Arch Virol (2015) 160:1565–1571 DOI 10.1007/s00705-015-2412-4
BRIEF REPORT
Experimental infection of mice with bovine viral diarrhea virus Giyong Seong1 • Jae-Ku Oem2 • Kyung-Hyun Lee2 Kyoung-Seong Choi1
•
Received: 22 June 2014 / Accepted: 29 March 2015 / Published online: 9 April 2015 Springer-Verlag Wien 2015
Abstract The objective of this study was to test the ability of bovine viral diarrhea virus (BVDV) to infect mice. Two mice each were either mock infected or inoculated with one of three BVDV strains by the intraperitoneal (IP) (n = 8) or intranasal (IN) (n = 8) route. All mice were euthanized at day 7 postinfection (p.i.). None of the infected mice exhibited any clinical signs of illness; however, the tissues harvested after BVDV challenge showed significant histopathological changes. Blood samples from five mice that were injected IP and one mouse that was inoculated IN were positive for BVDV by reverse transcription polymerase chain reaction (RT-PCR). Immunohistochemistry (IHC) was used to assess the presence of viral antigen in the organs of mice infected with three BVDV strains. In IP-injected mice, BVDV antigen was detected in the spleen (5/6), mesenteric lymph nodes (4/6), lymphatic tissue of the lung (3/6), lung (1/6), and stomach (1/6) of the infected mice; however, it was not detected in the liver (0/6) or kidney (0/6). In IN-inoculated mice, BVDV antigen was detected in the lung and mesenteric lymph nodes of one BVDV-infected mouse but was not detected in other tissues. The results of this study suggest that the spleen is the most reliable tissue for BVDV antigen detection using IHC in the IP-injected group. Our
G. Seong, J.-K. Oem, and K.-H. Lee contributed equally to this work. & Kyoung-Seong Choi [email protected] 1
Department of Animal Biotechnology, College of Ecology and Environmental Science, Kyungpook National University, Sangju 742-711, Republic of Korea
2
Animal Disease Diagnostic Division, Animal and Plant Quarantine Agency, Anyang 430-824, Republic of Korea
study demonstrates that mice can be infected by BVDV. This is the first report of BVDV infection in mice. Keywords Bovine viral diarrhea virus Mouse Intraperitoneal Intranasal Immunohistochemistry Bovine viral diarrhea virus (BVDV) is an important pathogen that can affect cattle productivity and lead to substantial economic losses in the livestock industry worldwide. BVDV is a single-stranded RNA virus that belongs to the family Flaviviridae, genus Pestivirus, along with border disease virus (BDV) and classical swine fever virus (CSFV). BVDV is divided into two distinct species, Bovine viral diarrhea virus 1 and Bovine viral diarrhea virus 2. Both species include viruses of two biotypes, cytopathic (cp) and non-cytopathic (ncp), which vary according to their activity in cultured cells [1]. BVDV infection is associated with diarrhea, respiratory disease, hemorrhagic syndrome, abortion, weak calf syndrome, teratogenic effects on fetuses, and mucosal disease (MD) [2–4]. BVDV is known to infect a wide range of wild and domesticated ruminant and porcine species [5–9]. In a previous study, a wide range of non-artiodactyls, including horses, cats, dogs, several small laboratory animals, and embryonated chicken eggs, were inoculated with the virus in order to determine the host range [10]. The authors reported that, of the species investigated, BVDV could only be propagated in rabbits [10]. Recent studies have also shown that BVDV infection occurs in rabbits [11, 12]; however, none of the rabbits that were inoculated intravenously showed any signs of illness or any BVDV infection lesions. However, calves inoculated with a spleen homogenate from these rabbits, by carrying out three alternating passages between the calves and rabbits and by
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75 successive passages within rabbits using a spleen cell suspension, developed typical signs of transient BVDV infection [10]. Whether rabbits serve as a natural reservoir for BVDV infection is not yet clear. Thus, to investigate whether mice could be infected with BVDV and to evaluate the outcome of experimental BVDV infection in mice, in this study, we inoculated mice with three different BVDV strains by two different routes: intraperitoneal (IP) injection and intranasal (IN) inoculation. Specific-pathogen-free BALB/c mice (6-8 weeks old) were purchased from Daehan Experimental Animal Center (Daejeon, Korea). All animals were maintained under pathogen-free conditions and handled in strict accordance with the guidelines and protocols approved for these experiments by the Kyungpook National University Animal Care and Use Committee. All experiments were repeated twice to confirm reproducibility. A total of 16 mice received IP injection, and 16 mice received IN inoculation. The BVDV1 and BVDV2 strains used in this study were isolated in the Republic of Korea. BVDV1 isolate (11Q472) was obtained from a 2-year-old Korean indigenous cow presenting with emaciation, inappetence, and diarrhea. An ear notch test and consecutive blood samples collected at 3-week intervals showed this animal to be persistently infected (PI). BVDV2 isolate (11F011) was obtained from a 2-year-old Korean indigenous steer with downer cow syndrome. The steer’s brain tissue tested positive for BVDV by reverse transcription polymerase chain reaction (RT-PCR). The BVDV1 and BVDV2 strains were confirmed as BVDV1b and BVDV2a, respectively, by sequencing analysis. Both viruses were classified as ncp based on their propagation in cultured Madin Darby bovine kidney (MDBK) cells and were used for BVDV inoculation. A cp BVDV1 strain, NADL, obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) was cultured in MDBK cells and then used for BVDV inoculation. Each virus was titrated in MDBK cells, and a multiplicity of infection (MOI) of 0.01 was used for subsequent passages. Two mice each were assigned to the ncp BVDV1-, ncp BVDV2-, cp BVDV1-infected, and mock-infected groups. A total of eight mice were used for each experiment. Two groups of eight mice were administered BVDV via two different routes (either IP or IN). The challenge was performed by IP injection or IN inoculation of 0.4 mL of tissue culture fluid containing 5 9 105 TCID50 of each virus. IN inoculation of cattle with this virus inoculum induced a transient infection [13]. Mock-infected mice were IP- or IN-administered 0.4 mL of tissue culture medium (Minimum Essential Medium; Life Technologies Corp.; Carlsbad, CA, USA).
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At day 7 postinfection (p.i.), when active viral replication is expected in BVDV-infected animals, all mice (ncp BVDV1-, ncp BVDV2-, cp BVDV1-infected, and mockinfected) were euthanized with CO2 gas to collect blood and tissues. The spleen, liver, kidney, lung, mesenteric lymph nodes, and stomach were removed at necropsy and fixed in 10 % buffered formalin. The tissues were routinely processed and embedded in paraffin for hematoxylin and eosin (H&E) staining. The severity of the histopathological changes in BVDV-infected mice was investigated. All evaluations were performed by investigators blinded to the treatment and were conducted separately by two pathologists. Histopathological severity was scored such that higher numbers reflected a higher degree of tissue inflammation, injury, and necrosis. Severity was determined by (i) the extent of necrotic cells and (ii) the number and relative area of inflammatory cell infiltration. RNA was extracted from blood using a PureLink Total RNA Blood Kit (Invitrogen, Carlsbad, CA, USA). RT-PCR was performed in a one-tube system using the pan-pestivirus primer pair V324-326 (INtRON Biotechnology, Inc.; Daejeon, Korea); this system amplifies a portion of the 50 -untranslated region (UTR) of pestivirus genomes [14]. The predicted size of the amplified PCR product was 297 bp. Immunohistochemical staining was performed on formalin-fixed paraffin-embedded blocks of mouse tissues, including the spleen, liver, kidney, lung, mesenteric lymph nodes, and stomach. Briefly, 5-lm-thick paraffin-embedded tissue sections were deparaffinized in xylene, hydrated through a graded alcohol series, and then washed in distilled water. To enhance antigen retrieval, the tissue sections were soaked in heat-induced sodium citrate buffer (pH 6.0) for 30 min, cooled to room temperature, and then incubated with 3 % H2O2 in methanol for 10 min to block endogenous peroxidase activity. After blocking, the primary monoclonal anti-BVDV antibody (Ab) 15C5 (Syracuse Bioanalytical; Ithaca, NY, USA) was incubated with the tissue sections overnight. The next day, tissue sections were stained with biotinylated anti-mouse IgG (Vector Laboratories, Inc.; Burlingame, CA, USA) for 1 h at room temperature, washed, and incubated with VECTASTAIN ABC Reagent (Vector) for 30 min. After washing, sections were reacted with peroxidase substrate solution (Vector), rinsed, counterstained, mounted, and photographed. Negative control slides were prepared using isotype-matched IgG at the same dilution as the primary antibody. In this study, mice were infected with BVDV by IP injection or IN inoculation. As anticipated, none of the mice exhibited clinical signs of illness. The presence or absence of viral RNA in whole blood samples obtained at day 7 p.i. was determined by RT-PCR. In the IP-injected group, blood samples from five animals were BVDV
The cp BVDV1 strain used in the study is NADL
*RT-PCR for viral RNA detection was performed using whole blood
Mock 2
‘‘?’’, positive, ‘‘-’’, negative
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-
-
Mock 1
-
-
-
? Ncp BVDV2 2
-
-
-
-
-
-
?
? -
-
-
-
? -
? Ncp BVDV2 1
-
?
?
-
? ? ? -
?
Ncp BVDV1 2
-
? ? ? Ncp BVDV1 1
-
-
-
-
? ?
-
-
? -
-
-
-
? ?
? ? Cp BVDV1 2
RT-PCR* Kidney Liver Stomach Mesenteric lymph node Lymphatic tissue of lung Lung
? Cp BVDV1 1
RT-PCR* Kidney Liver Mesenteric Lymph node Spleen Spleen
Lung
IN-inoculated group IP-injected group Animal
positive (Table 1), whilst in the IN-inoculated group, only one mouse infected with cp BVDV1 was shown to be BVDV positive by RT-PCR (Table 1). Viral RNA was not detected in the mock-infected animals. Immunohistochemical (IHC) analysis of each BVDVinfected mouse was evaluated at day 7 p.i. The distribution of viral antigen in different tissues of the mice is summarized in Table 1. No BVDV antigen was detected in any tissue samples of mock-infected mice. In the IP-injected group, BVDV antigen was detected in the spleen (5/6, 83.3 %) of all of the mice in this group, except in one ncp BVDV1-infected mouse. BVDV antigen was also detected in the mesenteric lymph nodes (4/6, 67 %), lymphatic tissue (3/6, 50 %) of the lung, lung of one mouse infected with ncp BVDV2 (1/6, 16.7 %), and stomach (1/6, 16.7 %) of one ncp BVDV1-infected mouse. In the spleen, BVDV antigen was detected in the lymphocytes (Fig. 1a), and in the mesenteric lymph nodes, BVDV antigen was detected in the lymphocytes of necrotic lesions and in the cortex (Fig. 1b). In the lung, BVDV antigen was detected in the alveolar macrophages (Fig. 1c). In the stomach, BVDV antigen was present in the stratified squamous epithelial cells (Fig. 1d). In the IN-inoculated group, BVDV antigen was detected in the lung (1/6, 16.7 %) and mesenteric lymph node (1/6, 16.7 %) of one cp BVDV1-infected mouse (Table 1). BVDV antigen was present in the bronchial epithelial cells of the lung (Fig. 1e) and the lymphocytes of the mesenteric lymph nodes (Fig. 1f). In contrast to the IP-injected group, BVDV antigen was not present in the spleen of IN-inoculated mice (Table 1). The liver or kidney of mice infected by IP injection or IN inoculation both demonstrated negative results for BVDV antigen. Histopathological examinations were performed on the liver, lung, kidney, spleen, and mesenteric lymph nodes of infected mice at day 7 p.i. All animals infected by IP injection showed mild to severe histopathological lesions, whereas mock-infected mice showed no lesions (Table 2). Histopathological changes were only observed in one mouse for the IN-inoculated group (Table 2). The livers of ncp BVDV1-infected mice showed small localized accumulations of macrophages and neutrophils, without apoptotic cells in the portal triad (Fig. 2a), and the livers of cp BVDV1-infected mice showed moderate hepatic lesions. In contrast, the livers of ncp BVDV2-infected mice showed more severe inflammatory lesions and tissue injury, including inflammatory cell infiltrates throughout the periportal regions. In addition, a greater degree of focal necrosis and large numbers of apoptotic cells were observed in ncp BVDV2-infected mice than in ncp BVDV1infected mice (Fig 2b). In ncp BVDV2-infected mice, a significant number of inflammatory cells were found in the histopathological hepatic lesions, including numerous
1567 Table 1 Summary of BVDV antigen detection in mice at day 7 pi after infection by intraperitoneal (IP) injection or intranasal (IN) inoculation. Results are representative of two independent experiments
Experimental infection of mice with…
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Fig. 1 Detection of BVDV antigen by immunohistochemistry at day 7 p.i. after IP injection or IN inoculation. Viral antigen was detected in the lymphocytes of the spleen (a), mesenteric lymph nodes (b), alveolar macrophages of the lymphatic tissue in the lung (c), stratified
Table 2 Summary of the histopathological findings in BVDV-infected mice. Results are representative of two independent experiments
Animal
squamous epithelial cells (d), bronchial epithelial cells of the lung (e), and the lymphocytes of the mesenteric lymph nodes (f) (original magnification, 2009). a–d, IP injection; e–f, IN injection
IP-injected group
IN-inoculated group
Spleen
Lung
Mesenteric Lymph node
Liver
Kidney
Spleen
Lung
Mesenteric Lymph node
Cp BVDV1 1
?
???
??
??
???
-
-
-
Cp BVDV1 2 Ncp BVDV1 1
? ???
??? ?
?
?? ?
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? -
? -
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Ncp BVDV1 2
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?
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-
-
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Ncp BVDV2 1
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Ncp BVDV2 2
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-
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Mock 1
-
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Mock 2
-
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‘‘-‘‘, no lesion; ‘‘?’’, mild; ‘‘??’’, moderate; ‘‘???’’, severe The cp BVDV strain used in the study is NADL
neutrophil and macrophage infiltrates. The kidneys of cp BVDV1-infected mice exhibited marked atrophy of the glomerulus compared to ncp BVDV1-infected or ncp BVDV2-infected mice (Fig. 3). The most common lesion observed in the lungs was thickening of the alveolar wall,
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which was most significant in cp BVDV1-infected mice (Fig. 4a), whereas the lungs of IN-inoculated mice showed only mild histopathological lesions (Fig. 4b). The spleens of ncp BVDV1-infected mice showed extramedullary hematogenesis (Fig. 5a) and more intense necrosis of the
Experimental infection of mice with…
Fig. 2 Hepatic histopathology at day 7 p.i. after IP injection. (a) The ncp BVDV2-infected mice showed more intense infiltrates around the bile duct, including intense lobular infiltrates and collections of mononuclear cells with abundant neutrophils (black arrows). (b) The
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intensive inflammatory infiltrates were also associated with a greater degree of focal tissue necrosis and apoptosis of hepatocytes (black arrows). (c) Mock-infected mice showed no lesions (H&E; original magnification, 4009)
Fig. 3 Kidney histopathology at day 7 p.i. after IP injection. (a) Compared to ncp BVDV1and ncp BVDV2-infected mice, cp BVDV1-infected mice showed marked atrophy of the glomerulus (black arrows.) (b) A mock-infected mouse showed a normal glomerulus (H&E; original magnification, 2009)
Fig. 4 Lung histopathology at day 7 p.i. after IP injection or IN inoculation. (a) Compared to ncp BVDV1- and ncp BVDV2-infected mice, mice infected with cp BVDV1 by IP injection showed hyperplasia of the alveoli wall (black arrows). (b) An IN-injected
mouse showed interstitial thickening and alveolar macrophage infiltration in the alveolar lumen (blue arrow). (c) Mock-infected mice showed no lesions (H&E, 2009)
lymphocytes within the lymphatic nodule (Fig. 5b) compared to cp BVDV1 or ncp BVDV2-infected mice. Although BVDV antigen was not detected in one mouse with ncp BVDV1 infection, the histopathological lesions showed a more significant change in the spleen of this mouse than in the other ncp BVDV1-infected mice. In the IP-injected group, the histological changes in the mesenteric lymph node differed somewhat among the mice. IPinjected mice showed lympholysis and moderate to severe
lymphoid depletion (Fig. 6a), while IN-inoculated mice showed moderate lymphoid depletion (Fig. 6b). Viral RNA was detected in the whole blood of five out of six IP-injected mice, whereas it was detected in only one out of six mice in the IN-inoculated group (Table 1). This suggests that the reason for the lower number of viremic mice in the IN-inoculated group might be the restriction of viral entry via this mode of administration. Therefore, further studies are required to evaluate viremia at several
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Fig. 5 Splenic histopathology at 7 days p.i. after IP injection. (a) The ncp BVDV1-infected mice showed more extramedullary hematogenesis (black arrows) and more intense lymphocyte necrosis within
the lymphatic nodule (black arrows). (b) Mock-infected mice showed no lesions (H&E; original magnification, 4009)
Fig. 6 Histopathology of mesenteric lymph nodes at 7 day p.i. after IP injection or IN inoculation. (a) Mice in the IP-injected group showed lympholysis and moderate to severe lymphoid depletion
(black arrow). (b) The IN-inoculated mouse showed moderate lymphoid depletion in the cortex (blue arrow). (c) A mock-infected mouse showed no lesions (H&E; original magnification, 4009)
different time points, using a larger number of infected mice. In the IP-injected group, BVDV antigen was most commonly detected in the spleen and was detected in the mesenteric lymph nodes of four out of six infected mice and in the lymphatic tissue of the lung in cp BVDV1 and ncp BVDV2-infected mice, but not in ncp BVDV1-infected mice (Table 1). In addition, BVDV antigen was detected in the lung and stomach of only one infected mouse (Table 1). In the IN-inoculated group, BVDV antigen was detected only in the lung and mesenteric lymph nodes of one cp BVDV1-infected mouse (Table 1). Interestingly, the presence of BVDV antigen in the stratified squamous epithelial cells of the stomach and the bronchial epithelial cells of the lung may be explained by the preferred location for antigen deposition in the mouse model of BVDV infection. These results show evidence of BVDV infection in mice after IP injection and IN inoculation. Our findings confirmed those of previous studies, which showed that natural infection of non-artiodactyls was accompanied with
changes in histopathology [11]. Bachofen et al. showed that after exposure of rabbits to BVDV, their lymphoid organs had histological changes typical of transient infection with a pestivirus and were BVDV positive by RT-PCR [11]. Similar to other studies of rabbits, our results showed mild to severe histopathological changes in all tissues of BVDV-infected mice that were examined (Table 2). The reason for the differences in these histopathological changes of the various BVDV-infected mice is not clear. However, it may be postulated to be associated with differences in the virulence and tissue tropism of the viral strains used in this study. The correlation between the distribution of viral antigen and histopathological lesions was not as clear. For example, despite the significant histopathological changes in the liver and kidney, no viral antigen was observed. The reason for these apparent differences in the distribution of viral antigen in mice is not known; however, it may be hypothesized to be related to low viral replication in mice and the restriction of replication to small clusters of cells in
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Experimental infection of mice with…
certain organ systems. Therefore, we can speculate that the reason for the variation in the distribution of viral antigen observed in this study may be attributable to the susceptibility of infected tissue to the virus, or to being the result of only transient BVDV infection. Taken together, these results suggest that the spleen is the most reliable tissue for detection of BVDV antigen using IHC in mice. Baker et al. showed that mice injected subcutaneously with spleen homogenate from a BVDV-infected calf did not demonstrate infection [10]. However, this result is not consistent with our observations. This difference may be due to variations in the route of infection, virus dosage, or duration of infection. Our findings demonstrated that mice can be infected by BVDV following IP injection or IN inoculation and that viremia, BVDV antigen, and pathologic findings were detected at day 7 p.i. However, BVDV infection after either IP injection or IN inoculation in mice resulted in considerably different outcomes. While in the IN-inoculated group, viremia did not occur in all infected mice and BVDV antigen was not detected in the spleen, in the IP-injected group, whilst five out of six mice showed viremia and the presence of BVDV antigen, all six mice demonstrated mild to severe histopathological changes. These results suggest that in mice, IN inoculation might restrict virus entry and replication. Thus, further investigations of IN inoculation are necessary. In conclusion, this is the first report of BVDV infection in mice. Our results indicate that mice can be infected with BVDV. Even though none of the infected mice showed clinically apparent disease, the presence of viremia provided evidence that the mice were infected with BVDV. BVDV antigen was primarily detected in the spleen of IPinjected mice by IHC. Histologically, all IP-injected mice demonstrated more severe pathological changes than those in the IN-inoculated groups. Overall, our results provide new information about BVDV infection. Infection was reliably established after IP injection, but not after IN inoculation; only one out of six mice was found to have viremia and the presence of BVDV antigen. To determine the applicability of mice as experimental hosts for BVDV, additional studies are needed in order to understand the pathogenesis and mechanism of BVDV infection in mice.
1571 funded by the Ministry of Education, Science, and Technology (NRF 2012R1A1A3011238).
References 1. Zhang G, Aldridge S, Clarke MC, McCauley JW (1996) Cell death induced by cytopathic bovine viral diarrhoea virus is mediated by apoptosis. J Gen Virol 77:1677–1681 2. Baker JC (1995) The clinical manifestations of bovine viral diarrhea infection. Vet Clin North Am Food Anim Pract 11:425–445 3. Dubovi EJ (1994) Impact of bovine viral diarrhea virus on reproductive performance in cattle. Vet Clin North Am Food Anim Pract 10:503–514 4. Brownlie J, Clarke MC, Howard CJ (1984) Experimental production of fatal mucosal disease in cattle. Vet Rec 114:535–536 5. Bachofen C, Vogt HR, Stalder H, Mathys T, Zanoni R, Hilbe M, Schweizer M, Peterhans E (2013) Persistent infections after natural transmission of bovine viral diarrhoea virus from cattle to goats and among goats. Vet Res 44:32 6. Uttenthal A, Grøndahl C, Hoyer MJ, Houe H, van Maanen C, Rasmussen TB, Larsen LE (2005) Persistent BVDV infection in mousedeer infects calves. Do we know the reservoirs for BVDV? Prev Vet Med 72:87–91 7. Van Campen H, Ridpath J, Williams E, Cavender J, Edwards J, Smith S, Sawyer H (2001) Isolation of bovine viral diarrhea virus from a free-ranging mule deer in Wyoming. J Wildl Dis 37:306–311 8. Becher P, Orlich M, Shannon AD, Horner G, Ko¨nig M, Thiel HJ (1997) Phylogenetic analysis of pestiviruses from domestic and wild ruminants. J Gen Virol 78:1357–1366 9. Terpstra C, Wensvoort G (1997) A congenital persistent infection of bovine virus diarrhoea virus in pigs: clinical, virological and immunological observations. Vet Q 19:97–101 10. Baker JA, York CJ, Gillepsie JH, Mitchell GB (1954) Virus diarrhea in cattle. Am J Vet Res 15:525–531 11. Bachofen C, Grant DM, Willoughby K, Zadoks RN, Dagleish MP, Russell GC (2014) Experimental infection of rabbits with bovine viral diarrhoea virus by a natural route of exposure. Vet Res 45:34 12. Fro¨lich K, Streich WJ (1998) Serologic evidence of bovine viral diarrhea virus in free-ranging rabbits from Germany. J Wildl Dis 34:173–178 13. Seong G, Oem JK, Choi KS (2013) Pathogenetic differences after experimental infection of calves with Korean non-cytopathic BVDV-1 and BVDV-2 isolates. Vet Immunol Immunopathol 156:147–152 14. Vilcek S, Herring AJ, Herring JA, Nettleton PF, Lowings JP, Paton DJ (1994) Pestiviruses isolated from pigs, cattle and sheep can be allocated into at least three genogroups using polymerase chain reaction and restriction endonuclease analysis. Arch Virol 136:309–323
Acknowledgments This work was supported by the Basic Science Research Program of the National Research Foundation of Korea,
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BRIEF REPORT
Experimental infection of mice with bovine viral diarrhea virus Giyong Seong1 • Jae-Ku Oem2 • Kyung-Hyun Lee2 Kyoung-Seong Choi1
•
Received: 22 June 2014 / Accepted: 29 March 2015 / Published online: 9 April 2015 Springer-Verlag Wien 2015
Abstract The objective of this study was to test the ability of bovine viral diarrhea virus (BVDV) to infect mice. Two mice each were either mock infected or inoculated with one of three BVDV strains by the intraperitoneal (IP) (n = 8) or intranasal (IN) (n = 8) route. All mice were euthanized at day 7 postinfection (p.i.). None of the infected mice exhibited any clinical signs of illness; however, the tissues harvested after BVDV challenge showed significant histopathological changes. Blood samples from five mice that were injected IP and one mouse that was inoculated IN were positive for BVDV by reverse transcription polymerase chain reaction (RT-PCR). Immunohistochemistry (IHC) was used to assess the presence of viral antigen in the organs of mice infected with three BVDV strains. In IP-injected mice, BVDV antigen was detected in the spleen (5/6), mesenteric lymph nodes (4/6), lymphatic tissue of the lung (3/6), lung (1/6), and stomach (1/6) of the infected mice; however, it was not detected in the liver (0/6) or kidney (0/6). In IN-inoculated mice, BVDV antigen was detected in the lung and mesenteric lymph nodes of one BVDV-infected mouse but was not detected in other tissues. The results of this study suggest that the spleen is the most reliable tissue for BVDV antigen detection using IHC in the IP-injected group. Our
G. Seong, J.-K. Oem, and K.-H. Lee contributed equally to this work. & Kyoung-Seong Choi [email protected] 1
Department of Animal Biotechnology, College of Ecology and Environmental Science, Kyungpook National University, Sangju 742-711, Republic of Korea
2
Animal Disease Diagnostic Division, Animal and Plant Quarantine Agency, Anyang 430-824, Republic of Korea
study demonstrates that mice can be infected by BVDV. This is the first report of BVDV infection in mice. Keywords Bovine viral diarrhea virus Mouse Intraperitoneal Intranasal Immunohistochemistry Bovine viral diarrhea virus (BVDV) is an important pathogen that can affect cattle productivity and lead to substantial economic losses in the livestock industry worldwide. BVDV is a single-stranded RNA virus that belongs to the family Flaviviridae, genus Pestivirus, along with border disease virus (BDV) and classical swine fever virus (CSFV). BVDV is divided into two distinct species, Bovine viral diarrhea virus 1 and Bovine viral diarrhea virus 2. Both species include viruses of two biotypes, cytopathic (cp) and non-cytopathic (ncp), which vary according to their activity in cultured cells [1]. BVDV infection is associated with diarrhea, respiratory disease, hemorrhagic syndrome, abortion, weak calf syndrome, teratogenic effects on fetuses, and mucosal disease (MD) [2–4]. BVDV is known to infect a wide range of wild and domesticated ruminant and porcine species [5–9]. In a previous study, a wide range of non-artiodactyls, including horses, cats, dogs, several small laboratory animals, and embryonated chicken eggs, were inoculated with the virus in order to determine the host range [10]. The authors reported that, of the species investigated, BVDV could only be propagated in rabbits [10]. Recent studies have also shown that BVDV infection occurs in rabbits [11, 12]; however, none of the rabbits that were inoculated intravenously showed any signs of illness or any BVDV infection lesions. However, calves inoculated with a spleen homogenate from these rabbits, by carrying out three alternating passages between the calves and rabbits and by
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75 successive passages within rabbits using a spleen cell suspension, developed typical signs of transient BVDV infection [10]. Whether rabbits serve as a natural reservoir for BVDV infection is not yet clear. Thus, to investigate whether mice could be infected with BVDV and to evaluate the outcome of experimental BVDV infection in mice, in this study, we inoculated mice with three different BVDV strains by two different routes: intraperitoneal (IP) injection and intranasal (IN) inoculation. Specific-pathogen-free BALB/c mice (6-8 weeks old) were purchased from Daehan Experimental Animal Center (Daejeon, Korea). All animals were maintained under pathogen-free conditions and handled in strict accordance with the guidelines and protocols approved for these experiments by the Kyungpook National University Animal Care and Use Committee. All experiments were repeated twice to confirm reproducibility. A total of 16 mice received IP injection, and 16 mice received IN inoculation. The BVDV1 and BVDV2 strains used in this study were isolated in the Republic of Korea. BVDV1 isolate (11Q472) was obtained from a 2-year-old Korean indigenous cow presenting with emaciation, inappetence, and diarrhea. An ear notch test and consecutive blood samples collected at 3-week intervals showed this animal to be persistently infected (PI). BVDV2 isolate (11F011) was obtained from a 2-year-old Korean indigenous steer with downer cow syndrome. The steer’s brain tissue tested positive for BVDV by reverse transcription polymerase chain reaction (RT-PCR). The BVDV1 and BVDV2 strains were confirmed as BVDV1b and BVDV2a, respectively, by sequencing analysis. Both viruses were classified as ncp based on their propagation in cultured Madin Darby bovine kidney (MDBK) cells and were used for BVDV inoculation. A cp BVDV1 strain, NADL, obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) was cultured in MDBK cells and then used for BVDV inoculation. Each virus was titrated in MDBK cells, and a multiplicity of infection (MOI) of 0.01 was used for subsequent passages. Two mice each were assigned to the ncp BVDV1-, ncp BVDV2-, cp BVDV1-infected, and mock-infected groups. A total of eight mice were used for each experiment. Two groups of eight mice were administered BVDV via two different routes (either IP or IN). The challenge was performed by IP injection or IN inoculation of 0.4 mL of tissue culture fluid containing 5 9 105 TCID50 of each virus. IN inoculation of cattle with this virus inoculum induced a transient infection [13]. Mock-infected mice were IP- or IN-administered 0.4 mL of tissue culture medium (Minimum Essential Medium; Life Technologies Corp.; Carlsbad, CA, USA).
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At day 7 postinfection (p.i.), when active viral replication is expected in BVDV-infected animals, all mice (ncp BVDV1-, ncp BVDV2-, cp BVDV1-infected, and mockinfected) were euthanized with CO2 gas to collect blood and tissues. The spleen, liver, kidney, lung, mesenteric lymph nodes, and stomach were removed at necropsy and fixed in 10 % buffered formalin. The tissues were routinely processed and embedded in paraffin for hematoxylin and eosin (H&E) staining. The severity of the histopathological changes in BVDV-infected mice was investigated. All evaluations were performed by investigators blinded to the treatment and were conducted separately by two pathologists. Histopathological severity was scored such that higher numbers reflected a higher degree of tissue inflammation, injury, and necrosis. Severity was determined by (i) the extent of necrotic cells and (ii) the number and relative area of inflammatory cell infiltration. RNA was extracted from blood using a PureLink Total RNA Blood Kit (Invitrogen, Carlsbad, CA, USA). RT-PCR was performed in a one-tube system using the pan-pestivirus primer pair V324-326 (INtRON Biotechnology, Inc.; Daejeon, Korea); this system amplifies a portion of the 50 -untranslated region (UTR) of pestivirus genomes [14]. The predicted size of the amplified PCR product was 297 bp. Immunohistochemical staining was performed on formalin-fixed paraffin-embedded blocks of mouse tissues, including the spleen, liver, kidney, lung, mesenteric lymph nodes, and stomach. Briefly, 5-lm-thick paraffin-embedded tissue sections were deparaffinized in xylene, hydrated through a graded alcohol series, and then washed in distilled water. To enhance antigen retrieval, the tissue sections were soaked in heat-induced sodium citrate buffer (pH 6.0) for 30 min, cooled to room temperature, and then incubated with 3 % H2O2 in methanol for 10 min to block endogenous peroxidase activity. After blocking, the primary monoclonal anti-BVDV antibody (Ab) 15C5 (Syracuse Bioanalytical; Ithaca, NY, USA) was incubated with the tissue sections overnight. The next day, tissue sections were stained with biotinylated anti-mouse IgG (Vector Laboratories, Inc.; Burlingame, CA, USA) for 1 h at room temperature, washed, and incubated with VECTASTAIN ABC Reagent (Vector) for 30 min. After washing, sections were reacted with peroxidase substrate solution (Vector), rinsed, counterstained, mounted, and photographed. Negative control slides were prepared using isotype-matched IgG at the same dilution as the primary antibody. In this study, mice were infected with BVDV by IP injection or IN inoculation. As anticipated, none of the mice exhibited clinical signs of illness. The presence or absence of viral RNA in whole blood samples obtained at day 7 p.i. was determined by RT-PCR. In the IP-injected group, blood samples from five animals were BVDV
The cp BVDV1 strain used in the study is NADL
*RT-PCR for viral RNA detection was performed using whole blood
Mock 2
‘‘?’’, positive, ‘‘-’’, negative
-
-
-
Mock 1
-
-
-
? Ncp BVDV2 2
-
-
-
-
-
-
?
? -
-
-
-
? -
? Ncp BVDV2 1
-
?
?
-
? ? ? -
?
Ncp BVDV1 2
-
? ? ? Ncp BVDV1 1
-
-
-
-
? ?
-
-
? -
-
-
-
? ?
? ? Cp BVDV1 2
RT-PCR* Kidney Liver Stomach Mesenteric lymph node Lymphatic tissue of lung Lung
? Cp BVDV1 1
RT-PCR* Kidney Liver Mesenteric Lymph node Spleen Spleen
Lung
IN-inoculated group IP-injected group Animal
positive (Table 1), whilst in the IN-inoculated group, only one mouse infected with cp BVDV1 was shown to be BVDV positive by RT-PCR (Table 1). Viral RNA was not detected in the mock-infected animals. Immunohistochemical (IHC) analysis of each BVDVinfected mouse was evaluated at day 7 p.i. The distribution of viral antigen in different tissues of the mice is summarized in Table 1. No BVDV antigen was detected in any tissue samples of mock-infected mice. In the IP-injected group, BVDV antigen was detected in the spleen (5/6, 83.3 %) of all of the mice in this group, except in one ncp BVDV1-infected mouse. BVDV antigen was also detected in the mesenteric lymph nodes (4/6, 67 %), lymphatic tissue (3/6, 50 %) of the lung, lung of one mouse infected with ncp BVDV2 (1/6, 16.7 %), and stomach (1/6, 16.7 %) of one ncp BVDV1-infected mouse. In the spleen, BVDV antigen was detected in the lymphocytes (Fig. 1a), and in the mesenteric lymph nodes, BVDV antigen was detected in the lymphocytes of necrotic lesions and in the cortex (Fig. 1b). In the lung, BVDV antigen was detected in the alveolar macrophages (Fig. 1c). In the stomach, BVDV antigen was present in the stratified squamous epithelial cells (Fig. 1d). In the IN-inoculated group, BVDV antigen was detected in the lung (1/6, 16.7 %) and mesenteric lymph node (1/6, 16.7 %) of one cp BVDV1-infected mouse (Table 1). BVDV antigen was present in the bronchial epithelial cells of the lung (Fig. 1e) and the lymphocytes of the mesenteric lymph nodes (Fig. 1f). In contrast to the IP-injected group, BVDV antigen was not present in the spleen of IN-inoculated mice (Table 1). The liver or kidney of mice infected by IP injection or IN inoculation both demonstrated negative results for BVDV antigen. Histopathological examinations were performed on the liver, lung, kidney, spleen, and mesenteric lymph nodes of infected mice at day 7 p.i. All animals infected by IP injection showed mild to severe histopathological lesions, whereas mock-infected mice showed no lesions (Table 2). Histopathological changes were only observed in one mouse for the IN-inoculated group (Table 2). The livers of ncp BVDV1-infected mice showed small localized accumulations of macrophages and neutrophils, without apoptotic cells in the portal triad (Fig. 2a), and the livers of cp BVDV1-infected mice showed moderate hepatic lesions. In contrast, the livers of ncp BVDV2-infected mice showed more severe inflammatory lesions and tissue injury, including inflammatory cell infiltrates throughout the periportal regions. In addition, a greater degree of focal necrosis and large numbers of apoptotic cells were observed in ncp BVDV2-infected mice than in ncp BVDV1infected mice (Fig 2b). In ncp BVDV2-infected mice, a significant number of inflammatory cells were found in the histopathological hepatic lesions, including numerous
1567 Table 1 Summary of BVDV antigen detection in mice at day 7 pi after infection by intraperitoneal (IP) injection or intranasal (IN) inoculation. Results are representative of two independent experiments
Experimental infection of mice with…
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Fig. 1 Detection of BVDV antigen by immunohistochemistry at day 7 p.i. after IP injection or IN inoculation. Viral antigen was detected in the lymphocytes of the spleen (a), mesenteric lymph nodes (b), alveolar macrophages of the lymphatic tissue in the lung (c), stratified
Table 2 Summary of the histopathological findings in BVDV-infected mice. Results are representative of two independent experiments
Animal
squamous epithelial cells (d), bronchial epithelial cells of the lung (e), and the lymphocytes of the mesenteric lymph nodes (f) (original magnification, 2009). a–d, IP injection; e–f, IN injection
IP-injected group
IN-inoculated group
Spleen
Lung
Mesenteric Lymph node
Liver
Kidney
Spleen
Lung
Mesenteric Lymph node
Cp BVDV1 1
?
???
??
??
???
-
-
-
Cp BVDV1 2 Ncp BVDV1 1
? ???
??? ?
?
?? ?
??? ?
? -
? -
?? -
Ncp BVDV1 2
???
?
???
?
?
-
-
-
Ncp BVDV2 1
??
??
-
???
??
-
-
-
Ncp BVDV2 2
??
??
???
???
??
-
-
-
Mock 1
-
-
-
-
-
-
-
-
Mock 2
-
-
-
-
-
-
-
-
‘‘-‘‘, no lesion; ‘‘?’’, mild; ‘‘??’’, moderate; ‘‘???’’, severe The cp BVDV strain used in the study is NADL
neutrophil and macrophage infiltrates. The kidneys of cp BVDV1-infected mice exhibited marked atrophy of the glomerulus compared to ncp BVDV1-infected or ncp BVDV2-infected mice (Fig. 3). The most common lesion observed in the lungs was thickening of the alveolar wall,
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which was most significant in cp BVDV1-infected mice (Fig. 4a), whereas the lungs of IN-inoculated mice showed only mild histopathological lesions (Fig. 4b). The spleens of ncp BVDV1-infected mice showed extramedullary hematogenesis (Fig. 5a) and more intense necrosis of the
Experimental infection of mice with…
Fig. 2 Hepatic histopathology at day 7 p.i. after IP injection. (a) The ncp BVDV2-infected mice showed more intense infiltrates around the bile duct, including intense lobular infiltrates and collections of mononuclear cells with abundant neutrophils (black arrows). (b) The
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intensive inflammatory infiltrates were also associated with a greater degree of focal tissue necrosis and apoptosis of hepatocytes (black arrows). (c) Mock-infected mice showed no lesions (H&E; original magnification, 4009)
Fig. 3 Kidney histopathology at day 7 p.i. after IP injection. (a) Compared to ncp BVDV1and ncp BVDV2-infected mice, cp BVDV1-infected mice showed marked atrophy of the glomerulus (black arrows.) (b) A mock-infected mouse showed a normal glomerulus (H&E; original magnification, 2009)
Fig. 4 Lung histopathology at day 7 p.i. after IP injection or IN inoculation. (a) Compared to ncp BVDV1- and ncp BVDV2-infected mice, mice infected with cp BVDV1 by IP injection showed hyperplasia of the alveoli wall (black arrows). (b) An IN-injected
mouse showed interstitial thickening and alveolar macrophage infiltration in the alveolar lumen (blue arrow). (c) Mock-infected mice showed no lesions (H&E, 2009)
lymphocytes within the lymphatic nodule (Fig. 5b) compared to cp BVDV1 or ncp BVDV2-infected mice. Although BVDV antigen was not detected in one mouse with ncp BVDV1 infection, the histopathological lesions showed a more significant change in the spleen of this mouse than in the other ncp BVDV1-infected mice. In the IP-injected group, the histological changes in the mesenteric lymph node differed somewhat among the mice. IPinjected mice showed lympholysis and moderate to severe
lymphoid depletion (Fig. 6a), while IN-inoculated mice showed moderate lymphoid depletion (Fig. 6b). Viral RNA was detected in the whole blood of five out of six IP-injected mice, whereas it was detected in only one out of six mice in the IN-inoculated group (Table 1). This suggests that the reason for the lower number of viremic mice in the IN-inoculated group might be the restriction of viral entry via this mode of administration. Therefore, further studies are required to evaluate viremia at several
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Fig. 5 Splenic histopathology at 7 days p.i. after IP injection. (a) The ncp BVDV1-infected mice showed more extramedullary hematogenesis (black arrows) and more intense lymphocyte necrosis within
the lymphatic nodule (black arrows). (b) Mock-infected mice showed no lesions (H&E; original magnification, 4009)
Fig. 6 Histopathology of mesenteric lymph nodes at 7 day p.i. after IP injection or IN inoculation. (a) Mice in the IP-injected group showed lympholysis and moderate to severe lymphoid depletion
(black arrow). (b) The IN-inoculated mouse showed moderate lymphoid depletion in the cortex (blue arrow). (c) A mock-infected mouse showed no lesions (H&E; original magnification, 4009)
different time points, using a larger number of infected mice. In the IP-injected group, BVDV antigen was most commonly detected in the spleen and was detected in the mesenteric lymph nodes of four out of six infected mice and in the lymphatic tissue of the lung in cp BVDV1 and ncp BVDV2-infected mice, but not in ncp BVDV1-infected mice (Table 1). In addition, BVDV antigen was detected in the lung and stomach of only one infected mouse (Table 1). In the IN-inoculated group, BVDV antigen was detected only in the lung and mesenteric lymph nodes of one cp BVDV1-infected mouse (Table 1). Interestingly, the presence of BVDV antigen in the stratified squamous epithelial cells of the stomach and the bronchial epithelial cells of the lung may be explained by the preferred location for antigen deposition in the mouse model of BVDV infection. These results show evidence of BVDV infection in mice after IP injection and IN inoculation. Our findings confirmed those of previous studies, which showed that natural infection of non-artiodactyls was accompanied with
changes in histopathology [11]. Bachofen et al. showed that after exposure of rabbits to BVDV, their lymphoid organs had histological changes typical of transient infection with a pestivirus and were BVDV positive by RT-PCR [11]. Similar to other studies of rabbits, our results showed mild to severe histopathological changes in all tissues of BVDV-infected mice that were examined (Table 2). The reason for the differences in these histopathological changes of the various BVDV-infected mice is not clear. However, it may be postulated to be associated with differences in the virulence and tissue tropism of the viral strains used in this study. The correlation between the distribution of viral antigen and histopathological lesions was not as clear. For example, despite the significant histopathological changes in the liver and kidney, no viral antigen was observed. The reason for these apparent differences in the distribution of viral antigen in mice is not known; however, it may be hypothesized to be related to low viral replication in mice and the restriction of replication to small clusters of cells in
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Experimental infection of mice with…
certain organ systems. Therefore, we can speculate that the reason for the variation in the distribution of viral antigen observed in this study may be attributable to the susceptibility of infected tissue to the virus, or to being the result of only transient BVDV infection. Taken together, these results suggest that the spleen is the most reliable tissue for detection of BVDV antigen using IHC in mice. Baker et al. showed that mice injected subcutaneously with spleen homogenate from a BVDV-infected calf did not demonstrate infection [10]. However, this result is not consistent with our observations. This difference may be due to variations in the route of infection, virus dosage, or duration of infection. Our findings demonstrated that mice can be infected by BVDV following IP injection or IN inoculation and that viremia, BVDV antigen, and pathologic findings were detected at day 7 p.i. However, BVDV infection after either IP injection or IN inoculation in mice resulted in considerably different outcomes. While in the IN-inoculated group, viremia did not occur in all infected mice and BVDV antigen was not detected in the spleen, in the IP-injected group, whilst five out of six mice showed viremia and the presence of BVDV antigen, all six mice demonstrated mild to severe histopathological changes. These results suggest that in mice, IN inoculation might restrict virus entry and replication. Thus, further investigations of IN inoculation are necessary. In conclusion, this is the first report of BVDV infection in mice. Our results indicate that mice can be infected with BVDV. Even though none of the infected mice showed clinically apparent disease, the presence of viremia provided evidence that the mice were infected with BVDV. BVDV antigen was primarily detected in the spleen of IPinjected mice by IHC. Histologically, all IP-injected mice demonstrated more severe pathological changes than those in the IN-inoculated groups. Overall, our results provide new information about BVDV infection. Infection was reliably established after IP injection, but not after IN inoculation; only one out of six mice was found to have viremia and the presence of BVDV antigen. To determine the applicability of mice as experimental hosts for BVDV, additional studies are needed in order to understand the pathogenesis and mechanism of BVDV infection in mice.
1571 funded by the Ministry of Education, Science, and Technology (NRF 2012R1A1A3011238).
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Acknowledgments This work was supported by the Basic Science Research Program of the National Research Foundation of Korea,
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