NOTE Virology
Field Distribution of END Phenomenon-Negative Bovine Viral Diarrhea Virus Kaoru NISHINE1), Hiroshi AOKI1)*, Yoshihiro SAKODA 2) and Akio FUKUSHO1) 1)School
of Veterinary Nursing and Technology, Faculty of Veterinary Science, Nippon Veterinary and Life Science University, 1–7–1 Kyonan-cho, Musashino, Tokyo 180-8602, Japan 2)Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, North 18, West 9, Kita-ku, Sapporo 060-0818, Japan (Received 3 May 2014/Accepted 8 August 2014/Published online in J-STAGE 2 September 2014) Field isolates of BVDV which do not show the exaltation of Newcastle disease virus (END) phenomenon (END–) are rarely reported. In this study, 45 BVDV field isolates from cattle in Hokkaido prefecture in Japan were analyzed by the reverse plaque formation method, the END method and observation of cytopathic effects. END– virus was detected in 34 of 45 isolates (75.6%), although 35 of 45 field isolates contained END phenomenon positive virus as the predominant virus population. We propose that END– viruses are widely distributed in the field and that it is possible that the mixture of biologically distinct BVDV correlates with the appearance of disease in infected animals. KEY WORDS: bovine viral diarrhea virus, END phenomenon, pestivirus, quasispecies, reverse plaque formation ABSTRACT.
doi: 10.1292/jvms.14-0220; J. Vet. Med. Sci. 76(12): 1635–1639, 2014
Bovine viral diarrhea virus (BVDV) is an enveloped positive-stranded RNA virus that is classified in the Pestivirus genus within the family Flaviviridae. Infection with BVDV leads to significant economic losses for cattle producers worldwide [8, 28]. Generally, acute BVDV infections cause transient fever and diarrhea, nasal discharge and reduced milk yield, from which animals naturally recover. However, there are also cases where infected cattle show diverse symptoms ranging from clinically inapparent to severe [1, 3]. Moreover, transplacental infection can result in abortion, significant fetal abnormalities or the birth of persistently infected (PI) calves [21]. PI calves continuously excrete large amounts of virus throughout their lives, showing few symptoms, and are a continual source of infection in a herd. Furthermore, they are at risk of developing fatal mucosal disease (MD) [2]. BVDV is classified into cytopathogenic (CP) and noncytopathogenic (NCP) biotypes on the basis of morphological changes in virus-infected cultured cells. Most BVDV field strains are NCP [7], and only NCP virus can establish a persistent infection [5, 9]. A pair of closely related NCP and CP viruses can be isolated from animals that have developed MD. Therefore, it is considered that the appearance of CP virus in a PI animal strongly correlates with the progression to development of MD [4]. Moreover, NCP BVDV and classical swine fever virus (CSFV) can be further divided into two biotypes, one of which shows the exaltation of New*Correspondence to: Aoki, H., School of Veterinary Nursing and Technology, Faculty of Veterinary Science, Nippon Veterinary and Life Science University, 1–7–1 Kyonan-cho, Musashino, Tokyo 180-8602, Japan. e-mail: [email protected] ©2014 The Japanese Society of Veterinary Science This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives (by-nc-nd) License .
castle disease virus phenomenon (END+) [10, 11, 15] and the other which does not (END–) but which interferes with vesicular stomatitis virus (VSV) and western equine encephalitis virus [6, 12, 18, 26]. However, END– virus has been rarely recognized, because most have been isolated from a few virus strains which have been passaged in vitro and are detected at a lower ratio than END+ phenotypes. Therefore, the epidemiological significance of END– virus in the field is unknown, and it is unclear whether END– virus plays a part in the variation in symptoms of BVDV during infection. Accordingly, we formulated a hypothesis that the ratio of biologically distinct viruses present as quasispecies within the same isolate can influence the clinical condition of an infected host. The purpose of this study was, as an initial step, to detect and quantify BVDV quasispecies in field isolates. Forty-five BVDV isolates were collected between 2006 and 2009 at three livestock hygiene service centers in Hokkaido prefecture. Epidemiological data about the viral genotype, host and disease status were based on the records collected by the livestock hygiene service centers (Table 1). Although the passage history of each isolate in the livestock hygiene center was unclear, all isolates were propagated in bovine testicular (BT) cells in our laboratory. The supernatants were collected as virus suspensions and used for this study. BT cells prepared from the testes of a BVDV-free calf were grown as monolayers in Eagle’s minimum essential medium (MEM) (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% inactivated fetal bovine serum (FBS) and 0.1125% NaHCO3, 2 mM L-glutamine and antibiotics. FBS was confirmed as being free from pestiviruses and BVDV antibodies by quality control tests [14]. The Miyadera strain of Newcastle disease virus (NDV) and the New Jersey serotype of VSV were used as challenge viruses in the END and reverse plaque formation (RPF) methods, respectively. CP virus and END+ viruses in field isolates were quantified by the END method following observation
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K. NISHINE, H. AOKI, Y. SAKODA AND A. FUKUSHO
Table 1. Profiles of BVDV isolates examined in this research Host Isolates KM-2 KM-3 KM-4 KM-5 KM-6 KM-7 KM-8 KM-9 KM-10 KM-11 KM-12 KM-13 KM-14 KM-15 NM-1 NM-2 NM-3 NM-4 NM-5 NM-6 NM-7 NM-8 NM-9 NM-10 NM-11 AB-1 AB-2 AB-3 AB-4 AB-5 AB-6 AB-7 AB-8 AB-9 AB-10 AB-11 AB-12 AB-13 AB-14 AB-15 AB-16 AB-17 AB-18 AB-19 AB-20
Breed
Sex
Age (months)
HS HS HS HS HS HS HS HS HS HS HS HS HS CB HS CB HS HS HS HS HS HS HS HS HS HS HS HS HS HS HS HS HS HS HS Unknown HS HS HS HS HS HS HS HS HS
F F Unknown F F F F F F F F F F Unknown F M F F F F F F F F F Unknown F F F F F F F F F Unknown F F F F F F F F F
1.4 19 Unknown 7.9 10.7 39.2 0.9 18.3 15.3 24.3 3.8 3 44.6 0.1 17.6 0.8 20.6 23.9 23.6 22.9 22.8 6.1 9.9 9.7 19.3 Unknown 11.8 15.3 27.2 4.3 5.1 5.7 10.4 0.1 0.1 Unknown 14.5 19.2 8.3 0.3 1.8 6.2 23.6 3.2 42.5
BVDV Disease status
Genotype
Biotype
PI PI sPI sPI PI PI sPI PI PI sPI sPI sPI PI Abnormal labor Unknown Death Death PI PI PI PI AI PI PI PI Aborted fetus sPI sPI sPI PI sPI sPI sPI sPI sPI Aborted fetus PI PI PI sPI sPI PI PI PI PI
1b 1b 1b 2a 1c 1c 1b 1b 1b 2a 1a 1a 1a 1b 1b 1a 1b 1b 1b 1b 1b 1a 2a 2a 2a 2a 2a 2a 2a 1b 1b 1b 1b 2a 2a 1b 1b 1b 1c 1c 1c 1c 1a 1c 1c
NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP CP NCP NCP
END+ log10 (0.69TCID50)/ml
Field Distribution of END Phenomenon-Negative Bovine Viral Diarrhea Virus Kaoru NISHINE1), Hiroshi AOKI1)*, Yoshihiro SAKODA 2) and Akio FUKUSHO1) 1)School
of Veterinary Nursing and Technology, Faculty of Veterinary Science, Nippon Veterinary and Life Science University, 1–7–1 Kyonan-cho, Musashino, Tokyo 180-8602, Japan 2)Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, North 18, West 9, Kita-ku, Sapporo 060-0818, Japan (Received 3 May 2014/Accepted 8 August 2014/Published online in J-STAGE 2 September 2014) Field isolates of BVDV which do not show the exaltation of Newcastle disease virus (END) phenomenon (END–) are rarely reported. In this study, 45 BVDV field isolates from cattle in Hokkaido prefecture in Japan were analyzed by the reverse plaque formation method, the END method and observation of cytopathic effects. END– virus was detected in 34 of 45 isolates (75.6%), although 35 of 45 field isolates contained END phenomenon positive virus as the predominant virus population. We propose that END– viruses are widely distributed in the field and that it is possible that the mixture of biologically distinct BVDV correlates with the appearance of disease in infected animals. KEY WORDS: bovine viral diarrhea virus, END phenomenon, pestivirus, quasispecies, reverse plaque formation ABSTRACT.
doi: 10.1292/jvms.14-0220; J. Vet. Med. Sci. 76(12): 1635–1639, 2014
Bovine viral diarrhea virus (BVDV) is an enveloped positive-stranded RNA virus that is classified in the Pestivirus genus within the family Flaviviridae. Infection with BVDV leads to significant economic losses for cattle producers worldwide [8, 28]. Generally, acute BVDV infections cause transient fever and diarrhea, nasal discharge and reduced milk yield, from which animals naturally recover. However, there are also cases where infected cattle show diverse symptoms ranging from clinically inapparent to severe [1, 3]. Moreover, transplacental infection can result in abortion, significant fetal abnormalities or the birth of persistently infected (PI) calves [21]. PI calves continuously excrete large amounts of virus throughout their lives, showing few symptoms, and are a continual source of infection in a herd. Furthermore, they are at risk of developing fatal mucosal disease (MD) [2]. BVDV is classified into cytopathogenic (CP) and noncytopathogenic (NCP) biotypes on the basis of morphological changes in virus-infected cultured cells. Most BVDV field strains are NCP [7], and only NCP virus can establish a persistent infection [5, 9]. A pair of closely related NCP and CP viruses can be isolated from animals that have developed MD. Therefore, it is considered that the appearance of CP virus in a PI animal strongly correlates with the progression to development of MD [4]. Moreover, NCP BVDV and classical swine fever virus (CSFV) can be further divided into two biotypes, one of which shows the exaltation of New*Correspondence to: Aoki, H., School of Veterinary Nursing and Technology, Faculty of Veterinary Science, Nippon Veterinary and Life Science University, 1–7–1 Kyonan-cho, Musashino, Tokyo 180-8602, Japan. e-mail: [email protected] ©2014 The Japanese Society of Veterinary Science This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives (by-nc-nd) License .
castle disease virus phenomenon (END+) [10, 11, 15] and the other which does not (END–) but which interferes with vesicular stomatitis virus (VSV) and western equine encephalitis virus [6, 12, 18, 26]. However, END– virus has been rarely recognized, because most have been isolated from a few virus strains which have been passaged in vitro and are detected at a lower ratio than END+ phenotypes. Therefore, the epidemiological significance of END– virus in the field is unknown, and it is unclear whether END– virus plays a part in the variation in symptoms of BVDV during infection. Accordingly, we formulated a hypothesis that the ratio of biologically distinct viruses present as quasispecies within the same isolate can influence the clinical condition of an infected host. The purpose of this study was, as an initial step, to detect and quantify BVDV quasispecies in field isolates. Forty-five BVDV isolates were collected between 2006 and 2009 at three livestock hygiene service centers in Hokkaido prefecture. Epidemiological data about the viral genotype, host and disease status were based on the records collected by the livestock hygiene service centers (Table 1). Although the passage history of each isolate in the livestock hygiene center was unclear, all isolates were propagated in bovine testicular (BT) cells in our laboratory. The supernatants were collected as virus suspensions and used for this study. BT cells prepared from the testes of a BVDV-free calf were grown as monolayers in Eagle’s minimum essential medium (MEM) (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% inactivated fetal bovine serum (FBS) and 0.1125% NaHCO3, 2 mM L-glutamine and antibiotics. FBS was confirmed as being free from pestiviruses and BVDV antibodies by quality control tests [14]. The Miyadera strain of Newcastle disease virus (NDV) and the New Jersey serotype of VSV were used as challenge viruses in the END and reverse plaque formation (RPF) methods, respectively. CP virus and END+ viruses in field isolates were quantified by the END method following observation
1636
K. NISHINE, H. AOKI, Y. SAKODA AND A. FUKUSHO
Table 1. Profiles of BVDV isolates examined in this research Host Isolates KM-2 KM-3 KM-4 KM-5 KM-6 KM-7 KM-8 KM-9 KM-10 KM-11 KM-12 KM-13 KM-14 KM-15 NM-1 NM-2 NM-3 NM-4 NM-5 NM-6 NM-7 NM-8 NM-9 NM-10 NM-11 AB-1 AB-2 AB-3 AB-4 AB-5 AB-6 AB-7 AB-8 AB-9 AB-10 AB-11 AB-12 AB-13 AB-14 AB-15 AB-16 AB-17 AB-18 AB-19 AB-20
Breed
Sex
Age (months)
HS HS HS HS HS HS HS HS HS HS HS HS HS CB HS CB HS HS HS HS HS HS HS HS HS HS HS HS HS HS HS HS HS HS HS Unknown HS HS HS HS HS HS HS HS HS
F F Unknown F F F F F F F F F F Unknown F M F F F F F F F F F Unknown F F F F F F F F F Unknown F F F F F F F F F
1.4 19 Unknown 7.9 10.7 39.2 0.9 18.3 15.3 24.3 3.8 3 44.6 0.1 17.6 0.8 20.6 23.9 23.6 22.9 22.8 6.1 9.9 9.7 19.3 Unknown 11.8 15.3 27.2 4.3 5.1 5.7 10.4 0.1 0.1 Unknown 14.5 19.2 8.3 0.3 1.8 6.2 23.6 3.2 42.5
BVDV Disease status
Genotype
Biotype
PI PI sPI sPI PI PI sPI PI PI sPI sPI sPI PI Abnormal labor Unknown Death Death PI PI PI PI AI PI PI PI Aborted fetus sPI sPI sPI PI sPI sPI sPI sPI sPI Aborted fetus PI PI PI sPI sPI PI PI PI PI
1b 1b 1b 2a 1c 1c 1b 1b 1b 2a 1a 1a 1a 1b 1b 1a 1b 1b 1b 1b 1b 1a 2a 2a 2a 2a 2a 2a 2a 1b 1b 1b 1b 2a 2a 1b 1b 1b 1c 1c 1c 1c 1a 1c 1c
NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP NCP CP NCP NCP
END+ log10 (0.69TCID50)/ml
