A study from Japan assessed the onset of SARS-CoV-2 virology upon infection in Syrian hamsters. Severe respiratory pathology was noted, in both the upper and lower respiratory tracts, along with a significant immune response that was protective against reinfection in previously-affected mice as well as other non-infected hamsters upon serum administration. These effects were demonstrated with both a low and high dose.

Application of Syrian Hamsters for Studying SARS-CoV-2 Virology

A study in Beijing, China was conducted on rhesus macaques and found that the monkeys were protected against subsequent infection by the identical strain of the SARS-CoV-2 virus once they entered the early recovery phase. 28 days post primary infection, researchers re-exposed the virus to four monkeys and detected no changes in viral load. Antibody titers were elevated at 14 days post re-exposure, and no viral RNA or infected cells were found in necropsy specimens following euthanasia.

Application of Rhesus Macaques for Studying SARS-CoV-2 Virology

A study from Wuhan, China investigated the potential routes of infection of COVID-19, focusing on mosquitoes as they are vectors for a number of other infectious diseases. The study conducted an experiment on cell lines from Aedes aegypti and Aedes albopictus and found no viral replication in the insect cell lines. Researchers also collected Culex and Anopheles mosquitoes from around Wuhan, China and found no infection by SARS-CoV-2 using a qRT-PCR detection assay. The study concludes that mosquitoes in general do not transmit SARS-CoV-2.

Application of Mosquitoes for Studying SARS-CoV-2 Virology

In this study performed by the Tulane National Primate Research Center, reports of physiological symptoms similar to patients with severe infections of SARS-CoV-2 were confirmed. Two cases of ARDS in African green monkeys (AGMs) and mild infections in 2 AGMs and 4 RMs infected with Covid-19 were observed. All the AGMs had increased levels of plasma IL-6 and three of the four infected AGMs had high levels of circulating cytokines. All of these factors indicate an immune response similar to those in humans and support the use of AGMs as animal models useful for observing the in vivo effects of ARDS. 

Study of the Application of Green African Monkeys

- Application of Rhesus Macaques for Studying SARS-CoV-2 Virology
- Application of Syrian Hamsters for Studying SARS-CoV-2 Virology
- Application of Mosquitoes for Studying SARS-CoV-2 Virology
- Application of African Green Monkeys for Studying SARS-CoV-2 Virology

Boston University

University of California, Davis

Tulane University

- Animal Studies on SARS-CoV-2 Virology
- Antigen Detection
- Classification
- Evolution and Mutations
- Methods of Host Cell Infection and Exploitation
- Stability in different environmental conditions
- Structure
- Vaccine Candidates, Immunogenicity, and Adaptive Immunity
- Reinfection

Focuses of Virology of COVID-19

While many animals have been used in laboratory studies of SARS-CoV-2, no model has been created that replicates the severity of the disease similar to hospitalized COVID-19 patients. In order to reduce COVID-19 morbidity and mortality, a reliable animal model is necessary to understand the biology of the infection as well as possible interventions. Animal models that produce severe infections, specifically pathogenesis of Covid-19 induced ARDS, are neccesary to develop reliable therapeutics. 

Potential animal models include:

- K18-hACE2 Mice
- AAV-hACE2 Mice
- HFH4-hACE2 Mice
- African Green Monkeys
- Rhesus Macaques

Animal Models Used to Study COVID-19

SARS-CoV-2 is comprised of four main structural components: the spike glycoprotein (S), the small envelope glycoprotein (E), the membrane glycoprotein (M), and the nucleocapsid protein (N). There are also several accesory proteins. 

Structure of SARS-CoV-2

- Manipulation of Innate Immune and Pro-inflammatory Response

Methods of Host Cell Infection and Exploitation

A study found that SARS-CoV-2 can be highly sensitive to heat. The virus was still detectable after being left out for 14 days in a 4°C room, while the virus inactive 5 minutes after being left in a 70°C. The study also found a difference in stability on different surfaces, such as paper, plastic, and metal.  At room temperature, the virus is highly stable in a range of pH3 - pH10. However, the standard disinfectant method was still able to affect the virus, with the researchers finding no active infection in different disinfectants after an incubation of 5 minutes (except for hand soap).

Stability of SARS-CoV-2 in different environmental conditions

Investigations are still ongoing in determining the current evolution and mutating nature of SARS-CoV-2, which will aid scientists in understanding more about this virus and in developing effective treatments and vaccines against the disease. 

Evolution and Mutations of SARS-CoV-2

Animal Studies on SARS-CoV-2 Virology

So far, vaccines consist of either inactivated or weakened versions of antigens or virions. Many pharmaceutical companies like Pfizer and Moderna are developing inactivated antigens in the form of mRNA, DNA, and proteins from the viral envelope or capsid that are often fragmented. 

Vaccine Candidates, Immunogenicity, and Adaptive Immunity

Until vaccine has passed all preclinical and clinical trials as well as FDA approval, the best approach to containing the pathogenicity and infectivity of COVID-19 will involve testing. The Safra center at Harvard advised to have up to 5 million tests being conducted within a day. Testing will not only slow down the spread, but also allow reopening of businesses and schools. So far, many testing methods involve viral RNAs or antigens. For detecting viral RNAs, most involve using RT-qPCR conjugated with Next Generation Sequencing to identify specific unique viral sequences. Recently, SHERLOCK detection technique utilizing CRISPR-Cas13 was tested in Broad Institute using a catalytically dead Cas13 to bind to specific viral sequences. On the other hand, detection of viral antigens involve either using ELISA with selected antibodies against viral antigens or using lateral flow assay.

Antigen Detection

Until August 28, 2020, there has been several cases of reinfections around the world (Hong Kong, Belgium, the Netherlands, and the US). 

Reinfection of SARS-CoV-2

The coronaviruses belong to the broad viral order Nidovirales within the family, Coronaviridae, notoriously known to be infecting mammals and humans in the recent years. In general, they can be commonly distinguished by their unique morphological structures of spherical or disk-like envelopes embedded with glycoproteins (Burrell et al., 2017). In terms of its genetic composition, it has a large positive sense RNA (33.5 kb) belonging to Group VI in the Baltimore classification. Other shared similarities include highly conserved genomes, Replicase gene upstream of structural and accessory genes as well as the translation of many nonstructural mRNAs from ribosomal frameshift. These viruses are very diverse and can be further subdivided into 4 subfamilies: alpha-, beta-, delta-, and gamma-coronaviruses based on serology (Fehr et al., 2015). 

Coronaviruses Classification

The heterozygous mouse K18-hACE2 (strain: 2B6.Cg-Tg(K18-ACE2)2Prlmn/J) is a transgenic line that expresses human ACE2 (hACE2).  When infected with SARS-CoV-2, this strain was shown to:

- support SARS-CoV-2 infection and replication
- undergo severe pulmonary disease
- lose weight
- have increased concentrations of pro-inflammatory cytokines and chemokines in the lungs
- experience histopathological changes in lung tissue that correlate with disease severity

Many of these observations mirror what is seen in severe SARS-CoV-2 infections in humans.  

K18-hACE2 Mouse Model of COVID-19

While no laboratory method or model is infallible, it has proven to be exceedingly difficult to create an accurate animal model for COVID-19 that replicates a severe human infection.  Reasons include:

- model organisms typically do not have the risk factors for COVID-19 that humans do (ex: old age, diabetes, cardiovascular disease)
- animals, including hamsters, ferrets, and nonhuman primates, seem to only develop mild symptoms and recover when infected with SARS-CoV-2
- conventional mouse models are ineffective because mouse ACE2 does not efficiently bind with SARS-CoV-2 S protein

Challenges in Creating an Animal Model of COVID-19

Currently, human entry receptor angiotensin-converting enzyme 2 (hACE2) transgenic mice are being used to study SARS-CoV-2 infection and pathogenesis, but these transgenic mice are limited to only one genetic background and are not widely available.  Thus, another mouse model of SARS-CoV-2 using adeno-associated virus (AAV)-mediated expression of hACE2 has been developed.  These AAV-hACE2 mice are capable of allowing for viral replication and demonstrate the pathogenicity of the virus as seen in patients with COVID-19.  

AAV-hACE2 Mouse Model of SARS-CoV-2

HFH4-hACE2 is a transgenic mouse line that has FoxJ1 promoter driven human ACE2 (hACE2) over-expression.  This model organism was designed in an attempt to overcome the issue of SARS-CoV-2 S protein not efficiently binding to murine ACE2.  hACE2 was found to be expressed in ciliated epithelial cells in the respiratory tract as well as in central nervous system tissue.  When infected with SARS-CoV-2, the following key results were observed:

- mice lost minimal weight, but only 60% were alive 5 days post infection (dpi)
- SARS-CoV-2 virus could be detected in the lung 2 dpi
- death is presumably caused by viral invasion of the brain
- whole body plethysmography (WBP) reveals that respiratory infection is not the likely cause of death

These findings suggest that HFH4-hACE2 mice are not an accurate model for COVID-19 pathogenesis.  

HFH4-hACE2 Mouse Model of COVID-19

Nonhuman primates (NHPs) successfully model respiratory viral infections in humans because of the vast similarities to human respiratory anatomy and immunology. Historically, NHP species have been used to model pathogenesis and develop vaccines. NHPs are vulnerable to infection and develop moderate SARS-CoV-2 disease phenotypes; however, prior studies have not been able to replicate severe infections and ARDS. Experiments performed by Tulane National Primate Research Center have observed ARDS in older RMs and AGMs that were infected with SARS-CoV-2. 

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