Hamsters inoculated with BA.5 lose less body weight compared to D614G inoculated hamsters.
Given the importance of viral shedding in the context of transmission studies, we determined a minimized infectious dose required to achieve reproducible infection and viral shedding for D614G and Omicron BA.5. Groups of female and male hamsters were intranasally inoculated with 20 µl containing a low (103 TCID50 per hamster) or moderate (104 TCID50 per hamster) dose (Supplementary Fig. 1A, B). For an initial conformation of productive infection of hamsters, seroconversion was assessed at 7 DPI; all hamsters seroconverted (Supplementary Fig. 1C, D). Overall, no significant difference in body weight loss was observed between the low and moderate dose for either variant (Fig. 1A, B). Compared to animals inoculated with Omicron BA.5, hamsters inoculated with D614G lost more body weight.
Male hamsters lose more body weight than female hamsters.
After inoculation with D614G, male hamsters lost approximately 10% of their body weight between 0 and 6 DPI. The body weight of female hamsters remained stable. Relative body weights of female hamsters were significantly higher than those of male hamsters between 1 DPI and 7 DPI (low dose) and between 6 DPI and 7 DPI (moderate dose), indicating a sex-dependent effect of SARS-CoV-2 infections (Fig. 1A). Almost no body weight loss was observed in either female or male hamsters inoculated with Omicron BA.5, but female hamsters gained more weight compared to males (Fig. 1B).
Hamsters inoculated with BA.5 have lower viral genome loads compared to D614G inoculated hamsters.
Throat swabs were obtained daily and viral replication kinetics were assessed by RT-qPCR and virus isolation. SARS-CoV-2 genomes were detected in all inoculated hamsters by RT-qPCR (Fig. 1C, D). Viral genomes in the throats peaked on day 1 after SARS-CoV-2 D614G inoculation (comparable for low and moderate dose), and declined until the end of the experiment on 7 DPI (Fig. 1C). Viral genome levels were significantly higher in D614G-inoculated male then in female hamsters 1 DPI (moderate dose). Additionally, viral genome levels were significantly higher in male hamsters after inoculation with a moderate dose then with a low dose 1DPI. Viral genome levels of BA.5 were overall lower than those of D614G. After Omicron BA.5 inoculation, viral genomes peaked at 1 DPI (moderate dose) or 2 DPI (low dose) and declined towards the end of the study period (Fig. 1D). Male hamsters inoculated with a low dose of BA.5 had significantly higher viral genome levels 2 and 3 DPI than male hamsters inoculated with a moderate dose of BA.5. No significant difference in viral genome levels was detected between male and female hamsters after inoculation with a low or moderate dose during the following days.
Male hamsters shed more infectious D614G and BA.5 than female hamsters.
Virus isolations were performed to determine the infectious viral load in the throats of hamsters. All hamsters inoculated with a moderate dose, either D614G or BA.5, shed infectious virus (Fig. 1E, F). For the low dose, all hamsters shed infectious virus except for 1 out of 3 females inoculated with BA.5. Similar to the RT-qPCR data, infectious virus shedding peaked on day 1 after D614G inoculation, and on day 1 or 2 after BA.5 inoculation. Shedding of infectious D614G was up to 100-fold higher than that of BA.5. Notable discrepancies between the measurement of virus genomes and infections virus levels were observed for two aspects: (1) viral clearance, and (2) difference between male and female hamsters. In contrast to the genome levels, infectious virus loads declined fast and were no longer detected at 7 DPI. Notably, based on virus isolation data, the virus replication kinetics differed strongly between male and female hamsters in all groups. Up to 1,000-fold more infectious virus was shed by male hamsters, independent of dose and challenge virus. Taken together, hamsters can be reproducibly infected with a low dose and volume inoculum of D614G or Omicron BA.5. These experimental conditions were used for subsequent experiments. Compared to D614G, Omicron BA.5 led to lower viral loads and lower and delayed shedding of infectious virus. Shedding of infectious virus was always significantly higher in male than in female hamsters, independent of variant.
24 hour co-housing-time starting 20 HPI ensures consistent BA.5 direct-contact transmission.
Based on the virus isolation data, male hamsters were used to establish the Omicron BA.5 transmission model. As this variant was circulating at the time of experiment, we performed the subsequent experiments exclusively with this variant. To determine the optimal co-housing duration leading to consistent direct-contact transmission, each inoculated donor hamster was co-housed with 3 naïve hamsters starting 20 HPI of donor hamsters. At 6, 12 or 24 hours post co-housing start, acceptor animals were moved from infection cages to clean isolators (Supplementary Fig. 2A).
Similar to the dose-finding experiment, no body weight loss was observed in either donor or acceptor hamsters (Supplementary Fig. 2B). All donor hamsters proved productively infected based on RT-qPCR and virus isolation from throat swabs. Viral loads peaked on day 2 and declined until the donor hamsters were sacrificed on day 12 and 13 (Fig. 2A, D). Donor hamsters did not transmit Omicron BA.5 to acceptor hamsters when co-housed for 6 hours, and only 1 out of 6 acceptor hamsters was infected by transmission after 12 hours of co-housing. The viral replication kinetics in this hamster were similar to those observed in the donor hamsters, but with a delayed peak on day 4 post co-housing (6 DPI). Interestingly, the infected acceptor hamster did not transmit to his cage mates in the subsequent two weeks. A 24 hour co-housing period led to consistent transmission to acceptor hamsters. In comparison to the donor hamsters, the peak of viral genome in the throat was delayed, on day 3 post co-housing (5 DPI). Similar to the donor hamsters, viral loads decreased until the end of the experiment (Fig. 2B, E). Viral loads calculated as area under the curve (AUC) observed in donor hamsters were significantly higher than in acceptor hamsters of the 6 hour and 12 hour co-housing groups, and viral loads observed in the 24 hour co-housing groups were significantly higher than in the 6h co-housing groups (Fig. 2C, F). All inoculated donor and acceptor hamsters in which virus replication was detected seroconverted (Supplementary Fig. 2C).
Viral loads in the respiratory tract are lower after direct-contact transmission when compared to direct inoculation.
To characterize potential differences in pathogenesis after direct inoculation (donor hamsters) and infection by direct-contact transmission (acceptor hamsters), we performed a virological and histopathological comparison between the respiratory tissues. To that end, Omicron BA.5-inoculated donor hamsters were co-housed with three immunologically naïve acceptor hamsters for 24 hours starting 20 HPI (Supplementary Fig. 3A). No body weight loss was observed in donor and acceptor hamsters (Supplementary Fig. 3B). Viral loads in throat swabs as measured by RT-qPCR and virus isolation peaked on day 2 (donor) or day 3 (acceptor) post exposure (DPE; donor exposure = inoculation, acceptor exposure = co-housing end) and declined towards the end of the study period (Supplementary Fig. 3C, D). Donors (n = 4 per timepoint) and acceptors (n = 2 per timepoint) were sacrificed on 1 DPE, 2 DPE, 4 DPE, 7 DPE and at the end of experiment (EOE).
The nasal turbinates from donor and acceptor hamsters were RT-qPCR positive at all necropsy timepoints. Viral genome levels of donor hamsters peaked 2 DPE (geometric mean (gm): 1.8 x 107 TCID50eq/g tissue) and declined at later timepoints (EOE gm: 3.5 x 103 TCID50eq/g tissue). In comparison, viral genome loads in the nasal turbinates of acceptor animals were low 2 DPE (gm: 1.5 x 103 TCID50eq/g tissue), peaked 7 DPE (gm: 3.1 x 105 TCID50eq/g tissue) and decreased until EOE (gm: 1.88 x 102 TCID50eq/g tissue) (Fig. 3A). Viral genome loads in the trachea of donor and acceptor hamsters peaked 4 DPE with a gm of 1.5 x 105 TCID50eq/g tissue for donors and 4.5 x 103 TCID50eq/g tissue for acceptors. At EOE, the viral genome loads in the trachea from donor and acceptor hamsters decreased to low and undetectable RNA levels, respectively (Fig. 3B). Comparatively, low levels of RNA were detected in the lungs from donor and acceptor hamster, which peaked 4 DPE (gm: 1.2 x 103 TCID50eq/g tissue) and 7 DPE (gm: 4.0 x 103 TCID50eq/g tissue), respectively, and declined to low and undetectable RNA levels at EOE (Fig. 3C). Infectious virus levels in the nasal turbinates of directly-inoculated donors were high at 1–4 DPE, and decreased to undetectable levels at EOE. In the nasal turbinates of the acceptor hamsters viral titers remained below the lower limit of detection, with the exception of low-level replication at 4 DPE (gm: 3.5 x 102 TCID50/g tissue) and 7 DPE (gm: 7.5 x 101 TCID50/g tissue) (Fig. 3D). In the trachea, infectious virus was detectable in all hamsters on 1–4 DPE, but with a higher titer for the donor hamsters compared to the acceptors on all 4 days (peak gm: 2.1 x 104 TCID50/g tissue and 1.10 x 102 TCID50/g tissue, respectively) (Fig. 3E). Infectious virus was only detected in the lungs of donor hamsters 4 DPE at gm 1.4 x 102 TCID50/g tissue and no infectious virus was found in the lungs of acceptor hamsters (Fig. 3F).
Taken together, we detected virus (RT-qPCR, virus isolation, and antigen) in the upper respiratory tract of both donor and acceptor hamsters, and to a lower extent in the lower respiratory tract. Viral genome loads were overall higher and peaked earlier in the donor hamsters compared to the acceptors. Similarly, infectious virus levels were higher in the directly-inoculated donors and were overall only detected in the first days after exposure.
Mild inflammation in the respiratory tract of Omicron BA.5 infected donor and acceptor hamsters
Histopathological evaluation (Supplementary Table 1) of the nasal mucosae showed slight to mild inflammation in all donor and acceptor hamsters from 2 DPE onwards. This rhinitis was evidenced by intra-epithelial infiltrates of neutrophils and degenerated to sloughed epithelial cells (Supplementary Fig. 4A), that co-localized with cytoplasmic positivity for SARS-CoV-2 antigen stained by immunohistochemistry (IHC). Despite the absence of inflammation at 1 DPE, SARS-CoV-2 antigen staining was also positive at that timepoint (Fig. 4). Antigen was predominantly detected in the respiratory epithelium of the nasal mucosa, whereas in the 2 and 4 DPE donor hamsters the nasal olfactory mucosa was also positive. Regardless of belonging to donor or acceptor groups, nearly all hamsters showed faint ciliary to mild cytoplasmic viral positivity of tracheal mucosal epithelial cells, in conjunction with slight to mild concomitant inflammation consisting of infiltrating neutrophils only present in a few 4 and 7 DPE donor hamsters and in 7 DPE acceptor hamsters (Supplementary Fig. 4B). Likewise, the majority of hamsters in both the donor and acceptor groups showed mostly faint ciliary viral positivity of bronchial mucosal epithelial cells, devoid of inflammation. Minor chronic inflammatory consolidated alveolar foci in few lungs of 7 DPE donor and acceptor animals were interpreted as background lesions since these, and all others hamster lungs, were devoid of viral positivity by IHC, as were all other organs examined. In line with viral loads detected by RT-qPCR and virus isolation, donor hamsters overall showed (slightly) more inflammation and higher antigen positivity compared to acceptors (Supplementary Fig. 5). Taken together, histopathological evaluation indicated mild inflammation and viral antigen positivity in the upper respiratory tract and viral antigen negativity in the lower respiratory tract.