vilcek et al., 2001; Vijayaraghavan et al., 2012).
Variability of BVDV is distinct. A growing number of BVDV-1 and BVDV-2 subgenotypes based on phylogenetic analysis indicated the BVDV is genetically highly heterogeneous. The highest number of various BVDV subgenotypes has been documented in European countries (Yesilbag et al., 2017). Different genomic regions, i.e., 5’UTR (Beer et al., 2002; Becher et al., 2003; O’Brien et al., 2017), Npro (Maya et al., 2016; O’Brien et al., 2017), E2 (Couvreur et al., 2002; Yilmaz et al., 2012), have been used for genotyping and classification of BVDV. Partial 5’UTR sequences have been most frequently used for phylogenetic analyses and genotyping of BVDV isolates. A 288bp size of 5’UTR was used for epidemiology of BVDV in present study.
The epidemiology and genetic variations analysis of BVDV can implicate in disease control as diagnostics and vaccines that work well against homologous strains can be less efficacious for geneticallydistinct viruses.

Materials And Methods

Sample collection and processing
The 167 whole blood, lung tissue and intestinal contents samples were collected from the cattle of six bovine slaughters and calf and bovine of 26 farms, which were healthy or showed minor respiratory disease, in Huhhot and its surrounding rural breeding area of Inner Mongolia Autonomous Region, China during 2017 and 2018. Blood samples were collected for diagnostic purposes by venipuncture into silicon-coated vacutainer tubes and were immediately transported to the laboratory and stored at -20OC in tubes appropriate for freezing. Other tissue samples were homogenized in 2 ml of PBS and centrifuged at a 2,000×g for 3 min to remove the suspended solids. The supernatants were stored at −80OC until testing.
RNA extraction and RT-PCR
Total RNA was extracted from whole blood or tissues using the TRIzol reagent (Life Technologies, U.S.) according to the manufacturer’s instructions. Viral cDNA was constructed using reverse transcriptase (M-MLV, Invitrogen) with 10 μL of RNA. The polymerase chain reaction (PCR) amplification was performed with 5 μL of cDNA as the template using previous detection primers BVP1 (5’TAGCCATGCCCTTAGTAGGAC-3’) and BVP2 (5’CTCCATGTGCCATGTACAGCA-3’) (Vilcek et al., 2003) flank a 288-bp DNA fragment for BVDV 5’UTR with DNA polymerase Pfu (NEB, US). The PCR amplification was performed for 35 cycles: denaturation at 94°C for 30 s, annealed at 57°C for 30 s, and elongation at 72°C for 30 s. The samples were then incubated for an additional 10 min at 72°C and cooled to 4°C until further processing. Five microliters of the PCR products were analyzed on agarose gel (1.5%) electrophoresis at 120 V for 20 min.
Sequencing and Phylogenetic analysis of nucleotides
Twenty-eight isolates were randomly chosen from RNA positive samples for sequencing within the 5’UTR of the genome. The amplicons were purified using an Omega gel extraction kit (Omega, U.S.) and sequenced. These nucleotide sequences were assembled and proof read used the SeqMan program of Lasergene package (DNASTAR Inc., USA). For phylogenetic analysis, a total of 79 related reference sequences of 5’UTR (Listed in Table 1) were retrieved from the NCBI GenBank database (http://www.ncbi.nlm.nih.gov/genbank) and field strains in present study were also included for comparison (28 strains). Some of reference strains are reported as classified subgenotypes. A phylogenetic tree was constructed by the UPGMA method with MEGA7.0. The evolutionary distances were computed using the Maximum Composite Likelihood method. The GenBank accession numbers for the sequences of 5’UTR genes of the 28 BVDV isolates in present paper are listed in Table 1.
Table 1: BVDV isolates and reference strains described in this study.

No

Strain

Country ? year of isolation

5’UTR reference

E2

reference

Genotype

Host

1

NM1

China-2017

MK204893

 

BVDV-1 Cattle

2

NM2

China-2017

MK204906   BVDV-2

Cattle

3

NM4

China-2017

MK204907  

BVDV-2

Cattle

4

NM5

China-2017

MK204894   BVDV-1

Cattle

5

NM6

China-2017

MK204895   BVDV-1

Cattle

6

NM7

China-2017

MK204908   BVDV-2

Cattle

7

NM15

China-2017

MK204909   BVDV-2

Cattle

8

NM19

China-2017

MK204896   BVDV-1

Cattle

9

NM21

China-2017

MK204897  

BVDV-1

Cattle

10

NM23

China-2017

MK204910  

BVDV-2

Cattle

11

NM24

China-2017

MK204911   BVDV-2

Cattle

12

NM25

China-2017

MK204912  

BVDV-2

Cattle

13

NM29

China-2017

MK204913

 

BVDV-2

Cattle

14

NM40

China-2017

MK204898

 

BVDV-1

Cattle

15

NM42

China-2017

MK204899

 

BVDV-1

Cattle

16

NM44

China-2017

MK204914

 

BVDV-2

Cattle

17

NM45

China-2017

MK204900

 

BVDV-1

Cattle

18

NM46

China-2017

MK204901

 

BVDV-1

Cattle

19

NM51

China-2017

MK204902

 

BVDV-1

Cattle

20

NM57

China-2017

MK204915

 

BVDV-2

Cattle

21

NM100

China-2017

MK204903

 

BVDV-1

Cattle

22

NM109

China-2017

MK204916

 

BVDV-2

Cattle

23

NM123

China-2017

MK204917

 

BVDV-2

Cattle

24

NM125

China-2017

MK204918

 

BVDV-2

Cattle

25

NM135

China-2017

MK204904

 

BVDV-1

Cattle

26

NM157

China-2017

MK204919

 

BVDV-2

Cattle

27

NMy1

China-2017

MK204920

 

BVDV-2

Cattle

28

810763

China-2018

MK204905

 

BVDV-1

Cattle

29

BVDV1/Serbia/2008

Serbia/2008

KY941186

 

BVDV-1

cattle

30

BVDV1/WB4/Serbia/2017

Serbia/2017

KY941185

 

BVDV-1

wild boar

31

4-81

South Korea/2009

GQ985460

 

BVDV-1a

calf

32

UEL7-BR/11

Brazil/2011

KJ188147

 

BVDV-1a

Bovine

33

435FaUY/032014

Uruguay/2014

KT833794

 

BVDV-1a

Bovine

34

181

Argentina/2016

MF120592

 

BVDV-1b

fetal bovine serum

35

4-2

South Korea/2009

 GQ985459

 

BVDV-1b

calf

36

SC China/2016

KX280711

 

BVDV-1b

calf

37

Bega-like

Australia/2012

KF896608

 

BVDV-1c

Bovine

38

Crookwell

Australia/1989

JQ743606

 

BVDV-1c

Bovine

39

Grafton Australia/1989 JQ743607

 

BVDV-1c

Bovine

40

Mogilla Australia/1987

JQ743605

 

BVDV-1c

Bovine

41

Bov_preto

Brazil/2015

KU564958

 

BVDV -1d

Bos taurus

42

fecal

China/2015

MF166858

 

BVDV -1d

yak

43

ELV_ca_10

Denmark/2010

JX966090

 

BVDV -1d

cattle

44

MSGOCAE110 Iraq/2016 MF347404

 

BVDV-1e

calf

45

MSKOCOE13 Iraq/2016 MF347405

 

BVDV-1e

Cow

46

[email protected] Italy/2016 KX766447

 

BVDV-1f

cattle

47

[email protected] Italy/2016 KX766452

 

BVDV-1f

cattle

48

MRI2569 South Korea/2006 LT902694

 

BVDV-1g

Bos taurus

49

48/08 Poland/2008 JN715036

 

BVDV-1g

cattle

50

L-AT Austria/1998 FJ493483

 

BVDV-1g

cattle

51

[email protected] Italy/2002 MG434576

 

BVDV-1h

Bovine

52

UM/126/07 Italy/2007 LT631725

 

BVDV-1h

Bos taurus

53

436FaUY/052014 Uruguay/2014 KT833795

 

BVDV-1i

bovine

54

MRI2021 United Kingdom/2017 LT902249

 

BVDV-1i

Bos taurus

55

A469 Chile/2010 GU987129

 

BVDV-1j

Vicugna pacos

56

LL795 Chile/2010 GU987133

 

BVDV-1j

Lama glama

57

SuwaCp Switzerland/1993 KC853441

 

BVDV-1k

Bovine

58

71-03 France/2005 KF205294

 

BVDV-1l

Bos taurus

59

TR84 Turkey/2012 MH753470

 

BVDV-1l

Bos taurus

60

XC China/2015 MH166806

 

BVDV-1m

Bos taurus

61

KB01 South Korea/2007 GQ495676

 

BVDV-1n

Cattle

62

Shitara/02/06 Japan/2006 LC089876

 

BVDV-1n

Bos taurus

63

IS26/01ncp Japan/2001 LC089875

 

BVDV-1o

Bos taurus

64

HY-3 China/2016 KY865366

 

BVDV-1o

dairy cattle

65

TJ06 China/2006 GU120246

 

BVDV-1p

Cattle

66

BJ0701 China/2007 GU120247

 

BVDV-1p

Cattle

67

SD0803 China/2008 JN400273

 

BVDV-1q

Pig

 

VE/245/12 Italy/2010 LM994671

 

BVDV-1r

cattle

 

UM/136/08 Italy/2008 LN515612

 

BVDV-1s

cattle

 

SI/207/12 Italy/2012 LN515611

 

BVDV-1t

 

68

08Q723 South Korea/2012 JQ418632

 

BVDV-2a

cattle

69

5-80 South Korea/2009 GQ985458

 

BVDV-2a calf

70

HB-1511

China/2015

KX096718

 

BVDV-2a

cattle

71

NY-93

USA/1993

KR093034

 

BVDV-2a

cattle

72

10-636

Argentina/2010

MH294527

 

BVDV-2b Bos taurus

73

MSGPCAB233

Iraq/2017

MF491397

 

BVDV-2b

calf

74

MSGLCAB226

Iraq/2017

MF491396

 

BVDV-2b

calf

75

439RvUY/082014

Uruguay/2014

KT833799

 

BVDV-2b

Bovine

76

4p

Brazil/2015

MG436782

 

BVDV-2b

Homo sapiens

77

Bov/Ita/124.15-28

Italy/2015

KX890138

 

BVDV-2c

Bos taurus

78

Bov/Ita/856.14-578

Italy/2014

KX350085

 

BVDV-2c

Bos taurus

79

Bov/Ita/124.15-7

Italy/2015

 KX350081

 

BVDV-2c

Bos taurus

80

354

Argentina/2000

AF244959

 

BVDV-2d

Bovine

81

BJ6(09) China/2009 GU385894

 

BVDV-2

Bovine

82

Bangwa South Korea/2008 GQ985457

 

BVDV-2 calf

83

IT-1732 Italy/2001 AJ416018

 

BVDV-2 Bovine

84

TR-2006-3 Turkey/2006 EU542423

 

BVDV-2

cattle

85

M10 3432 USA/2010 JN377413

 

BVDV-2 Bovine

86

890 USA/1994 NC_039237

 

BVDV-2 Bovine

87

Ind 5197 India/2007 EF547201

 

BVDV-2 Goat

88

58 USA/2007 FJ431191

 

BVDV-2 Goat

89

73 USA/2007 FJ431194

 

BVDV-2 Goat

90

0602ja South Korea/2006 DQ973184

 

BVDV-2 Goat

91

LN01

China/2009

KC176779

 

BVDV-2

cattle

92

BVDV2/Serbia/2008

Serbia/2008

KY941187

 

BVDV-2

cattle

93

58-10

France/2005

KF205291

 

BVDV-2

Bos taurus

94

BVDV2-125c

USA/2013

KC596020

 

BVDV-2

Bos taurus

95

XJ-04

China/2004

FJ527854

 

BVDV-2

cattle

96

76/08

Argentina/2008

JX848364

 

BVDV-2 Bovine

97

106

Argentina/2014

MF120586

 

BVDV-2 Bovine

98

101

Argentina/2014

MF120585

 

BVDV-2 Bovine

99

Buffalo 17 band A

Argentina/2005

FM165309

 

BVDV-2 Buffalo

100

HI4463

Germany/2003

AY379546

 

BVDV-2 Not mentioned

101

13148906

USA/2006

FJ387325

 

BVDV-2 Bos taurus

102

86275

Argentina/2008

JX679696

 

BVDV-2 Bovine

103

LV-96 USA/2001 AF410787

 

BVDV-2 Not mentioned

104

Glessen 6 Germany/2003 AY379547

 

BVDV-2 Not mentioned

105

VS-6 USA/2001 AF410789

 

BVDV-2 Not mentioned

106

UEL12-BR/17 Brazil/2017 MG004720

 

BVDV-2 Bovine

Notes: The bold letters are strains isolated in present study.

Results

RT-PCR survey of clinical samples
RT-PCR assays were performed to determine the BVDV species with 5’UTR specific primers responsible for the infection. Of the 167 samples, 68 (40.72%) were BVDV-RNA-positive by RT-PCR. Of the products 28 were sequenced and blasted in NCBI GenBank. Thirteen BVDV-1 isolates (47.83%) were genotyped. Fifteen (52.17%) were classified as BVDV-2 in all the sequenced28 antigen positive samples. The electrophoresis results of RT-PCR products of some samples were shown in figure 1a. A summary of the isolates and reference strains is presented in Table 1. The 5’UTR genes of these isolates were used for further phylogenetic analysis.


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Figure 1: RT-PCR detection of clinical samples by BVDV 5’UTR special primers. M, DL1000 Marker; 1-15, Clinical samples collected after 5 passages; 16, Negative control; 17, Positive control.


Subgenotypes based on phylogenetic analysis of 5’UTR of BVDV-1 in Inner Mongolia
The nucleotide sequences of field viruses and a number of known reference strains representative of all known species and subtypes of BVDV were aligned and phylogenetic trees were constructed. It displayed phylogenetic trees for a selection of the 5’UTR in Fig. 2. The specified fragments of 5’UTR region (288bp) from cDNA preparations were detected and sequenced. Phylogenetic analysis was performed based on the 288bp fragments of 13 BVDV-1 strains of 68 BVDV-positive samples collected from cattle’s in Inner Mongolia between 2017 and 2018. Thirty-six B reference strains from GenBank were used. BVDV-1 isolates analyzed in this work were deposited in GenBank under following accession numbers: MK204893-MK204905 (Listed in Table 1). The topology of the tree (Figure 2a) showed that all 49 BVDV-1 strains belonged to 20 distinct subgenotypes, namely BVDV-1a (n=2),BVDV-1b (n=4), BVDV-1c (n=8), BVDV-1d (n=4), BVDV-1e (n=2), BVDV-1f (n=2), BVDV-1g (n=4), BVDV-1h (n=2), BVDV-1i (n=2), BVDV-1j (n=3), BVDV-1k (n=1), BVDV-1l (n=1), BVDV-1m (n=4), BVDV-1n (n=2), BVDV-1o (n=1), BVDV-1p (n=2), BVDV-1q (n=1), BVDV-1r (n=2), BVDV-1s(n=1), BVDV-1t(n=1), BVDV-1u(n=1), BVDV-1v(n=2), BVDV-1w(n=2). The subgenotypes BVDV-1m, -1n, -1o, -1p, and -1q had been detected exclusively in Asia. Similarly, BVDV -1f, -1g,-1h, -1k, -1l, -1r, 1s, and -1t have not been reported to occur in countries outside Europe(Giammarioli et al., 2008; Yilmaz et al., 2012; Factor et al., 2016; Gomez-Romero et al., 2017; Silveira et al., 2017; Yesilbag et al., 2017). However, phylogenetic analysis clustered the 13 BVDV-1 isolates into six subgenotypes in present study: BVDV-1a, BVDV-1c, BVDV-1d, BVDV-1m, and two potentially novel subgenotypes, tentatively designated as ‘BVDV-1v’ and ‘BVDV-1w’ in present study (Figure 2).


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UEL-BR/11 isolated from Brazil, which was belong to BVDV-1a. There are 4 strains: NM1, NM5, NM19 and NM46 in the BVDV-1c group, which also including 4 strains from Australia. The NM21 and NM45 were classified as BVDV-1d. ELV_ca_10 strain from Denmark belonging to BVDV-1d in a previous work was not clustered into BVDV-1d but BVDV-1b in present work. 810763 and NM135 strains isolated from bovine herd located west of Inner Mongolia were classified as BVDV-1m. The first BVDV-1m strain, ZM-95, was initially isolated from swine herds in Inner Mongolia in 1995(Wang X, 1996). Six BVDV-1m isolates from Beijing were detected in 2015(Weng et al., 2015). Accumulating evidence is indicating that BVDV-1m also is a predominant subgenotype in cattle herd (Deng Y. et al., 2012; Gao et al., 2013).
Interesting, the other four strains NM6, NM51, NM40 and NM42 were shown clustering into two novel distinct phylogenetic groups from BVDV-1a~1u. Here, we first tentatively named them as ‘BVDV-1v’ and ‘BVDV-1w’. When NM6 is blasted with other strains in GenBank, the homology is up to 90%. There is highest identity between NM6 and MF-2, a BVDV-1c strain from China. Though NM51 is belong to same cluster with NM6, it has an 84% identity with BJ09_21 strain, which is also from China. NM40 only has a homology up to 82% with other strains in GenBank, which are belong to BVDV-1d and BVDV-1a when blasting. NM40 is likely origin from has most similar with [email protected], a BVDV-1d isolate from Italy. We deduced NM40 are likely origin from recombination with BVDV-1a and BVDV-1d.
Subgenotypes based on phylogenetic analysis of 5’UTR of BVDV-2 in Inner Mongolia
The 15 positive samples belonged to BVDV2, which were divided into 2 different subgenotypes (Figure 2b). Phylogenetic analysis results showed that 60% (9/15) of the sequences (NM2, NM4, NM7, NM23, NM24, NM25, NM29, NM157, NMy1) were typed into a single subgroup BVDV-2a. Specially, other 6 strains (NM15, NM44, NM57, NM109, NM123, NM125) were characterized as a new subgroup that is different from BVDV-2a~BVDV-2d. These six isolates could not be assigned to any known BVDV-2 subgenotype. We appointed it as ‘BVDV-2e’. Subgenotypes BVDV-2b, -2c, and -2d strains, which have been reported in South America, were not detected in current study. The BVDV-2a subgenotype appears to be more common than the other subgenotypes worldwide.


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Figure 2: Phylogenetic tree based on the 5’UTR sequences of reference BVDV strains and isolates from Huhhot, China. Evolutionary analyses were conducted in MEGA7, supported by 1,000 bootstrap replications.


a. Phylogenetic tree showing the genetic relationship between Bovine viral diarrhea virus 1 isolates based on analysis of 49 nucleotides derived from the 5’UTR. The tree contains 13 BVDV-1 isolates sequenced in this study plus 36 reference strains (shown in Table 2). The studied strains were labeled with a symbol.
b. The phylogenetic tree was generated based on comparison of nucleotide sequences of the 5’UTR of 15 BVDV-2 isolates with 39 reference sequences (shown in Table 2) downloaded from the GenBank database. The 15 BVDV-2 isolates are indicated in symbol Numbers at the phylogenetic branches indicate branch lengths (next to the branches) in the same units. The sequences for reference strains were listed with strain name and GenBank no.
 

Discussion

Were detected more than other 4 groups 1a, 1m, 1v and 1w.
Deng et al. investigated BVDV genotypes in four bovine species to found BVDV-1b, BVDV-1m and a new cluster BVDV-1u were dominant subtypes in China (Deng M. et al., 2015). However, they collected the samples from east but not west of Inner Mongolia. We mainly located in west of Inner Mongolia to supervise the prevalence of BVDV. Importantly, we found not only the subgenotypes BVDV1m but also two novel clusters BVDV-1v and BVDV1w. It indicated that more complicated BVDV strains were spread in west of Inner Mongolia.
Infection with BVDV-2 was first described in North America in the early of 1990s (Corapi et al., 1989). BVDV-2 was found in cattle in Xinjiang Autonomous Region and in Qinghai province of China (Gong et al., 2014). The BVDV-2a subgenotype appears to be more common than the other subgenotypes worldwide (Giammarioli et al., 2008; Oguzoglu et al., 2010; Behera et al., 2011; Han et al., 2016; Gomez-Romero et al., 2017). A recent phylogenetic analysis of BVDV in Mongolia revealed BVDV-1a and BVDV-2a were dominant genotype (Ochirkhuu et al., 2016). Though Inner Mongolia of China locates nearby Mongolia, the BVDV genotype based on epidemiological studies showed more subgenotypes appeared in Inner Mongolia of China. Two BVDV-2 subgenotypes were found in present study.
Here, we present the results of an epidemiological survey for Bovine viral diarrhea virus (BVDV) in Inner Mongolia, China, especially in Huhhot and surrounding areas of Inner Mongolia Autonomous Region. Our investigation describes the genetic diversity of BVDV from cattle in Huhhot of Inner Mongolia. It provides important information for the clinician and diagnostician responsible for diagnosis of BVDV.
To summarize, the presence of novel subgenotypes BVDV-1v and BVDV-1w and BVDV-2e were described in China dairy cattle for the first time. Further studies are required to investigate the prevalence of BVDV infection in a larger cattle population as well as the role in various clinical conditions and economical losses. The presence of new species is important for the evaluation of current diagnostic protocols and for the development of control programs, such as vaccination. Furthermore, it underlines the necessity to quarantine and test imported cattle thoroughly before introduction into local herds.

Acknowledgements

This work has been funded in whole or in part with Introducing Talents Scientific Research Project of Inner Mongolia Agricultural University ‘Research on prevention and control of herbivore animal diseases and biological products’ (NDGCC2016-22 and NDYB2018- 2) and National Natural Science Foundation of China (NSFC) ‘Molecular mechanism for synaptic recycling dysfunction induced by G protein of street rabies virus in primary mouse neural cells’ (31572505). The study was also supported with Key Laboratory of Clinical Diagnosis and Treatment Technology in Animal Disease, Ministry of Agriculture, P.R China and Ruminant Animal Disease Diagnosis center, Inner Mongolia Agricultural University.

Author contributions:

thor contributions: FXW, YJW contributed to the design of the work; JHF, WDGW, KY, XPL, ZPM, MJ HZ, and YY performed the experiments in the study. FXW, YML, YMS, HW, CYL, BWZ, HZZ, and QJS analyzed the data. FXW, YJW wrote the manuscript. All authors read and approved the final manuscript.

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