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. 2019 Apr 24;11(4):381.
doi: 10.3390/v11040381.

Lack of Middle East Respiratory Syndrome Coronavirus Transmission in Rabbits

W Widagdo  1 , Nisreen M A Okba  2 , Mathilde Richard  3 , Dennis de Meulder  4 , Theo M Bestebroer  5 , Pascal Lexmond  6 , Elmoubasher A B A Farag  7 , Mohammed Al-Hajri  8 , Koert J Stittelaar  9 , Leon de Waal  10 , Geert van Amerongen  11 , Judith M A van den Brand  12 , Bart L Haagmans  13 , Sander Herfst  14
Affiliations

Lack of Middle East Respiratory Syndrome Coronavirus Transmission in Rabbits

W Widagdo et al. Viruses. .

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) transmission from dromedaries to humans has resulted in major outbreaks in the Middle East. Although some other livestock animal species have been shown to be susceptible to MERS-CoV, it is not fully understood why the spread of the virus in these animal species has not been observed in the field. In this study, we used rabbits to further characterize the transmission potential of MERS-CoV. In line with the presence of MERS-CoV receptor in the rabbit nasal epithelium, high levels of viral RNA were shed from the nose following virus inoculation. However, unlike MERS-CoV-infected dromedaries, these rabbits did not develop clinical manifestations including nasal discharge and did shed only limited amounts of infectious virus from the nose. Consistently, no transmission by contact or airborne routes was observed in rabbits. Our data indicate that despite relatively high viral RNA levels produced, low levels of infectious virus are excreted in the upper respiratory tract of rabbits as compared to dromedary camels, thus resulting in a lack of viral transmission.

Keywords: MERS-coronavirus; rabbits; transmission.

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Conflict of interest statement

K.S., L.d.W., and G.J.A. are full time employees at Viroclinics Biosciences BV. The other authors have no conflict of interest to disclose. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Dipeptidyl peptidase-1 (DPP4) expression in the respiratory tract tissues of rabbits. DPP4 is detected using immunohistochemistry and indicated in red in the figure. Type II cells are indicated with arrows, and type I cells with an arrowhead. Nasal epithelium and bronchiolar epithelium pictures were taken at a 400× magnification, and alveolar epithelium at 1000×. The isotype control showed no background signal in our assay.
Figure 2
Figure 2
Middle East respiratory syndrome coronavirus (MERS-CoV) inoculation in rabbits with different routes and volumes. Three New Zealand rabbits were each infected either with (a) 200 µL of 1 × 106 TCID50/mL MERS-CoV intranasal (I.N.); (b) 1 mL of 1 × 106 TCID50/mL MERS-CoV I.N.; (c) 200 µL of 1 × 106 TCID50/mL MERS-CoV I.N. combined with 3 mL of 4 × 106 TCID50/mL MERS-CoV intratracheal (I.T.); or (d) 1mL of PBS I.N as negative control. These animals were sacrificed at day 4 post inoculation. Viral RNA shed by the MERS-CoV-inoculated rabbits in the nasal swabs (A) and throat swabs (B) are reported in genome equivalents per ml (GE/mL). Viral RNA detected in the lungs of these rabbits are reported in genome equivalents per gram tissues (GE/g). Dashed lines depict the detection limit of the assays. All error bars represent standard deviations. Statistical analysis is performed using Kruskal wallis test (**, p value < 0.01; ***, p value < 0.001). Representative figures of immunohistochemistry detecting MERS-CoV nucleoprotein (displayed in red) in the lungs of these rabbits were taken at a 200× magnification (D). All MERS-CoV-inoculated rabbits shed a relatively equal amount of viral RNA in the nasal and throat swabs (A,B). However, there was significantly less viral RNA in the lungs of rabbits in the group (a) in comparison to the other MERS-CoV-inoculated groups (C). A similar finding is observed in our immunohistochemistry analysis detecting MERS-CoV nucleoprotein (displayed in red) in the lungs of these rabbits (D). Pictures were taken in 200× magnification. The amount of viral RNA is displayed either in genome equivalent per mL (GE/mL) or per gram tissues (GE/g). Dashed lines depict the detection limit of the assays. All error bars represent standard deviations. Statistical analysis is performed using Kruskal–Wallis test (**, p value < 0.01; ***, p value < 0.001).
Figure 3
Figure 3
Middle East respiratory syndrome coronavirus (MERS-CoV) EMC strain and Qatar15 strain replicate equally in the upper respiratory tract of rabbits. Five New Zealand rabbits were each intranasally inoculated either with 1 mL of 1 × 106 TCID50/mL MERS-CoV EMC strain or Qatar15 strain. Nasal and throat swabs were obtained from day 0 (before inoculation) until day 4 post inoculation. The amount of viral RNA is displayed in genome equivalents per mL (GE/mL). Dashed lines depict the detection limit of the assay. All error bars represent standard deviations.
Figure 4
Figure 4
Middle East respiratory syndrome coronavirus (MERS-CoV) infects the upper and lower respiratory tract of Netherland dwarf rabbits. The amount of viral RNA in the nasal and throat swabs (A) as well as in the respiratory tract tissues (B) are displayed in genome equivalents per mL (GE/mL) and genome equivalents per gram tissues (GE/g), respectively. Dashed lines depict the detection limit of the assays. All error bars represent standard error of means.
Figure 5
Figure 5
Shedding of Middle East respiratory syndrome coronavirus (MERS-CoV) RNA and infectious virus in directly inoculated, contact-exposed and airborne-exposed rabbits. MERS-CoV RNA and infectious virus were measured in the nasal (A,B) and throat swabs (C,D). The amount of viral RNA is displayed in genome equivalents per mL (GE/mL), while infectious virus is shown as 50% tissue culture infective dose per ml (TCID50/mL). Dashed lines depict the detection limit of each assay.
Figure 6
Figure 6
Middle East respiratory syndrome coronavirus (MERS-CoV)-specific antibody response in rabbits. S1-specific MERS-CoV antibodies were measured with ELISA and displayed as optical density (OD) value (A), while MERS-CoV neutralizing antibodies were measured with the virus neutralization test (VNT) and displayed in titers (B). Dashed lines depict the detection limit of the assay.
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