Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2020 Nov 20;72(12):e978–e992. doi: 10.1093/cid/ciaa1747

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America.

This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

PMC Copyright notice

Figure 4. — Viral replication profiles in the lung and nasal turbinate (NT) tissue of hamsters after monoinfection, simultaneous, or sequential coinfection. A, Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load in lung and NT tissues. SARS-CoV-2 RdRp gene copies determined by real-time reverse-transcription polymerase chain reaction (RT-qPCR) (left panel). SARS-CoV-2 infectious viral titers were determined by plaque assays in Vero E6 cells (right). n = 3 each group. P values are calculated by one-way analysis of variance (ANOVA). Error bars indicate mean ± standard deviation (SD). Horizontal dashed line indicates detection limit of the assays. B, Mouse-adapted A/Hongkong/415742/2009 H1N1 (pH1N1) viral load in lung and NT tissues by RT-qPCR (influenza A matrix gene, left panel) or plaque assays in Madin-Darby canine kidney cells (right). n = 3 each group. P values are calculated by one-way ANOVA. Error bars indicate mean ± SD. Horizontal dashed line indicates detection limit of the assays. C, Representative images of immunohistochemistry-stained SARS-CoV-2 N protein in the lung tissues after monoinfection or coinfection. SARS-CoV-2 N protein Continued was stained in brown. The images of SARS-CoV-2 monoinfection shows N protein expression in regional alveoli and bronchiolar epithelium (arrows); simultaneously coinfected lung has small patches of N protein expression in a large area of inflammatory consolidated lung tissue (arrows); the lung coinfected sequentially by SARS-CoV-2 prior to pH1N1 (SARS-CoV-2 || pH1N1) shows extensive N protein expression distributed diffusely in the lung, whereas in sequential coinfection by pH1N1 prior to SARS-CoV-2 (pH1N1 || SARS-CoV-2), the large area of inflammatory consolidation in lung tissue shows a few N protein–positive cells. D, Immunofluorescence images illustrate pH1N1 NP antigen and SARS-CoV-2 N antigen in single virus–infected hamster lung tissues. pH1N1 nucleoprotein (NP) is only seen in the epithelium of bronchiolar epithelium, not in alveoli (NP, green). SARS-CoV-2 N antigen is seen diffusely distributed in alveoli (N, red). E, Dual immunofluorescence images illustrate the distribution of pH1N1 NP and SARS-CoV-2 N in simultaneously coinfected hamster NT (upper panels) and lung tissues (lower panels). pH1N1 NP antigen was rarely seen in the coinfected NT tissues, whereas SARS-CoV-2 N protein was abundantly detected (arrows, N stained red). In the lung tissue, the 2 viral antigens also distributed differently. These images show pH1N1 NP expression in a few cells in the bronchiolar epithelium, but not in alveoli (arrows, green), while SARS-CoV-2 N is seen in alveoli (red) but not in bronchiole. Co-localization of SARS-CoV-2 N and pH1N1 NP antigen is shown in a macrophage (magnified image, open arrow). F, Relative expression of inflammatory cytokines/chemokines in hamster lung tissues determined by RT-qPCR using gene-specific primers. G, RT-qPCR determined SARS-CoV-2 viral load (left) and pH1N1 viral load (right) in the lung and NT tissues taken at 7 days postinfection (dpi) or 14 dpi after simultaneous coinfection or monoinfection. n = 3 each group. Error bars indicate mean ± SD. Horizontal dashed line indicates detection limit of the assays. H, SARS-CoV-2 (left) and pH1N1 (right) virus shedding from oral swabs determined by RT-qPCR. Oral swab samples were collected every other day during the 14 days after virus challenge. Error bars indicate mean ± SD. Horizontal dashed line indicates detection limit of the assays. Double vertical lines indicate sequential inoculation (24 hours apart); + indicates simultaneous inoculation. Abbreviations: IP-10, interferon-γ induced protein 10; M gene, influenza A matrix gene; MIP-1α, macrophage inflammatory protein 1 alpha; N, severe acute respiratory syndrome coronavirus 2 nucleocapsid protein; NP, influenza A nucleoprotein; NT, nasal turbinate; pH1N1, mouse-adapted A/Hongkong/415742/2009 H1N1; PFU, plaque-forming unit; RANTES, regulated upon activation, normal T-cell expressed protein; RdRp, RNA-dependent RNA polymerase; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; TNF-α, tumor necrosis factor alpha.

Figure 4. — Viral replication profiles in the lung and nasal turbinate (NT) tissue of hamsters after monoinfection, simultaneous, or sequential coinfection. A, Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load in lung and NT tissues. SARS-CoV-2 RdRp gene copies determined by real-time reverse-transcription polymerase chain reaction (RT-qPCR) (left panel). SARS-CoV-2 infectious viral titers were determined by plaque assays in Vero E6 cells (right). n = 3 each group. P values are calculated by one-way analysis of variance (ANOVA). Error bars indicate mean ± standard deviation (SD). Horizontal dashed line indicates detection limit of the assays. B, Mouse-adapted A/Hongkong/415742/2009 H1N1 (pH1N1) viral load in lung and NT tissues by RT-qPCR (influenza A matrix gene, left panel) or plaque assays in Madin-Darby canine kidney cells (right). n = 3 each group. P values are calculated by one-way ANOVA. Error bars indicate mean ± SD. Horizontal dashed line indicates detection limit of the assays. C, Representative images of immunohistochemistry-stained SARS-CoV-2 N protein in the lung tissues after monoinfection or coinfection. SARS-CoV-2 N protein Continued was stained in brown. The images of SARS-CoV-2 monoinfection shows N protein expression in regional alveoli and bronchiolar epithelium (arrows); simultaneously coinfected lung has small patches of N protein expression in a large area of inflammatory consolidated lung tissue (arrows); the lung coinfected sequentially by SARS-CoV-2 prior to pH1N1 (SARS-CoV-2 || pH1N1) shows extensive N protein expression distributed diffusely in the lung, whereas in sequential coinfection by pH1N1 prior to SARS-CoV-2 (pH1N1 || SARS-CoV-2), the large area of inflammatory consolidation in lung tissue shows a few N protein–positive cells. D, Immunofluorescence images illustrate pH1N1 NP antigen and SARS-CoV-2 N antigen in single virus–infected hamster lung tissues. pH1N1 nucleoprotein (NP) is only seen in the epithelium of bronchiolar epithelium, not in alveoli (NP, green). SARS-CoV-2 N antigen is seen diffusely distributed in alveoli (N, red). E, Dual immunofluorescence images illustrate the distribution of pH1N1 NP and SARS-CoV-2 N in simultaneously coinfected hamster NT (upper panels) and lung tissues (lower panels). pH1N1 NP antigen was rarely seen in the coinfected NT tissues, whereas SARS-CoV-2 N protein was abundantly detected (arrows, N stained red). In the lung tissue, the 2 viral antigens also distributed differently. These images show pH1N1 NP expression in a few cells in the bronchiolar epithelium, but not in alveoli (arrows, green), while SARS-CoV-2 N is seen in alveoli (red) but not in bronchiole. Co-localization of SARS-CoV-2 N and pH1N1 NP antigen is shown in a macrophage (magnified image, open arrow). F, Relative expression of inflammatory cytokines/chemokines in hamster lung tissues determined by RT-qPCR using gene-specific primers. G, RT-qPCR determined SARS-CoV-2 viral load (left) and pH1N1 viral load (right) in the lung and NT tissues taken at 7 days postinfection (dpi) or 14 dpi after simultaneous coinfection or monoinfection. n = 3 each group. Error bars indicate mean ± SD. Horizontal dashed line indicates detection limit of the assays. H, SARS-CoV-2 (left) and pH1N1 (right) virus shedding from oral swabs determined by RT-qPCR. Oral swab samples were collected every other day during the 14 days after virus challenge. Error bars indicate mean ± SD. Horizontal dashed line indicates detection limit of the assays. Double vertical lines indicate sequential inoculation (24 hours apart); + indicates simultaneous inoculation. Abbreviations: IP-10, interferon-γ induced protein 10; M gene, influenza A matrix gene; MIP-1α, macrophage inflammatory protein 1 alpha; N, severe acute respiratory syndrome coronavirus 2 nucleocapsid protein; NP, influenza A nucleoprotein; NT, nasal turbinate; pH1N1, mouse-adapted A/Hongkong/415742/2009 H1N1; PFU, plaque-forming unit; RANTES, regulated upon activation, normal T-cell expressed protein; RdRp, RNA-dependent RNA polymerase; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; TNF-α, tumor necrosis factor alpha.