Selection of a Less Pathogenic BVDV Strain for the Construction of Avirulent Chimeric Pestivirus

Jaejo Kim; Seong-In Lim; Dong-Seob Tark; Jae-Young Song; Byounghan Kim

doi:10.4167/jbv.2010.40.1.39

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Kim, Lim, Tark, Song, and Kim: Selection of a Less Pathogenic BVDV Strain for the Construction of Avirulent Chimeric Pestivirus

Original Article

J Bacteriol Virol 2010;40(1):39-47.

Published online: 18 January 2010

DOI: https://doi.org/10.4167/jbv.2010.40.1.39

Selection of a Less Pathogenic BVDV Strain for the Construction of Avirulent Chimeric Pestivirus

Jaejo Kim, Seong-In Lim, Dong-Seob Tark, Jae-Young Song, Byounghan Kim^∗

National Veterinary Research and Quarantine Service, Anyang, Korea

^∗Corresponding author: Byoung-Han Kim. National Veterinary Research and Quarantine Service, 480, Anyang 6 dong, Manan-gu, Anyang City, Gyeonggi-do, 430-757, Korea. Phone: +82-31-467-1951, Fax: +82-31-467-1938 e-mail: kimbh@nvrqs.go.kr

Revised 25 August 20092 December 2009 Accepted 7 December 2009

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

To select a less pathogenic bovine viral diarrhea virus (BVDV) strain for the construction of chimeric pestivirus harboring classical swine fever virus (CSFV) E2 gene, five Korean BVDV isolates (four type 1 isolates and a type 2 isolate) were evaluated for their pathological and biological properties with respect to porcine infection. Each of five groups of 100-day-old pigs was inoculated intranasally with one of the five BVDV isolates. No clinical sign or leukopenia was observed in any pig throughout the duration of the experiment, but viruses were detected in blood, nasal discharges and postmortem samples using RT-PCR. These results indicated that although the five BVD viruses could infect pigs, they did not cause clinically apparent symptoms. Because of its proper infection dynamics shown in this preliminary animal study and its fast growth rate and quick CPE in cell culture, one isolate (KD26-1) was chosen among the five isolates to test its virulence and immunogenic properties in 40-day-old piglets. Neither clinical sign nor pathological lesion was observed in 40-day-old piglets during the course of infection of isolate KD26-1. The first neutralizing antibodies were detectable 14 days post-inoculation (PI) and increased to 1:128~1:256 28 days PI. A BVDV specific gene was detectable by RT-PCR in tonsil, spleen, inguinal lymph node and brain until 14 days PI. According to this study, it can be concluded that isolate KD26-1 has little pathological effect in pigs and is a candidate for construction of chimeric pestivirus harboring CSFV E2 gene.

Keywords: Bovine viral diarrhea virus, Classical swine fever virus, Animal study, Pigs, Pathological effect

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Figure 1.

Leukocyte counts (10³ cells/mm³) during the course of intranasal infection in the first animal study. Mean leukocyte counts in pigs inoculated with isolates KD26-1, 32669, 95589, 32527 and 95002 and those of contact pigs are given. Standard deviations are shown as error bars.

Figure 2.

Leukocyte counts (10³/mm³) of pigs after intramuscular inoculation with KD26-1 (10^7.5 TCID₅₀/head). Mean leukocyte counts in pigs inoculated with isolates KD26-1 and those of contact pigs are given. Standard deviations are shown as error bars.

Figure 3.

Virus neutralization titers of sera from pigs after intramuscular inoculation with KD26-1 (10^7.5 TCID₅₀/head). Mean virus neutralization titers in pigs inoculated with isolates KD26-1 and those of contact pigs are given. Standard deviations are shown as error bars.

Table 1.

The properties of five BVDV isolates used in animal experiments

BVD isolate	Origin	CPE (dpi^a)	Genotype^b
KD26-1	Diarrhea	3~4	1
32669	Persistent infection	4~5	1
95002	Respiratory infection	6~7	2
95589	Diarrhea	4~5	1
32527	Diarrhea	4~5	1

^a dpi: the number of days after which more than 80% cell death had occurred after inoculation with 0.1 MOI of each virus

^b Genotype determined by RT-PCR (19)

Table 2.

RT-PCR results for detection of viral antigen from nasal swabs of five pig groups during the course of intranasal infection

Group	Inoculated virus	Days post inoculation
Group	Inoculated virus	-4	0	1	2	3	4	5	7	9	11
I	KD26-1	0/6^a	0/6	0/6	2/6	0/6	1/6	0/6	2/6	0/4	0/2
	Contact	0/2	0/2	0/2	0/2	0/2	0/2	1/2	1/2	0/1	1/1
II	32669	0/6	0/6	1/6	1/6	0/6	0/6	0/6	0/6	0/4	0/2
	Contact	0/2	0/2	0/2	0/2	0/2	0/2	0/2	0/2	0/1	0/1
III	95002	0/6	0/6	2/6	0/6	0/6	1/6	1/6	2/6	0/4	0/2
	Contact	0/2	0/2	0/2	0/2	0/2	0/2	0/2	0/2	0/1	0/1
IV	95589	0/6	0/6	0/6	0/6	1/6	0/6	1/6	1/6	0/4	0/2
	Contact	0/2	0/2	0/2	1/2	1/2	0/2	1/2	0/2	0/1	0/1
V	32527	0/6	0/6	0/6	0/6	2/6	1/6	1/6	0/6	0/4	0/2
	Contact	0/2	0/2	0/2	0/2	0/2	0/2	1/2	1/2	1/1	0/1

^a Number of positive pigs / Total number of pigs

Table 3.

RT-PCR results for detection of viral antigen from blood samples of five pig groups during the course of intranasal infection

Group	Inoculated virus	Days post inoculation
Group	Inoculated virus	-4	0	1	2	3	4	5	7	9	11
I	KD26-1	0/6^a	0/6	0/6	0/6	1/6	1/6	1/6	0/6	0/4	0/2
	Contact	0/2	0/2	0/2	0/2	0/2	0/2	1/2	0/2	0/1	0/1
II	32669	0/6	0/6	0/6	0/6	0/6	1/6	1/6	1/6	0/4	0/2
	Contact	0/2	0/2	0/2	0/2	0/2	0/2	0/2	0/2	0/1	0/1
III	95002	0/6	0/6	0/6	0/6	0/6	0/6	1/6	0/6	0/4	0/2
	Contact	0/2	0/2	0/2	0/2	0/2	1/2	0/2	0/2	0/1	0/1
IV	95589	0/6	0/6	0/6	0/6	0/6	2/6	2/6	0/6	0/4	0/2
	Contact	0/2	0/2	0/2	0/2	0/2	0/2	0/2	0/2	0/1	0/1
V	32527	0/6	0/6	0/6	0/6	0/6	0/6	0/6	0/6	0/4	0/2
	Contact	0/2	0/2	0/2	0/2	0/2	0/2	0/2	1/2	0/1	0/1

^a Number of positive pigs / Total number of pigs

Table 4.

RT-PCR detection of viral antigen from tissues samples of five pig groups at 7, 9, and 11 days PI

Group	Inoculated virus	7 dpi^a	9 dpi	11 dpi
I	KD26-1	-^b	T^c, B^d, L^e	T, B, L
	Contact	–	NP^f	–
II	32669	T, 2B^g, L	L	L
	Contact	T	NP	–
III	95002	–	–	–
	Contact	–	NP	–
IV	95589	–	T	–
	Contact	–	NP	–
V	32527	–	–	T
	Contact	–	NP	–

^a dpi: days post inoculation,

^b –: negative in tissues

^c T: positive in tonsil

^d B: positive in bronchial lymph node

^e L: positive in lung

^f NP: not performed

^g 2B: Two pigs were positive in bronchial lymph node.

Table 5.

Summary of viral antigen positive pigs (%) in thirty 100-day-old pigs inoculated intranasally with BVDV type 1 and 2

Virus type	Antigen positive samples			No. of pigs tested
Virus type	Nasal swabs	Blood	Tissues	No. of pigs tested
Type 1	10 (42)	9 (38)	8 (33)	24 (100)
Type 2	4 (67)	1 (17)	0 (0)	6 (100)

Table 6.

RT-PCR results for detection of viral antigen from organs of euthanized pigs after intramuscular inoculation with KD26-1 (10^7.5 TCID₅₀/head)

Organ	KD26-1 inoculated pigs					Contact pigs
Organ	3 d^a	7 d	14 d	21 d	28 d	3 d	7 d	14 d	21 d	28 d
Tonsil	1/2^b	1/2	1/2	0/2	0/2	0/1	0/1	0/1	0/1	0/1
Lung	0/2	0/2	0/2	0/2	0/2	0/1	0/1	0/1	0/1	0/1
Heart	0/2	0/2	0/2	0/2	0/2	0/1	0/1	0/1	0/1	0/1
Spleen	0/2	1/2	0/2	0/2	0/2	0/1	0/1	0/1	0/1	0/1
Liver	0/2	0/2	0/2	0/2	0/2	0/1	0/1	0/1	0/1	0/1
Kidney	0/2	0/2	0/2	0/2	0/2	0/1	0/1	0/1	0/1	0/1
Mesenteric LN	0/2	0/2	0/2	0/2	0/2	0/1	0/1	0/1	0/1	0/1
Ileum	0/2	0/2	0/2	0/2	0/2	0/1	0/1	0/1	0/1	0/1
Caecum	0/2	0/2	0/2	0/2	0/2	0/1	0/1	0/1	0/1	0/1
Inguinal LN	0/2	0/2	1/2	0/2	0/2	0/1	0/1	0/1	0/1	0/1
Brain	1/2	0/2	0/2	0/2	0/2	0/1	0/1	0/1	0/1	0/1

^a d: days post-inoculation

^b Number of pigs positive to viral antigen / Number of pigs tested

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