Human Adenovirus and Influenza A Virus Exacerbate SARS-CoV-2 Infection in Animal Models

Abstract

In this study, we investigated the features of the infectious process by simulating co-infection with SARS-CoV-2 and human adenovirus type 5 (HAdV-5) or influenza A virus (IAV) in vitro and in vivo. The determination of infectious activity of viruses and digital PCR demonstrated that during simultaneous and sequential HAdV-5 followed by SARS-CoV-2 infection in vitro and in vivo, the HAdV-5 infection does not interfere with replication of SARS-CoV-2. The hamsters co-infected and mono-infected with SARS-CoV-2 exhibited nearly identical viral titers and viral loads of SARS-CoV-2 in the lungs. The hamsters and ferrets co-infected by SARS-CoV-2- and IAV demonstrated more pronounced clinical manifestations than mono-infected animals. Additionally, the lung histological data illustrate that HAdV-5 or IAV and SARS-CoV-2 co-infection induces more severe pathological changes in the lungs than mono-infection. The expression of several genes specific to interferon and cytokine signaling pathways in the lungs of co-infected hamsters was more upregulated compared to single infected with SARS-CoV-2 animals. Thus, co-infection with HAdV-5 or IAV and SARS-CoV-2 leads to more severe pulmonary disease in animals.

Keywords: SARS-CoV-2; coinfection; ferret; hamster; human adenovirus 5; influenza A virus.

Figure 1

Body weight changes in monoinfected and coinfected with HAdV-5 and SARS-CoV-2 Syrian hamsters. Body weight changes in monoinfected and coinfected with HAdV-5 and SARS-CoV-2 Syrian hamsters. “CoV (SARS-CoV-2)”—on Day 0, the Syrian hamsters were intranasally challenged with SARS-CoV-2 (10⁵ TCID₅₀); “ad (HAdV-5)/3-days/CoV”—on Day 0, the preinfected (3 days earlier) with HAdV-5 (10⁶ TCID₅₀) hamsters were intranasally challenged with SARS-CoV-2 (10⁵ TCID₅₀); “ad”—on Day 0, the Syrian hamsters were intranasally challenged with HAdV-5 (10⁶ TCID₅₀); “mock-infected”—on Day 0, the hamsters were intranasally inoculated wits PBS. n = 9 at 0 dpi to 4 dpi; n = 6 at 5 dpi to 6 dpi as 3 animals were sacrificed; n = 3 at 7 dpi to 12 dpi as 3 animals were sacrificed. The values represent the means ± SDs of individual animals. Student’s t-test was used for two-group comparisons, p < 0.05.

Figure 2

SARS-CoV-2 and HadV-5 replication in monoinfected and coinfected hamsters. (A) SARS-CoV-2 and HadV-5 viral infectious titers; (B) SARS-CoV-2 and HadV-5 viral genome loads. SARS-CoV-2—Syrian hamsters were intranasally challenged with SARS-CoV-2 (10⁵ TCID₅₀); SARS-CoV-2/HadV-5—Syrian hamsters were simultaneously intranasally challenged with SARS-CoV-2 (10⁵ TCID₅₀) and HadV-5 (10⁶ TCID₅₀); HadV-5/3-days/SARS-CoV-2—Syrian hamsters were intranasally challenged with HadV-5 (10⁶ TCID₅₀) and, 3 days later, with SARS-CoV-2 (10⁵ TCID₅₀); ad—Syrian hamsters were intranasally challenged with HadV-5 (10⁶ TCID₅₀). On Day 3 postinfection (for the ad/3 days/CoV group, this took place on Day 3 after the challenge with SARS-CoV-2), half of the lung tissues collected from hamsters were homogenized to determine the viral infectious titers and RNA/DNA genome loads. (A) The SARS-CoV-2 and HadV-5 titers, expressed as the 50% tissue culture infectious doses per gram (TCID₅₀/g), were determined by the CPE assay in Vero E6 (Table S2) and HEK293A (Table S3) cells, respectively. (B) The lung homogenates were analyzed for viral genome loads by dPCR. The SARS-CoV-2 and HadV-5 genome loads were determined using primers targeting the 1ab and hexon genes, respectively. The values represent the means ± SDs of three hamsters. Student’s t-test was used for two-group comparisons, * p< 0.05.

Figure 3

SARS-CoV-2 and IAV replication in monoinfected and coinfected hamsters and ferrets. (A,C) SARS-CoV-2 viral genome loads were determined using qPCR for the 1ab gene in nasopharyngeal washes of hamsters and ferrets, respectively. (B,D) IAV viral genome loads were determined using qPCR for the M gene in nasopharyngeal washes of hamsters and ferrets, respectively. SARS-CoV-2—the Syrian hamsters and ferrets were intranasally challenged with SARS-CoV-2 (10⁴ TCID₅₀ and 3 × 10⁵ TCID₅₀, respectively); IAV—the Syrian hamsters and ferrets were intranasally challenged with IAV (2 × 10³ TCID₅₀ and 10² TCID₅₀, respectively); IAV/SARS-CoV-2—the Syrian hamsters were intranasally challenged with IAV (2 × 10³ TCID₅₀) and, 4 days later, with SARS-CoV-2 (10⁴ TCID₅₀); the ferrets were intranasally challenged with IAV (10² TCID₅₀) and, 2 days later, with SARS-CoV-2 (3 × 10⁵ TCID₅₀). On Days 6, 8, and 10 after infection with IAV for the hamsters and on Days 4, 6, and 8 after infection with IAV for the ferrets, nasopharyngeal washes were collected from all animals to determine the viral genome loads. The nasopharyngeal washes were analyzed by qPCR. The SARS-CoV-2 and IAV viral genome loads were determined using primers targeting the 1ab and M genes, respectively. The values represent the means ± SDs of three individual animals. Student’s t-test was used for two-group comparisons, * p < 0.05.

Figure 4

Body weight changes in monoinfected and coinfected with IAV and SARS-CoV-2 animals. (A) Body weight changes in monoinfected and coinfected with IAV and SARS-CoV-2 ferrets. “CoV (SARS-CoV-2)”—on Day 0, the ferrets were intranasally challenged with SARS-CoV-2 (3 × 10⁵ TCID₅₀); “IAV”—on Day 0, the ferrets were intranasally challenged with IAV (10² TCID₅₀); “IAV/CoV”—on Day 0, the ferrets preinfected (2 days earlier) with IAV (10² TCID₅₀) were intranasally challenged with SARS-CoV-2 (3 × 10⁵ TCID₅₀); “mock-infected”—on Day 0, the ferrets were intranasally inoculated with PBS. n = 4. (B) Body weight changes in monoinfected and coinfected with IAV and SARS-CoV-2 hamsters. “CoV SARS-CoV-2)”—on Day 0, the hamsters were intranasally challenged with SARS-CoV-2 (10⁴ TCID₅₀); “IAV”—on Day 0, the hamsters were intranasally challenged with IAV (2 × 10³ TCID₅₀); “IAV/CoV”—on Day 0, the hamsters preinfected (4 days earlier) with IAV (2 × 10³ TCID₅₀) were intranasally challenged with SARS-CoV-2 (10⁴ TCID₅₀); “mock-infected”—on Day 0, the hamsters were intranasally inoculated with PBS. n = 8 at 0 dpi to 6 dpi; n = 4 at 7 dpi to 12 dpi, as 4 animals were sacrificed. The values represent the means ± SDs of individual animals. Student’s t-test was used for two-group comparisons. * p < 0.05.

Figure 5

Histopathological alterations in hamster lungs monoinfected (HAdV-5, SARS-CoV-2, IAV) and coinfected with HAdV-5 and SARS-CoV-2. (A) mock-infected control animal, normal lung. (B–E) Representative images showing pathological changes in lung tissue after infection: (B)—with HAdV-5; (C)—with HAdV-5 and SARS-CoV-2 simultaneously; (D)—with SARS-CoV-2; (E)—with HAdV-5 and, 3 days later, with SARS-CoV-2. In both cases of coinfection with two viruses (C,E), there was a significant increase in the consolidation zones and pronounced perifocal compensatory atelectasis. (F) Manifestations of adenovirus infection: edema and mixed inflammatory cell infiltration of the interalveolar septa and, in the upper part of the image, pronounced plethora of the capillaries. (G–K) Typical pathological changes found in mixed infections. (G) Blood (black arrows), macrophages, and syncytium (white arrow) in the lumen of the alveoli and the activation and hyperplasia of type II pneumocytes. (H) Hemorrhage in the alveoli (black arrows). (I) Spasm of small vessels of the arterial type (black arrow); perivascular edema and perivascular lymphocytic infiltration (white arrow); desquamation of the epithelial lining of the bronchi (red arrow). (J) Destruction of the small bronchial wall (bronchiolitis, black arrow); desquamated bronchial epithelium in the lumen (red arrow); peribronchial and perivascular infiltration (white arrow). (K) Zone of consolidation of lung tissue with spasmodic arterioles (black arrow). (L–N) Manifestations of IAV infection: epithelial cell degeneration, vasculitis, and perivascular lymphocyte infiltration, respectively (black arrows).

Figure 5

Histopathological alterations in hamster lungs monoinfected (HAdV-5, SARS-CoV-2, IAV) and coinfected with HAdV-5 and SARS-CoV-2. (A) mock-infected control animal, normal lung. (B–E) Representative images showing pathological changes in lung tissue after infection: (B)—with HAdV-5; (C)—with HAdV-5 and SARS-CoV-2 simultaneously; (D)—with SARS-CoV-2; (E)—with HAdV-5 and, 3 days later, with SARS-CoV-2. In both cases of coinfection with two viruses (C,E), there was a significant increase in the consolidation zones and pronounced perifocal compensatory atelectasis. (F) Manifestations of adenovirus infection: edema and mixed inflammatory cell infiltration of the interalveolar septa and, in the upper part of the image, pronounced plethora of the capillaries. (G–K) Typical pathological changes found in mixed infections. (G) Blood (black arrows), macrophages, and syncytium (white arrow) in the lumen of the alveoli and the activation and hyperplasia of type II pneumocytes. (H) Hemorrhage in the alveoli (black arrows). (I) Spasm of small vessels of the arterial type (black arrow); perivascular edema and perivascular lymphocytic infiltration (white arrow); desquamation of the epithelial lining of the bronchi (red arrow). (J) Destruction of the small bronchial wall (bronchiolitis, black arrow); desquamated bronchial epithelium in the lumen (red arrow); peribronchial and perivascular infiltration (white arrow). (K) Zone of consolidation of lung tissue with spasmodic arterioles (black arrow). (L–N) Manifestations of IAV infection: epithelial cell degeneration, vasculitis, and perivascular lymphocyte infiltration, respectively (black arrows).

Figure 6

Pathological lesions in hamster lungs 10 days after monoinfection with IAV (B,C) or on Day 10 after infection with IAV and Day 6 after infection with SARS-CoV-2 for the case of coinfection (D,E). Each of the six survey images shows the lung tissue of one animal (the three on the left—the left lung; the three on the right—the right lung) to give a representative idea of the nature and prevalence of the pathology. (A) Mock-infected animal. (D,E) In double-infected animals, zones of dense consolidation of lung tissue caused by edema and cellular infiltration, mainly lymphocytic, spread to the entire anatomical lobe. Staining with hematoxylin–eosin. All images were taken using a 20× objective and are presented at the same scale. The scale bar is shown in the pictures.

Figure 7

The chemokine/cytokine profile in the lungs of hamsters coinfected with SARS-CoV-2 and HAdV-5 or IAV. “CoV (SARS-CoV-2)”—on Day 0, the hamsters were intranasally challenged with SARS-CoV-2 (10⁵ TCID₅₀); “ad5 (HAdV-5)/CoV”—on Day 0, the hamsters preinfected (3 days earlier) with HAdV-5 (10⁶ TCID₅₀) were intranasally challenged with SARS-CoV-2 (10⁵ TCID₅₀); IAV/CoV—on Day 0, the hamsters preinfected (4 days earlier) with IAV (2 × 10³ TCID₅₀) were intranasally challenged with SARS-CoV-2 (10⁴ TCID₅₀); dpi—day postinfection with SARS-CoV-2. The data are expressed as fold changes relative to the mock-infected group. The values represent the means ± SDs of three individual animals. Student’s t-test was used for two-group comparisons, p < 0.05.

Figure 7

The chemokine/cytokine profile in the lungs of hamsters coinfected with SARS-CoV-2 and HAdV-5 or IAV. “CoV (SARS-CoV-2)”—on Day 0, the hamsters were intranasally challenged with SARS-CoV-2 (10⁵ TCID₅₀); “ad5 (HAdV-5)/CoV”—on Day 0, the hamsters preinfected (3 days earlier) with HAdV-5 (10⁶ TCID₅₀) were intranasally challenged with SARS-CoV-2 (10⁵ TCID₅₀); IAV/CoV—on Day 0, the hamsters preinfected (4 days earlier) with IAV (2 × 10³ TCID₅₀) were intranasally challenged with SARS-CoV-2 (10⁴ TCID₅₀); dpi—day postinfection with SARS-CoV-2. The data are expressed as fold changes relative to the mock-infected group. The values represent the means ± SDs of three individual animals. Student’s t-test was used for two-group comparisons, p < 0.05.