Age-related rhesus macaque models of COVID-19.

BACKGROUND: Since December 2019, an outbreak of the Corona Virus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2) in Wuhan, China, has become a public health emergency of international concern. The high fatality of aged cases caused by SARS-CoV-2 was a need to explore the possible age-related phenomena with non-human primate models. METHODS: Three 3-5 years old and two 15 years old rhesus macaques were intratracheally infected with SARS-CoV-2, and then analyzed by clinical signs, viral replication, chest X-ray, histopathological changes and immune response. RESULTS: Viral replication of nasopharyngeal swabs, anal swabs and lung in old monkeys was more active than that in young monkeys for 14 days after SARS-CoV-2 challenge. Monkeys developed typical interstitial pneumonia characterized by thickened alveolar septum accompanied with inflammation and edema, notably, old monkeys exhibited diffuse severe interstitial pneumonia. Viral antigens were detected mainly in alveolar epithelial cells and macrophages. CONCLUSION: SARS-CoV-2 caused more severe interstitial pneumonia in old monkeys than that in young monkeys. Rhesus macaque models infected with SARS-CoV-2 provided insight into the pathogenic mechanism and facilitated the development of vaccines and therapeutics against SARS-CoV-2 infection.

Sequential immunizations confer cross-protection against variants of SARS-CoV-2, including Omicron in Rhesus macaques.

Variants of concern (VOCs) like Delta and Omicron, harbor a high number of mutations, which aid these viruses in escaping a majority of known SARS-CoV-2 neutralizing antibodies (NAbs). In this study, Rhesus macaques immunized with 2-dose inactivated vaccines (Coronavac) were boosted with an additional dose of homologous vaccine or an RBD-subunit vaccine, or a bivalent inactivated vaccine (Beta and Delta) to determine the effectiveness of sequential immunization. The booster vaccination significantly enhanced the duration and levels of neutralizing antibody titers against wild-type, Beta, Delta, and Omicron. Animals administered with an indicated booster dose and subsequently challenged with Delta or Omicron variants showed markedly reduced viral loads and improved histopathological profiles compared to control animals, indicating that sequential immunization could protect primates against Omicron. These results suggest that sequential immunization of inactivated vaccines or polyvalent vaccines could be a potentially effective countermeasure against newly emerging variants.

Integrated histopathological, lipidomic, and metabolomic profiles reveal mink is a useful animal model to mimic the pathogenicity of severe COVID-19 patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is transmitted on mink farms between minks and humans in many countries. However, the systemic pathological features of SARS-CoV-2-infected minks are mostly unknown. Here, we demonstrated that minks were largely permissive to SARS-CoV-2, characterized by severe and diffuse alveolar damage, and lasted at least 14 days post inoculation (dpi). We first reported that infected minks displayed multiple organ-system lesions accompanied by an increased inflammatory response and widespread viral distribution in the cardiovascular, hepatobiliary, urinary, endocrine, digestive, and immune systems. The viral protein partially co-localized with activated Mac-2+ macrophages throughout the body. Moreover, we first found that the alterations in lipids and metabolites were correlated with the histological lesions in infected minks, especially at 6 dpi, and were similar to that of patients with severe and fatal COVID-19. Particularly, altered metabolic pathways, abnormal digestion, and absorption of vitamins, lipids, cholesterol, steroids, amino acids, and proteins, consistent with hepatic dysfunction, highlight metabolic and immune dysregulation. Enriched kynurenine in infected minks contributed to significant activation of the kynurenine pathway and was related to macrophage activation. Melatonin, which has significant anti-inflammatory and immunomodulating effects, was significantly downregulated at 6 dpi and displayed potential as a targeted medicine. Our data first illustrate systematic analyses of infected minks to recapitulate those observations in severe and fetal COVID-19 patients, delineating a useful animal model to mimic SARS-CoV-2-induced systematic and severe pathophysiological features and provide a reliable tool for the development of effective and targeted treatment strategies, vaccine research, and potential biomarkers.

SARS-CoV-2 crosses the blood-brain barrier accompanied with basement membrane disruption without tight junctions alteration.

SARS-CoV-2 has been reported to show a capacity for invading the brains of humans and model animals. However, it remains unclear whether and how SARS-CoV-2 crosses the blood-brain barrier (BBB). Herein, SARS-CoV-2 RNA was occasionally detected in the vascular wall and perivascular space, as well as in brain microvascular endothelial cells (BMECs) in the infected K18-hACE2 transgenic mice. Moreover, the permeability of the infected vessel was increased. Furthermore, disintegrity of BBB was discovered in the infected hamsters by administration of Evans blue. Interestingly, the expression of claudin5, ZO-1, occludin and the ultrastructure of tight junctions (TJs) showed unchanged, whereas, the basement membrane was disrupted in the infected animals. Using an in vitro BBB model that comprises primary BMECs with astrocytes, SARS-CoV-2 was found to infect and cross through the BMECs. Consistent with in vivo experiments, the expression of MMP9 was increased and collagen IV was decreased while the markers for TJs were not altered in the SARS-CoV-2-infected BMECs. Besides, inflammatory responses including vasculitis, glial activation, and upregulated inflammatory factors occurred after SARS-CoV-2 infection. Overall, our results provide evidence supporting that SARS-CoV-2 can cross the BBB in a transcellular pathway accompanied with basement membrane disrupted without obvious alteration of TJs.

Sequential infection with H1N1 and SARS-CoV-2 aggravated COVID-19 pathogenesis in a mammalian model, and co-vaccination as an effective method of prevention of COVID-19 and influenza.

Influenza A virus may circulate simultaneously with the SARS-CoV-2 virus, leading to more serious respiratory diseases during this winter. However, the influence of these viruses on disease outcome when both influenza A and SARS-CoV-2 are present in the host remains unclear. Using a mammalian model, sequential infection was performed in ferrets and in K18-hACE2 mice, with SARS-CoV-2 infection following H1N1. We found that co-infection with H1N1 and SARS-CoV-2 extended the duration of clinical manifestation of COVID-19, and enhanced pulmonary damage, but reduced viral shedding of throat swabs and viral loads in the lungs of ferrets. Moreover, mortality was increased in sequentially infected mice compared with single-infection mice. Compared with single-vaccine inoculation, co-inoculation of PiCoVacc (a SARS-CoV-2 vaccine) and the flu vaccine showed no significant differences in neutralizing antibody titers or virus-specific immune responses. Combined immunization effectively protected K18-hACE2 mice against both H1N1 and SARS-CoV-2 infection. Our findings indicated the development of systematic models of co-infection of H1N1 and SARS-CoV-2, which together notably enhanced pneumonia in ferrets and mice, as well as demonstrated that simultaneous vaccination against H1N1 and SARS-CoV-2 may be an effective prevention strategy for the coming winter.

Susceptibility and Attenuated Transmissibility of SARS-CoV-2 in Domestic Cats.

Domestic cats, an important companion animal, can be infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). This has aroused concern regarding the ability of domestic cats to spread the virus that causes coronavirus disease 2019. We systematically demonstrated the pathogenesis and transmissibility of SARS-CoV-2 in cats. Serial passaging of the virus between cats dramatically attenuated the viral transmissibility, likely owing to variations of the amino acids in the receptor-binding domain sites of angiotensin-converting enzyme 2 between humans and cats. These findings provide insight into the transmissibility of SARS-CoV-2 in cats and information for protecting the health of humans and cats.

SARS-CoV-2 Causes a Systemically Multiple Organs Damages and Dissemination in Hamsters.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has spread across the world and impacted global healthcare systems. For clinical patients, COVID-19 not only induces pulmonary lesions but also affects extrapulmonary organs. An ideal animal model that mimics COVID-19 in humans in terms of the induced systematic lesions is urgently needed. Here, we report that Syrian hamster is highly permissive to SARS-CoV-2 and exhibit diffuse alveolar damage and induced extrapulmonary multi-organs damage, including spleen, lymph nodes, different segments of alimentary tract, kidney, adrenal gland, ovary, vesicular gland and prostate damage, at 3-7 days post inoculation (dpi), based on qRT-PCR, in situ hybridization and immunohistochemistry detection. Notably, the adrenal gland is a novel target organ, with abundant viral RNA and antigen expression detected, accompanied by focal to diffuse inflammation. Additionally, viral RNA was also detected in the corpus luteum of the ovary, vesicular gland and prostate. Focal lesions in liver, gallbladder, myocardium, and lymph nodes were still present at 18 dpi, suggesting potential damage after disease. Our findings illustrate systemic histological observations and the viral RNA and antigen distribution in infected hamsters during disease and convalescence to recapitulate those observed in humans with COVID-19, providing helpful data to the pathophysiologic characterization of SARS-CoV-2-induced systemic disease and the development of effective treatment strategies.

Ocular conjunctival inoculation of SARS-CoV-2 can cause mild COVID-19 in rhesus macaques.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly transmitted through the respiratory route, but potential extra-respiratory routes of SARS-CoV-2 transmission remain uncertain. Here we inoculated five rhesus macaques with 1 × 106 TCID50 of SARS-CoV-2 conjunctivally (CJ), intratracheally (IT), and intragastrically (IG). Nasal and throat swabs collected from CJ and IT had detectable viral RNA at 1-7 days post-inoculation (dpi). Viral RNA was detected in anal swabs from only the IT group at 1-7 dpi. Viral RNA was undetectable in tested swabs and tissues after intragastric inoculation. The CJ infected animal had a higher viral load in the nasolacrimal system than the IT infected animal but also showed mild interstitial pneumonia, suggesting distinct virus distributions. This study shows that infection via the conjunctival route is possible in non-human primates; further studies are necessary to compare the relative risk and pathogenesis of infection through these different routes in more detail.

Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 via Close Contact and Respiratory Droplets Among Human Angiotensin-Converting Enzyme 2 Mice.

We simulated 3 transmission modes, including close-contact, respiratory droplets and aerosol routes, in the laboratory. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be highly transmitted among naive human angiotensin-converting enzyme 2 (hACE2) mice via close contact because 7 of 13 naive hACE2 mice were SARS-CoV-2 antibody seropositive 14 days after being introduced into the same cage with 3 infected-hACE2 mice. For respiratory droplets, SARS-CoV-2 antibodies from 3 of 10 naive hACE2 mice showed seropositivity 14 days after introduction into the same cage with 3 infected-hACE2 mice, separated by grids. In addition, hACE2 mice cannot be experimentally infected via aerosol inoculation until continued up to 25 minutes with high viral concentrations.

Primary exposure to SARS-CoV-2 protects against reinfection in rhesus macaques.

Coronavirus disease 2019 (COVID-19), which is caused by infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic. It is unclear whether convalescing patients have a risk of reinfection. We generated a rhesus macaque model of SARS-CoV-2 infection that was characterized by interstitial pneumonia and systemic viral dissemination mainly in the respiratory and gastrointestinal tracts. Rhesus macaques reinfected with the identical SARS-CoV-2 strain during the early recovery phase of the initial SARS-CoV-2 infection did not show detectable viral dissemination, clinical manifestations of viral disease, or histopathological changes. Comparing the humoral and cellular immunity between primary infection and rechallenge revealed notably enhanced neutralizing antibody and immune responses. Our results suggest that primary SARS-CoV-2 exposure protects against subsequent reinfection in rhesus macaques.

The pathogenicity of SARS-CoV-2 in hACE2 transgenic mice.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019 (COVID-19), which has become a public health emergency of international concern1. Angiotensin-converting enzyme 2 (ACE2) is the cell-entry receptor for severe acute respiratory syndrome coronavirus (SARS-CoV)2. Here we infected transgenic mice that express human ACE2 (hereafter, hACE2 mice) with SARS-CoV-2 and studied the pathogenicity of the virus. We observed weight loss as well as virus replication in the lungs of hACE2 mice infected with SARS-CoV-2. The typical histopathology was interstitial pneumonia with infiltration of considerable numbers of macrophages and lymphocytes into the alveolar interstitium, and the accumulation of macrophages in alveolar cavities. We observed viral antigens in bronchial epithelial cells, macrophages and alveolar epithelia. These phenomena were not found in wild-type mice infected with SARS-CoV-2. Notably, we have confirmed the pathogenicity of SARS-CoV-2 in hACE2 mice. This mouse model of SARS-CoV-2 infection will be valuable for evaluating antiviral therapeutic agents and vaccines, as well as understanding the pathogenesis of COVID-19.