Experimental infection of inbred BALB/c and A/J mice with Massachusetts and Brazilian strains of infectious bronchitis virus (IBV)

Abstract

The ability of avian coronaviruses to replicate in mice was investigated to investigate interspecies transmission. Two inbred mouse strains (BALB/c and A/J) with different genetic backgrounds were inoculated with the avian coronavirus strains Mass and BR-I and monitored for at least 10 days. Analysis of viral RNA, histopathological examinations, immunohistochemistry and serology were performed. After virus inoculation, neither clinical signs nor evident gross lesions were observed. Viral RNA, histopathological changes, and viral nucleoprotein were observed in the lung, trachea and sinus of all inoculated mice. Our study demonstrates the importance of elucidating the epidemiology of coronaviruses, including in rodents that are pests in poultry production.

Coronaviruses are a large family of viruses belonging to the order Nidovirales, family Coronaviridae. Recently, the discovery of novel coronaviruses resulted in the division of the family Coronaviridae into two subfamilies: Torovirinae and Coronavirinae. The subfamily Coronavirinae is further split into four genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus [9]. Members of the genera Alphacoronavirus and Betacoronavirus are found mainly in mammals, in which they can cause mild or severe diseases [5]. This group includes the recently identified Middle East respiratory syndrome coronavirus [4]. Members of the genera Gammacoronavirus and Deltacoronavirus are found in birds and mammals [14, 18, 19]. The ancestors of the different genetic groups have not been identified.

Gammacoronaviruses have been detected in many Galliformes and non-Galliformes bird species over the past years [2]. Some experimental evidence suggests that coronaviruses are not limited to replication in a single host, and this is especially true of those that infect mammals [20]. Indeed, close contact of animals of different species in domestic environments and wildlife markets as well as close contact with humans may allow interspecies jumping and could subsequently pose risks of further genetic changes if the virus adapts to human hosts, as has occurred in the case of SARS [3] and H5N1 virus [17]. In the intensive poultry industry, poultry are increasingly exposed to pathogens of other birds and mammals and vice versa. An experimental study showed that bovine coronavirus (BCV) is pathogenic for 1-day-old turkey poults [10]. In this context, the present study aimed to evaluate whether avian coronaviruses can replicate, are pathogenic, and induce an immune response in mice.

Two inbred mouse lineages, BALB/c (MHC haplotype: H-2d) and A/J (MHC haplotype H-2a), were used for the experiments. The animals, 6- to 8-week-old specific-pathogen-free females, were purchased from CEMIB (Multidisciplinary Centre for Biological Investigation, UNICAMP, Brazil). Mice were maintained under specific-pathogen-free conditions, and manipulations were performed according to the Brazilian Ethics Community guidelines and approved by the local ethics committee (CEUA-UNICAMP no. 2746-1, May, 21, 2012). Two IBV strains isolated previously [6], named 810 (BR-I) and 67T (Mass), and one vaccine strain (H120-Mass) (Bio-Bronk-Vet-H120, Laboratório Biovet, Vargem Grande Paulista, Brasil) were used. Thirty-six BALB/c mice were divided into four groups: a control group and three groups inoculated with virus (strains 67T, 810 or H120). Six A/J mice were inoculated with the H120 vaccine strain, and six A/J mice were kept as a control group. Mice were anesthetized and inoculated intranasally with 50 μL of a suspension containing 106 EID50 of IBV per mouse according to previous studies [16]. BALB/c and A/J mice were monitored daily for two weeks for weight loss and survival.

Subsequently, real-time RT-PCR (RRT-PCR) was employed to detect viral RNA in the sinus, trachea, lung, and duodenum. Tissues were homogenized in 400 µL of ice-cold Dulbecco’s modified Eagle medium (Life Technologies, Carlsbad, USA), and supernatants were harvested after centrifugation for 10 min at 1000 × g at 4 °C. Viral RNA from tissue suspensions were purified using a QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. RRT-PCR targeting the 5′ untranslated region (5′UTR) of IBV was used [1]. The copy number was calculated using a standard curve, and beta actin was used as an internal control as described previously [7]. One-way analysis of variance and Student’s t-test were carried out to compare differences among groups, and the differences were considered significant at P < 0.05. Histopathological evaluation was performed to assess whether the mice displayed any histopathological lesions related to infection. Trachea, lung, and duodenum of inoculated BALB/c mice were collected at 3 and 10 dpi and fixed in 4 % paraformaldehyde for 12 h at room temperature. The specimens were treated with xylene, dehydrated in graded ethanol, embedded in paraffin, and cut into 5-µm-thick sections. Histopathological changes were evaluated in sections stained with hematoxylin and eosin (H&E). Additionally, the slides were subjected to immunohistochemistry for detection of virus in the tissues. Immunostaining was performed according to a previous study [11]. After treatment with Triton X-100 (#93443, Sigma Aldrich, St. Louis, USA) to permeabilize the cells, the tissue sections were incubated for 12 hours with a 1:20 dilution of a monoclonal primary antibody targeting the IBV nucleoprotein (no. nAB90926, Abcam, Cambridge, UK). The slides were then incubated with a 1:20 dilution of goat anti-IgG biotin conjugate (no. B7264, Sigma Aldrich, St. Louis, USA). After washing, each slide was treated with a solution of avidin and biotinylated peroxidase. The reaction was visualized by adding a solution containing the substrate (H2O2) and the chromogen diaminobenzidine (DAB). The sections were counterstained with hematoxylin, mounted, and examined using an optical microscope (Zeiss).

After blocking the tissue sections, a 1:20 dilution of a monoclonal primary antibody targeting the IBV nucleoprotein (no. nAB90926, Abcam, Cambridge, UK) was added, and the sample was incubated for 12 hours; After incubation, the slides were incubated with a 1:20 dilution of goat anti-IgG biotin conjugate (no. B7264, Sigma Aldrich, St. Louis, USA).

To determine whether IBV induced a humoral immune response in the mice, sera were collected at 3 and 10 dpi from all of the inoculated mice and tested using a commercial competitive ELISA (IBVC-2P, Id Vet, Montpellier, France).

Although coronavirus infection usually results in weight loss in mice, no clinical signs or significant changes in weight were observed in the mice of either lineage during the entire experiment. In all cases, gross examination revealed normal tissue morphology with no noticeable gross lesions (data not shown).

After inoculation with virus, viral RNA was detected at different time points in the sinus, trachea, lung, and duodenum of mice of both inbred lineages (Fig. 1). In the sinus, the viral RNA level was significantly higher at 3 dpi in the A/J and BALB/c mice inoculated with the H120 strain than in mice inoculated with the 67T strain. Viral RNA was detected in the 67T- and 810-inoculated mice at 14 dpi (Fig. 1A). In the trachea, viral RNA was also detected in the mice inoculated with the 810 strain at 10 dpi and in the mice inoculated with the 810 strain at 14 dpi (Fig. 1B). For both inbred mouse lines, viral RNA was detected in the lungs of animals inoculated with each of the virus strains at 3 dpi (Fig. 1C). Viral RNA was also detected in the duodenum of all inoculated BALB/c mice at 3 dpi (Fig. 1D).

Fig. 1
figure1

Detection and quantification of viral RNA in organs of BALB/c and A/J inbred mice after inoculation with different IBV strains at different time points. A, sinus; B, trachea; C, lung; D, duodenum. Mean ± standard error at time points with no common superscript differ significantly (P < 0.05). Differences were only observed when more than one mouse was detected by the test. The limit of detection was 105.38 copies per mL

Mice inoculated with H120 virus had a large accumulation of mucus in the trachea at 3 dpi, but this was not observed until 10 dpi in the mice inoculated with strain 810. Mucus accumulation was not evident in the inoculated mice from the 67T group. Interestingly, nucleoprotein was detected in all of the inoculated groups at 3 dpi (Fig. 3), but not at 10 dpi. Intense perialveolar and perivascular infiltration was observed in the lungs of all inoculated groups at 3 dpi when compared to the control group (Fig. 2A-D). Furthermore, the intense infiltrate was substantially reduced at 10 dpi in the mice infected with 67T or H120, whereas the mice infected with the 810 virus showed persistent inflammatory infiltrate at 10 dpi. The viral nucleoprotein was detected in the lungs of all inoculated mice at 3 dpi (Fig. 3), especially in the mice inoculated with 67T virus, whereas the labeling decreased at 10 dpi. Moreover, in the duodenum, the increase in goblet cells with greater mucus production was more pronounced in mice inoculated with strain 810 than in the other groups at 3 dpi, but this was drastically reduced at 10 dpi. Nucleoprotein was also detected at 3 dpi in all of the inoculated mice; whereas this signal was no longer detectable at 10 dpi mice (Fig. 3). In addition to RNA viral detection by RRT-PCR, histopathological changes and changes in immunohistochemistry were observed in different tissues in the inoculated mice.

Fig. 2
figure2

Microscopic lesions in lungs of inoculated BALB/c mice. A) Control mice showing diffuse interstitial pneumonia. B) Severe interstitial pneumonia in mice inoculated with the H120 strain at 3 dpi. C) Moderate interstitial pneumonia induced by strain 810 at 3 dpi D). Mice inoculated with strain 67T had moderate interstitial pneumonia, diffuse oedema, and peribronchiolar mononuclear infiltration at 3 dpi. Arrowheads show interstitial pneumonia (haematoxylin and eosin staining; magnification, 40X)

Fig. 3
figure3

Viral nucleoprotein in duodenum, trachea, and lungs of inoculated mice. Viral nucleoprotein was detected (arrows) in lamina propria and intestinal glands of the duodenum at 3 dpi in mice inoculated with the H120, 810, and 67T strains. Viral nucleoprotein was detected (arrows) at 3 dpi in the tracheal epithelium of mice inoculated with the H120, 810, and 67T strains. Viral nucleoprotein was detected (arrows) in alveolar epithelial cells at 3 dpi in mice inoculated with the H120, 810, and 67T strains. No positive staining was seen in uninfected mice. Bars, 200 µm

Specific antibodies to IBV were detected at 3 dpi in the BALB/c mice inoculated with H120, 810, or 67T, with percentage inhibition above 40 %: 40.34 ± 0.22, 74.97 ± 0.04 and 42.62 ± 0.17, respectively. The percentage inhibition value in the A/J mice inoculated with H120 was 30.54 % at 3 dpi, which corresponded to a suspicious outcome. No specific antibody was detected at 10 dpi in either mouse lineage.

In this study, we investigated whether avian gammacoronavirus strains could replicate in a mammal model. Interestingly, viral RNA and histopathological changes in different organs were more evident in inoculated BALB/c mice, suggesting that these mice are more susceptible than A/J mice to avian coronavirus infection. BALB/c mice have been shown to be susceptible to experimental infection with avian paramyxoviruses and recombinant avian paramyxoviruses [11, 12]; but other studies have shown that A/J and C57BL/6 mice were more susceptible to infection with bovine respiratory syncytial virus than BALB/c mice [15] and that no difference was observed after infection with mouse hepatitis virus [13]. In our study, all tissue changes correlated to the detection of viral RNA by RRT-PCR or viral nucleoprotein by immunostaining, suggesting that virus replication had occurred, although virus isolation was not attempted. Viral nucleoprotein was strongly detected at 3 dpi, suggesting that the presence of virus correlated with severe histological changes. Those findings are in accordance with an acute inflammatory state [8]. Additionally, viral RNA, histopathological lesions and viral antigen were also more pronounced at 3 dpi than at 10 dpi, suggesting that replication occurred for a short time. Low levels of antibodies specific for IBV were detected at 3 dpi, suggesting slight IgM production, which is typically associated with the primary immune response. Nonetheless, no specific antibody was detected at 10 dpi, suggesting that this antibody response was not important given its short duration.

Our study demonstrates the importance of elucidating the epidemiology of coronaviruses that can infect a broad spectrum of hosts, including rodents that are pests in poultry production. Further studies should be performed to investigate whether rodents near poultry farms can carry avian coronavirus.

References

  1. 1.

    Callison SA, Hilt DA, Boynton TO, Sample BF, Robison R, Swayne DE, Jackwood MW (2006) Development and evaluation of a real-time Taqman RT-PCR assay for the detection of infectious bronchitis virus from infected chickens. J Virol Methods 138:60–65. doi:10.1016/j.jviromet.2006.07.018

    CAS  PubMed  Article  Google Scholar 

  2. 2.

    Cavanagh D (2005) Coronaviruses in poultry and other birds. Avian Pathol 34:439–448. doi:10.1080/03079450500367682

    CAS  PubMed  Article  Google Scholar 

  3. 3.

    Cheng VC, Lau SK, Woo PC, Yuen KY (2007) Severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection. Clin Microbiol Rev 20:660–694. doi:10.1128/CMR.00023-07

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  4. 4.

    de Groot RJ, Baker SC, Baric RS, Brown CS, Drosten C, Enjuanes L, Fouchier RA, Galiano M, Gorbalenya AE, Memish ZA, Perlman S, Poon LL, Snijder EJ, Stephens GM, Woo PC, Zaki AM, Zambon M, Ziebuhr J (2013) Middle East respiratory syndrome coronavirus (MERS-CoV): announcement of the Coronavirus Study Group. J Virol 87:7790–7792. doi:10.1128/JVI.01244-13

    PubMed Central  PubMed  Article  Google Scholar 

  5. 5.

    Dong BQ, Liu W, Fan XH, Vijaykrishna D, Tang XC, Gao F, Li LF, Li GJ, Zhang JX, Yang LQ, Poon LL, Zhang SY, Peiris JS, Smith GJ, Chen H, Guan Y (2007) Detection of a novel and highly divergent coronavirus from asian leopard cats and Chinese ferret badgers in Southern China. J Virol 81:6920–6926. doi:10.1128/JVI.00299-07

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  6. 6.

    Felippe PA, da Silva LH, Santos MM, Spilki FR, Arns CW (2010) Genetic diversity of avian infectious bronchitis virus isolated from domestic chicken flocks and coronaviruses from feral pigeons in Brazil between 2003 and 2009. Avian Dis 54:1191–1196. doi:10.1637/9371-041510-Reg.1

    CAS  PubMed  Article  Google Scholar 

  7. 7.

    Ferreira HL, Spilki FR, de Almeida RS, Santos MM, Arns CW (2007) Inhibition of avian metapneumovirus (AMPV) replication by RNA interference targeting nucleoprotein gene (N) in cultured cells. Antiviral Res 74:77–81. doi:10.1016/j.antiviral.2006.12.002

    CAS  PubMed  Article  Google Scholar 

  8. 8.

    Griffin DE (1984) The inflammatory response to acute viral infections. In: Notkins A, Oldstone MA (eds) Concepts in viral pathogenesis. Springer, New York, pp 46–52

    Google Scholar 

  9. 9.

    ICTV (2012) Virus taxonomy: classification and nomenclature of viruses: ninth report of the International Committee on Taxonomy of Viruses. Elsevier Academic Press, San Diego

    Google Scholar 

  10. 10.

    Ismail MM, Cho KO, Ward LA, Saif LJ, Saif YM (2001) Experimental bovine coronavirus in turkey poults and young chickens. Avian Dis 45:157–163

    CAS  PubMed  Article  Google Scholar 

  11. 11.

    Khattar SK, Kumar S, Xiao S, Collins PL, Samal SK (2011) Experimental infection of mice with avian paramyxovirus serotypes 1 to 9. PLoS ONE 6:e16776. doi:10.1371/journal.pone.0016776

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  12. 12.

    Kim SH, Chen S, Jiang X, Green KY, Samal SK (2014) Newcastle disease virus vector producing human norovirus-like particles induces serum, cellular, and mucosal immune responses in mice. J Virol 88:9718–9727. doi:10.1128/JVI.01570-14

    PubMed Central  PubMed  Article  Google Scholar 

  13. 13.

    Lucchiari MA, Martin JP, Modolell M, Pereira CA (1991) Acquired immunity of A/J mice to mouse hepatitis virus 3 infection: dependence on interferon-gamma synthesis and macrophage sensitivity to interferon-gamma. J Gen Virol 72(Pt 6):1317–1322. doi:10.1099/0022-1317-72-6-1317

    CAS  PubMed  Article  Google Scholar 

  14. 14.

    Mihindukulasuriya KA, Wu G, St Leger J, Nordhausen RW, Wang D (2008) Identification of a novel coronavirus from a beluga whale by using a panviral microarray. J Virol 82:5084–5088. doi:10.1128/JVI.02722-07

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  15. 15.

    Spilki FR, Almeida RS, Ferreira HL, Gameiro J, Verinaud L, Arns CW (2006) Effects of experimental inoculation of bovine respiratory syncytial virus in different inbred mice lineages: establishment of a murine model for BRSV infection. Vet Microbiol 118:161–168. doi:10.1016/j.vetmic.2006.07.011

    CAS  PubMed  Article  Google Scholar 

  16. 16.

    Torrieri-Dramard L, Lambrecht B, Ferreira HL, van den Berg T, Klatzmann D, Bellier B (2011) Intranasal DNA vaccination induces potent mucosal and systemic immune responses and cross-protective immunity against influenza viruses. Mol Ther 19:602–611. doi:10.1038/mt.2010.222

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  17. 17.

    Webster RG (1997) Influenza virus: transmission between species and relevance to emergence of the next human pandemic. Arch Virol Suppl 13:105–113. doi:10.1007/978-3-7091-6534-8_11

    CAS  PubMed  Google Scholar 

  18. 18.

    Woo PC, Lau SK, Lam CS, Lai KK, Huang Y, Lee P, Luk GS, Dyrting KC, Chan KH, Yuen KY (2009) Comparative analysis of complete genome sequences of three avian coronaviruses reveals a novel group 3c coronavirus. J Virol 83:908–917. doi:10.1128/JVI.01977-08

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  19. 19.

    Woo PC, Lau SK, Lam CS, Lau CC, Tsang AK, Lau JH, Bai R, Teng JL, Tsang CC, Wang M, Zheng BJ, Chan KH, Yuen KY (2012) Discovery of seven novel mammalian and avian coronaviruses in the Genus Deltacoronavirus supports bat coronaviruses as the gene source of Alphacoronavirus and Betacoronavirus and avian coronaviruses as the gene source of Gammacoronavirus and Deltacoronavirus. J Virol 86:3995–4008. doi:10.1128/JVI.06540-11

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  20. 20.

    Woods RD, Cheville NF, Gallagher JE (1981) Lesions in the small intestine of newborn pigs inoculated with porcine, feline, and canine coronaviruses. Am J Vet Res 42:1163–1169

    CAS  PubMed  Google Scholar 

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Acknowledgments

We are thankful to Julia Benassi and Guilherme S. Pereira for their excellent technical assistance. CWA and TCC are recipients of a fellowship from CNPq. The Houghton trust also supported this work (HT/02.14/0669). This work was done within the UNIVERSAL project supported by CNPq (number: 472840/2011-8).

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Correspondence to Helena L. Ferreira.

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M. C. Martini and J. Gameiro contributed equally.

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Martini, M.C., Gameiro, J., Cardoso, T.C. et al. Experimental infection of inbred BALB/c and A/J mice with Massachusetts and Brazilian strains of infectious bronchitis virus (IBV). Arch Virol 160, 1785–1790 (2015). https://doi.org/10.1007/s00705-015-2443-x

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Keywords

  • Virus
  • Mice
  • Avian
  • Pneumonia