2021 Volume 4 Issue 1 Pages 20-24
The COVID-19 is still raging worldwide. COVID-19 has shown severe pathogenicity in the elderly and people with underlying diseases. Thus, it is necessary to elucidate the pathology in patients with underlying diseases and in elderly patients. The use of an appropriate animal model is needed to overcome COVID-19; however, healthy young animals are usually used as experimental animals. Here, we discuss SARS-CoV-2 infection and related pathological conditions in cynomolgus monkeys including those with underlying diseases causing severe pathogenicity such as metabolic disease and advanced age. Cynomolgus macaques, with various clinical conditions and ages, were infected with SARS-CoV-2. There were differences between the pathologies of young and elderly macaques with underlying diseases. Consistent with humans, both viral RNA and infectious virus particles in pharyngeal swabs was higher in the aged macaques than in the young macaques. These were for a long period of time in the aged macaques. Inflammation due to pneumonia lasted for a longer period in the aged macaques. Therefore, the COVID-19 cynomolgus monkey model would be useful for elucidating COVID-19 pathophysiology, as well as, developing therapeutic and prophylactic agents against this disease.
The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in a miserable reality for global health and life with cases continually increasing worldwide. Although rapid vaccination has been progressing in the world as a countermeasure against COVID-19, there are many issues regarding this disease. This includes the emergence of variants and of their virulence that need to be elucidated. Thus, a useful animal model is needed.
The presentation of SARS-CoV-2 infection, which has been named coronavirus disease 2019 (COVID-19), ranges from none to severe symptoms. COVID-19 has shown severe pathogenicity in the elderly and people with underlying diseases [1, 2]. Many infectious diseases such as influenza are more severe in the elderly, but at the same time, young individuals are also highly susceptible. However, the pathology of COVID-19 is complicated and it is extremely mild in young people. Therefore, in addition to elucidating the pathological features in healthy individuals, it is necessary to elucidate its pathology in patients with underlying diseases and in elderly patients.
Cynomolgus monkeys (CMs), which are common laboratory animals among non-human primates, show various human-like characteristics. These include high brain functions, long life span, single pregnancy, and regular menstrual period, that are not found in other experimental animals. We have established COVID-19 model CMs using healthy young CMs and CMs of advanced age CMs (23−30 years of age, equivalent to 69−90 years of age in humans) with underlying diseases including diabetes and hyperlipidemia [3]. Here we discuss the differences in SARS-CoV-2 infection in the young and elderly CMs and the similarities of its pathophysiology in humans and our established COVID-19 CM models.
Several non-human primate (NHP) models of COVID-19 have been reported [4,5,6,7,8,9,10,11], and there have also been some reports on a middle-aged monkey model [5, 6, 8]. Unlike the models in those studies, elderly monkeys of more than 23 years of age and with metabolic diseases such as diabetes and hyperlipidemia were examined in our study [3]. CMs housed at the Tsukuba Primate Research Center, National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN, Ibaraki, Japan) were used following approval by the Committee on the Ethics of Animal Experiments of NIBIOHN in accordance with the guidelines for animal experiments at NIBIOHN. Two groups of CMs (a young group that included 4 CMs (2 males and 2 females) aged 3–8 years and an elderly group of 5 female CMs aged 23–29 years) were used in this comparison study (Table 1). All of the young CMs were healthy and some CMs in the elderly group had an underlying disease (Table 1). The nine CMs were infected with 1 × 106 TCID50 SARS-CoV-2 via a combination of intratracheal (IT) and intranasal (IN) routes (Table 1). After SARS-CoV-2 inoculation, all of the young CMs developed fever, which is a common COVID-19 symptoms (Fig. 1A). Several cytokines and chemokines have been associated with SARS-CoV-2 infection and disease severity [1, 12,13,14,15,16]. Changes in one cytokine (interleukin (IL)-6), one cytokine-related protein (IL-1 receptor antagonist (IL-1RA)), and three chemokines (Eotaxin, monocyte chemoattractant protein (MCP)-1, and I-TAC (CXCL11)) were observed immediately after infection in most of the CMs. Most of these cytokines and chemokines were transiently but significantly elevated in each animal compared to the levels between the indicated time points (Fig. 1B). These cytokine and chemokines are also aggravated in humans, suggesting that this COVID-19 CM model can be used in future pathological studies related to these factors.
| Group | Number | Sex | Age | Underlying disease | Infection dose | Infection route |
|---|---|---|---|---|---|---|
| Young | n=4 | M: 2, F: 2 | 3–8 | 1 × 106 TCID50 | IT, IN | |
| Elderly* | n=5 | F: 5 | 23–30 | DM§, HL||, Obesity | 1 × 106 TCID50 | IT, IN |
| Control | n=3 | M: 2, F: 1 | 7–8 | PBS | IT, IN |
§DM (diabetes mellitus), ||HL (hyperlipemia). *In the elderly group, one CM had DM and HL, one CM had obesity and HL, and one CM had HL and prediabetes.
Comparison of clinical symptoms, biomedical changes, and viral dynamics in SARS-CoV-2-infected young and elderly cynomolgus macaques. All CMs were infected with 1 × 106 TCID50 SARS-CoV-2 via a combination of IT and IN routes. (A) Daily body temperature was recorded by an intraperitoneally embedded data logger (DST milli-HRT; Star-Oddi) in young SARS-CoV-2-infected monkeys. Body temperature in PBS-administered control young animals is indicated by a blue line. (B) Levels of cytokines and chemokines in serum after SARS-CoV-2 infection. The cytokine and chemokines shown were increased in association with the infection. Statistical analyses between the indicated time points were performed using the Wilcoxon test. *P<0.05; **P<0.01. (C) Determination of viral shedding by measurements of viral loads and viral titers in nasal, pharynx and rectal swab samples. Viral loads determined by RT-PCR in the groups are indicated by circles (blue: young, red: elderly). Infectious viral titers determined by TCID50 using VeroE6/TMPRSS2 cells are indicated by squares (blue: young, red: elderly). (D–E) Representative chest CT 3D images analyzed by AZE Virtual Place (Canon). Inflammation areas are highlighted in red. Typical pneumonia images at 5 d.p.i (D) and lung inflammation images in young and elderly CM at 5 d.p.i and 12 d.p.i (E). (F) Detection of viral RNA in indicated tissues and organs in a SARS-CoV-2-infected CM. The heat map shows levels of detected viral RNA (left: equivalent TCID50/mg) and sgRNA (right: copy/g) at 7 or 14 days post infection.
Comparisons of viral shedding times and durations in the young and elderly groups are shown in (Fig. 1C). When the viral titer was examined using the mucosal swab samples in VeroE6/TMPRSS2 cells, infectious viruses were detected in the nasal and pharyngeal samples for longer periods in the elderly monkeys than in the young monkeys (Fig. 1D). Pneumonia was observed by computed tomography (CT) in all CMs except for one elderly CM with obesity due to the lack of a clear chest image probably caused by its obese status. Typically, the inflammation peaked at five days after infection (Fig. 1D). No significant differences were found after analyzing CT inflammatory images between the young and elderly monkeys. However, inflammation was observed at multiple locations and persisted for a longer period in elderly monkeys (Fig. 1E). This is consistent with the results from studies which showed that the incidence of multiple ground-glass opacities was higher in elderly patients than in young patients [17,18,19].
Viral distribution in systemic organs was analyzed (Fig. 1F). One monkey in each group was dissected at 7 or 14 days after infection. Samples were obtained from various organs for assessment of SARS-CoV-2 localization and were scaled for the weight of each organ piece. Loads of the virus detected by RT-qPCR in organs were measured by a previously reported method for assessing vaccine efficacy so that all individuals could be compared equally [20, 21]. To collect fragments of the same sizes from the same sites of the organs of each animal, a template using a biopsy punch instrument was placed over the organ. At seven days after infection, the virus was detected in various tissues and organs by qPCR of the N gene in both young and elderly CMs. Subgenome RNA (sgRNA) is thought to reflect the viral replication in infected cells [22, 23]. When subgenome qPCR was performed, higher positive reactions were detected in the lungs of the elderly CM. Moreover, sgRNA was also detected in mediastinal lymph nodes of the elderly CM (Fig. 1F). At 14 days after infection, both CMs showed positive reactions in qPCR of the N gene (Fig. 1F). Positive reactions were still observed in the lungs at the inoculation site and in many organs including the digestive organs in the elderly CM. However, sgRNA results were negative in all organs at 14 days after infection. The viral genome was detected in not only the respiratory region but also in the lymph nodes, major organs, and digestive tissues at day 7 or day 14. Since viral RNAs are often detected in the feces of patients with COVID-19, viral RNA in the rectal swab and the digestive tissues were detected in some CMs (Fig. 1C and 1F). However, sgRNA was not detected in regions other than the respiratory region in CMs tested in this study. Virus infection in intestinal enterocytes has been reported by several groups [24,25,26]. It is likely that the virus can infect intestinal tissue but its replication efficacy is not equivalent to that in the respiratory region.
Establishing appropriate animal models is essential for overcoming the COVID-19 pandemic. The cynomolgus macaque is considered to be one of the most suitable animals that reflect the pathological conditions of humans. In this study, we established animal models of COVID-19 using healthy CMs as well as CMs of advanced age and with pathological conditions such as metabolic diseases. The CMs infected with SARS-CoV-2 did not develop severe or fatal symptoms as in humans. Symptoms in the CMs were mild, but typical COVID-19 symptoms including fever, pneumonia, elevated viral titers, and inflammatory factors were observed. Notably, the viral load in pharyngeal swabs was higher in the aged CMs than in the young CMs. Moreover, both viral RNA and infectious virus were detected in nasal and pharyngeal swabs for a long period of time in the aged CMs, consistent with human infection (Fig. 1C) [27]. Generally, research on virus susceptibility and aggravation has been conducted in juvenile animals. Since advanced age is one of the risk factors for increased severity of several infectious diseases, we believe that studies using various unique CM models such as an advanced age CM model will be very important for understanding the variations of pathological conditions caused not only by SARS-CoV-2 but also by other pathogens.
Overall, the SARS-CoV-2-infected CM models reflected the pathology in humans. Studies using COVID-19 CM models should contribute to elucidating the complicated pathophysiology of COVID-19 and to the development of vaccines and therapeutic agents.
The authors have nothing to disclose.
