Basic Science

Introduction to SARS-CoV-2

Virus Biology

Immunologic Response

Clinical Course

References

Main points:

• • Currently, VeroE6, Huh7 and human airway epithelial cells have been popular cell lines to isolate SARS-CoV-2 in vitro

  • VeroE6 has been widely used in many labs across the world due to their high ACE2 receptor expressibility and their ease in cell maintenance

  • By allowing VeroE6 to coexpress TMPRSS2, a transmembrane protease, serine 2, the cell line becomes highly susceptible to SARS-CoV-2 infection, increasing viral RNA copies by over 100 times in comparison to VeroE6

  • Labs that require a higher titer of SARS-CoV-2 should isolate and propagate their virus in the engineered VeroE6/TMPRSS2 cell line

Introduction

The SARS-CoV-2 virus infects the respiratory system due to expression of the ACE2 receptor in the lungs. This receptor is present on many other organs and on endothelial cells. This allows for the direct viral infection of the endothelium which could explain issues seen in the vascular system in COVID-19 infections. This correspondence looked at three cases where endothelial pathology showed signs of infection and endotheliitis.

Main Points

• Post-mortem histology of patients with COVID-19 infections showed viral inclusion structures and accumulations of inflammatory cells in the endothelium of multiple organs, including lungs, kidneys, heart, liver, and small intestine.

• Viral infection of the endothelium can induce endothelial dysfunction including apoptosis. Dysfunction could also induce more vasoconstriction which could lead to more organ ischemia and inflammation.

• Stabilizing the endothelium could be a target of therapy with ACE inhibitors, statins, and anti-inflammatory drugs.

What is the purpose of this editorial?

• • To discuss whether biological sex plays a role in susceptability to COVID-19 infection

What did they find?

• • Females are less susceptible to COVID-19 infection in comparison due to different innate immunity, steroid hormones and various genes expressed in the sex chromosomes

  • The X chromosomes contain immune regulatory genes that are responsible for lower viral load levels, less inflammation, higher CD4+ T cell counts, regulatory factors like FOXP3, and transcription factors like Treg

    • Even though one of the two X chromosomes are inactivated in females, the inactivated X chromosome may still play a role in immunity

  • Women also produce antibodies with longer half-lives, express TLR7 more greatly than in men

Weaknesses

• • No qualitative or quantitative analyses were included in this paper

    • Difficult to completely believe all of these claims without data

    • Claims are probably true - the data may just be in the references

Take home message

• • Due to many of the various genes expressed in the X chromosome, there may be a protective effect in women against COVID-19 infection even if one of the two X chromosomes is inactivated

Background:

COVID-19 disease severity has previously been associated with hypertension, diabetes, chronic kidney disease, and cardiovascular disease; however, confounding variables such as age, sex, other comorbid conditions, and medication use were not considered in many studies reporting such an association. Given that SARS-CoV-2 gains entry to cells via the ACE2 enzyme, this review examines the relationship between COVID-19 severity, the renin-angiotensin system, and hypertension.

Key Points:

• ACE2 is the cell surface enzyme that converts Angiotensin II into Angiotensin-(1-7). It is also the enzyme to which SARS-CoV-2 attaches in order to gain entry into cells.

  • Normally, Ang-(1-7) acts on Mas receptors, lowering blood pressure through vasodilation and increased renal sodium and water secretion. It also attenuates inflammation through NO production.

• ACE is the enzyme that converts Angiotensin I into Angiotensin II.

  • Ang II acts on type 1 angiotensin receptors (AT1R), which increases blood pressure through vasoconstriction and increased renal sodium and water absorption, and promotes inflammation via increased oxidative stress.

    • Angiotensin-receptor blockers (ARBs) block the action of Ang II on AT1R.

  • ACE inhibitors block the conversion of Ang I to Ang II.

• Due to the opposing actions of Ang-(1-7) and Ang II, the balance between them partially determines the presence and extent of tissue injury in response to certain stimuli.

• Prior research has suggested that increased ACE activity relative to ACE2 activity might be partially responsible for acute lung injury in SARS-CoV, and thus also in SARS-CoV-2.

  • ACE2 is potentially suppressed in SARS-CoV-2 infection, possibly due to increased internalization of the enzyme, leading to decreased Ang-(1-7) levels and increased Ang II levels.

    • Increased Ang II levels can lead to increased activation of AT1R, possibly contributing to the inflammatory response in the lungs seen in some COVID-19 cases.

• There is some concern that ACE inhibitors or ARBs could increase the risk of SARS-CoV-2 infection, and lead to a more severe infection. Alternatively, ARBs could be protective by inhibiting the activation of AT1R by Ang II.

  • Studies using animal models have reported upregulation of ACE2 in heart and kidney tissue in response to ACE inhibitors and ARBs.

    • Increases in ACE2 would provide easier viral entry for SARS-CoV-2.

    • However, no studies have shown upregulation of ACE2 in the lungs

Conclusions:

Future research needs to investigate the relationship between medications modulating the renin-angiotensin system and COVID-19 severity. Until that relationship is better understood, cessation of such medications or changes in dosing should be avoided unless medically necessary.

Introduction:

While the clinical presentation of COVID-19 can vary from asymptomatic in some patients to severe respiratory distress in others, there is no completely effective biomarker that could be measured to determine the severity of the disease. This meta-analysis looked at 9 studies who reported IL-6 levels in patients and the severity of their disease to determine if there was any statistically significant correlation between IL-6 levels and disease severity.

Main Points:

• Cytokines, including IL-6, are important for mediating inflammatory responses in patients. IL-6 has been studied as a biomarker in Hepatitis B infections for disease severity.

• After meta-analysis of nine studies, the researchers concluded that IL-6 levels were significantly elevated in severe COVID-19 patients compared to non-severe ones.

• High levels of IL-6 could be related to cytokine-mediated lung damage seen in severe COVID-19 infections.

• Serial measurements of IL-6 of admitted COVID-19 patients could be beneficial in determining disease progression and clinical decision-making.

• Significance: T cells are decreased and exhausted in patients with COVID-19. Cytokines such as IL-10, IL-6 and TNF-α might directly mediate T cell reduction. Thus, new therapeutic measures are needed for treatment of ICU patients, and may even be necessary early on to preempt disease progression of non-ICU patients who are higher-risk patients and have low T cell counts.

• Background: T cells play a critical role in effective antiviral immune response but their numbers and functional state in COVID-19 patients remain largely unclear.

• Methods: Retrospectively review of total T cells, CD4+, CD8+ T cell subsets, and serum cytokine concentration from 522 hospital patients with laboratory-confirmed COVID-19 and compared to 40 healthy control patients. These 522 patients were admitted into two hospitals in Wuhan from December 2019 to January 2020. The expression of T cell exhaustion markers PD-1 and Tim-3 were measured by flow cytometry in the peripheral blood of 14 COVID-19 cases.

• Results:

  • There is a downtrend of total T cells, CD4+and CD8+ subsets that is inversely related to increased COVID-19 disease severity.

  • There is an age-dependent reduction of T cell numbers in COVID-19 patients, (lowest T cells numbers found in patients ≥60 years old).

  • T cell numbers declined as severity of disease in non-ICU patients increased.

  • TNF-α, IL-6 and IL-10 were significantly increased in infected patients; their levels in ICU patients are significantly higher than in non-ICU patients.The concentration of TNF-α, IL-6 and IL-10 cytokines was negatively correlated with total T cell counts, CD4+ counts, and CD8+ counts.

  • SARS-CoV-2 viruses induce T cell exhaustion in COVID-19 patients, particularly in those requiring ICU care, as indicated by elevated levels of exhaustion protein (PD-1).

• Conclusions:

  • 70.56% of non-ICU patients had a decrease in the total T cells and CD4+ T cells. However, 95% of ICU patients showed a decrease in both total T cells and CD4+ T cells. Most importantly, all of the patients displayed decreases in CD8+ T cells.

  • Lower T cell totals may be a potential cause for increased susceptibility to COVID-19 in elderly patients.

  • Aggressive interventions may be required for non-ICU patients even in the absence of more severe symptoms should their T cell counts fall below a “critical threshold”.

  • The decrease of T cells seen in COVID-19 patients is likely the result of high serum concentration of TNF-α, IL-6 and IL-10 negatively regulating T cell survival or proliferation.

  • T cells are exhausted in COVID-19 patients during SARS-CoV-2 infection.

• Limitations:

  • The age range of patients was between 5 days and 97 years old. The data may not be normalized for each age.

  • The figures and graphs they cite in the paper use median instead of mean and have overlapping confidence intervals. The p-values calculated in the data do not correlate with the figures. More data analysis and statistical critique is necessary.

• What was the purpose of this article?

  • To describe how arachidonic acids and other unsaturated fatty acids could be used to prevent COVID-19 infection

• What’s so special about arachidonic acids (AAs)?

  • AAs and other unsaturated fatty acids have shown properties to inactivate enveloped viruses and other microbial organisms

  • They also have pro-inflammatory and wound healing properties, rendering humans susceptible to infection if AA deficient

  • When cells are infected by bacteria, viruses, or any other infectious agent, the cells most likely release AAs and other unsaturated fatty acids to attempt to inactivate the invading organisms and viruses

    • AAs can induce leakage and lysis in the cell membranes of microbes, and disrupt the envelopes of viral proteins

  • AAs could potentially be used to prevent and even recover from viral infection

• Weaknesses of the paper

  • No evidence or data was provided in this paper to support the theory of AAs being used as a prophylactic agent or potential treatment against COVID-19

  • This paper simply implies that this idea works to a certain extent on microorganisms and viruses, and suggests that AAs would probably be effective against COVID-19 as well

• Take home message

  • Supplemental arachidonic acids and other unsaturated fatty acids could potentially be used to prevent COVID-19 infection but the data is lacking

What was the purpose of this study?

• • To develop and test an in vitro cell-cell fusion assay that can be used to identify various inhibitory compounds against SARS-CoV-2 infection in Huh7 and 293T/ACE2 cells

  • To test various derivatives of the group’s EK1 compound against SARS-CoV-2 infection in vitro and in a mouse model

How does this fusion assay work?

• • Rather than using actual SARS-CoV-2, this group cloned the S gene, which encodes for the proteins responsible for viral entry, and EGFP, a fluorescent protein, into a vector and transfected 293T cells to express these proteins

  • ACE2/293T cells were used a target cells

    • There have been many studies that confirmed that the S protein and the ACE2 receptor interact extensively and facilitate viral entry

  • The effector cells (transfected cells) and the target cells (ACE2/293T) were then co-cultured and fusion could then quantitatively be measured via fluorescence microscopy

  • Fusion inhibitors can then be added in this assay to measure inhibitory activity

What did they find with their in vitro assay?

• • After optimizing their EK1 lipoprotein compound via structure-activity relationship analysis, they developed EK1C4, which was the most potent inhibitor among all the derivatives

    • The IC₅₀ of EK1C4 was 4.3 nM while the IC₅₀ of the original EK1 was 409.3 nM

  • The CC₅₀ (half cytotoxic concentration) of EK1C4 was above 5 μM, which indicates very low toxic effects in vitro

  • EK1C4 targets the highly conserved HR1 and HR2 domains in SARS-CoV-2

    • There are currently 103 variants of SARS-CoV-2 but all reported genomes show 100% identity in the HR1 and HR2 region

      • EK1C4 thus has an incredibly high barrier to resistance

Can EK1C4 be used as a prophylactic agent or a treatment?

• • In an HCoV-OC43 infection mouse model, they administered one intranasal dose of EK1C4 in newborn mice at various time points (0.5 h, 2 h, 4 h, 12 h, and 24 h) before HCoV-OC43 infection (100 TCID₅₀)

    • TCID₅₀ stands for 50% tissue culture infectious dose

  • The survival rates of the mice in the 0.5 h, 2 h, 4 h, 12 h, and 24 h groups were 100%, 100%, 100%, 83%, and 0%, respectively

    • The survival rate of the mice in the control group (no drug) had a 100% mortality rate

  • EK1C4 was then administered 0.5 h and 2 h after HCoV-OC43 infection and the survival rates for each group was 100% and 16.7%, respectively

Take home message

• • EK1C4 could potentially be used as an intranasal prophylactic agent that could be found useful especially when in close contact with a high-risk population

  • EK1C4 could potentially reduce viral loads if taken shortly after SARS-CoV-2 exposure

  • EK1C4 is a peptide derivative and is administered locally (not systemically), so it may have a fewer off-target effects in humans

  • More and more promising agents are being discovered every day to fight SARS-CoV-2 and even if we do not have a vaccine ready for distribution in the next few months, new prophylactic drugs can be used to prevent transmission and to help “flatten the curve”

Introduction

This letter to the editor in the Journal of Medical Virology addresses the possibility of neuroinvasive infection from SARS-CoV2, which could play a role in the respiratory failure of some COVID-19 patients. Specifically, this letter highlights a possible alteration in the synthesis of dopamine due to the infection.

Key Points

• The Angiotensin 1 Converting Enzyme 2 (ACE2) gene encodes the main receptor by which SARS-CoV2 is able to enter cells;

  • ACE2 expression can be downregulated in SARS-CoV2 infection.

• Dopa Decarboxylase (DDC) is responsible for conversion of L-DOPA to dopamine, and L-5-HT to serotonin.

• It has previously been reported that ACE2 and DDC expression are co-regulated, i.e. alteration in expression of ACE2 may be associated with a similar alteration in expression in DDC.

  • Infusion of angiotensin 1-7 in the hypothalamus of rats has been associated with an increase in brain dopamine.

  • Angiotensin 1-7 stimulates renal dopamine synthesis.

  • ACE2 knockout mice exhibited low serotonin levels in both blood and the brain.

• It is possible that ACE2 and DDC expression are coregulated in both neuronal and some nonneuronal cell types.

• Similar high expression of ACE2 and DDC has been reported in intestinal epithelial cells, which are an important source of blood-circulating dopamine.

  • It is possible that downregulation of ACE2 in intestinal epithelial cells during SARS-CoV2 infection is associated with downregulation of DDC as well, leading to decreases in blood dopamine levels

• Dopamine may play an important role in shaping the immune response in the lungs, as dopamine receptors are expressed by alveolar epithelial cells, lung macrophages, and lung nerves.

  • A D1 receptor agonist, fenoldopam, has been shown to dampen lung inflammation and pulmonary edema in a model of endotoxin-induced lung injury.

Main Takeaway

Expression of ACE2 and DDC, which converts L-DOPA to dopamine, is often coregulated in both neuronal and nonneuronal tissues. Downregulation of ACE2 in nonneuronal cells during SARS-CoV2 infection may be associated with a similar downregulation of DDC in intestinal epithelial cells, resulting in lower blood dopamine levels. Dopamine may play a role in mediating the immune response of the lungs, and lower levels could be partially responsible for respiratory complications in SARS-CoV2 infections.

Introduction

Smoking has been known to have many adverse effects on health, particularly in the respiratory system. This places smokers at increased risk of all types of respiratory infections, including bacterial and viral. Smoking also contributes to chronic conditions, such as COPD. In China, smoking is very prevalent in the general population (45%), and most COVID-19 related deaths in China were in older men with chronic conditions, a population with the highest prevalence of smoking. This article looked at the association between smoking and ACE2 receptor expression in the lungs, which is a receptor for the SARS-CoV-2 virus.

Main Points

• The ACE2 receptor is a known cell-binding site for SARS-CoV, and studies now show that the SARS-CoV-2 virus has 10- to 20-fold increased affinity for the receptor. This allows the virus to attach for longer periods of time in the respiratory tract, promoting extended person-to-person spread.

• In both smokers and those with COPD, immunologic stains of lung in this study showed increased expression of the ACE2 receptor compared to normal lung tissue. This finding could explain the mechanism for poorer outcomes among smokers who are diagnosed with COVID-19.

• Further areas of research include investigating this relationship with vaping, continued monitoring of outcomes among the smoking population, and using the ACE2 receptors as a potential therapeutic target. Also, providers now have more reason than ever to promote smoking cessation in this vulnerable population.

The information presented in this article was gathered by the Standing Committee on Emerging Infectious Diseases and 21st Century Health Threats.

Laboratory data so far indicates reduced survival of SARS-CoV-2 at elevated temperatures.

• The virus’s temperature sensitivity is a function of the type of surface the virus is on.

• There have not been many well controlled studies on this topic at this time.

A report by Chin et al. studied the stability of SARS-CoV-2 as a function of temperature, type of surface, and following the use of disinfectants.

As a function of temperature: With a starting suspension of 6.7 log TCID50/ml (50% tissue culture infective dose per mL of virus transport medium):

• No virus was detected after 5 minutes at a temperature of 70°C and after 30 minutes at 56°C

• A 3-log unit reduction after 1 day and no virus detected afterward at 37°C

• A 3-log unit reduction after 7 days and no virus detection after 14 days at 22°C

• A 0.6 log-unit reduction at the end of 14 days at 4°C

With respect to survival on surfaces using a 5 microliter drop of virus culture at 7.8 log TCID50/ml:

• No virus was recovered from printing and tissue paper after 3 hours

• No virus was detected on cloth after 2 days

• No virus was detected on stainless steel after 7 days

• Outside of a surgical mask, 0.1% of the original inoculation dose was detected on day 7, presenting a concern for PPE reuse.

At Tulane University, preliminary results of a dynamic aerosol stability experiments with SARS-CoV-2 conducted over several weeks were released.

• In an aerosol with a fairly uniform distribution, at an ambient temperature of 23°C with 50% humidity, SARS-CoV-2 has a longer half-life than influenza virus, SARS-CoV-1, and Mycobacterium tuberculosis.

It was noted that many of these studies are experimental with preliminary results that may not be wholly reflective of real-world conditions.

Studies regarding potential seasonal effects so far have conflicting results, with no conclusive evidence pointing to a potential pattern of seasonality.

• Some evidence suggests that the virus transmits less effectively in environments with higher ambient temperature and humidity.

• A study conducted in Italy found highest rates in temperate regions, with lower rates in both warmer and colder climates.

• Multiple studies agree that lack of global host immunity means any seasonality found may not lead to significant reduction in disease spread without extensive public health interventions.

Summary

• This study investigated the susceptibility of ferrets and animals in close contact with humans to SARS-CoV-2. It was found that SARS-CoV-2 replicates poorly in dogs, pigs, chickens, and ducks, but efficiently in ferrets and cats.The virus does transmit in cats via respiratory droplets. However, these findings suggest that none of these species plays a part in the epidemiology of COVID-19.

Update

• Nadia, a tiger at the Bronx Zoo in New York, has become the first of her kind to test positive for the coronavirus.

Link: https://www.cnn.com/2020/04/05/us/tiger-coronavirus-new-york-trnd/index.html

Background

• SARS-CoV-2 is thought to have originated in bats; however, the intermediate animal sources of the virus are completely unknown. SARS-CoV-2 shares 96.2% identity at the nucleotide level with the coronavirus RaTG13, which was detected in horseshoe bats (Rhinolophus spp), but it had not previously been detected in humans or other animals. This pandemic raises many urgent questions, including: could the widely disseminated viruses transmit to other animal species which then become reservoirs of infection?

Method

• All experiments with infectious SARS-CoV-2 were performed in the biosafety level 4 and animal biosafety level 4 facilities. The protocols for animal study and animal welfare were reviewed and ethically approved. Animals were inoculated intranasally, grouped with uninfected animals, and then all were euthanized for organ viral RNA quantification.

Results

• • Ferrets: SARS-CoV-2 can replicate in the upper respiratory tract of ferrets, but its replication in other organs is undetectable (including rectal swabs). SARS-CoV-2 can replicate in the upper respiratory tract of ferrets for up to eight days without causing severe disease or death. Antibodies against SARS-CoV-2 were detected in all ferrets.

  • Cats: Infectious virus was detected in the upper respiratory tract of cats, but was not recovered from the small intestines. SARS-CoV-2 replicates efficiently in cats, with younger cats being more permissive and the virus can transmit between cats via respiratory droplets.

  • Dogs: Infectious virus was not detected in any swabs collected from dogs. The study results indicate dogs have low susceptibility to SARS-CoV-2.

  • Pigs, Chickens, and Ducks: Viral RNA was not detected in any swabs collected from any of these virus-inoculated animals or from naïve contact animals. All of the animals were seronegative for SARS-CoV-2 when tested by using the ELISA. These results indicate that pigs, chickens, and ducks are not susceptible to SARS-CoV-2.

Significance

• Ferrets and cats are highly susceptible to SARS-CoV-2, dogs have low susceptibility, and livestock including pigs, chickens, and ducks are not susceptible to the virus. The fact that SARS-CoV-2 replicates efficiently in the upper respiratory tract of ferrets makes them a candidate animal model for evaluating antiviral drugs or vaccine candidates against COVID-19. Surveillance for SARS-CoV-2 in cats should be considered as an adjunct to elimination of COVID-19 in humans.

What is the purpose of this letter?

• • To compare the stability of SARS-CoV-2 in aerosol and on various surfaces (plastic, stainless steel, copper, and cardboard) to the stability of SARS-CoV-1

What did they find?

• • Using a Bayesian regression model, they determined that SARS-CoV-2 remained viable in aerosols for 3 hours with a reduction in titer from 10^3.5 to 10^2.7 TCID₅₀ per mL

    • This reduction was similar to SARS-CoV-1 (10^4.3 to 10^3.5 TCID₅₀ per mL)

  • SARS-CoV-2 showed greater stability on plastic and stainless steel than on cardboard and copper

    • SARS-CoV-2 remained viable on these surfaces up to 72 hours

    • Virus titer greatly reduced on plastic after 72 hours, one stainless steel after 42 hours, on cardboard after 24 hours and on copper after 4 hours

      • SARS-CoV-1 showed similar results

Take home message

• • SARS-CoV-2 can remain infectious and viable in aerosols for hours and on surfaces for days

  • Take the quarantine seriously, wash your hands, and clean your surfaces!

What is the purpose of this review?

• • To determine whether coronaviruses (SARS-CoV, not SARS-CoV-2) could be killed via heat

  • If so, could personal protective equipment (e.g., face masks) be thermally disinfected for reuse?

What did they find?

• • A thermal disinfection cycle of 60 °C for 30 m, 65 °C for 15 m and 80 °C was able to reduce coronavirus infectivity by approximately 4 log10 (reduction of 99.99%)

  • The SARS-CoV nucleocapsid protein was completely denatured after 10 m at 55 °C

Limitations of this study

• • These tests were performed with isolated coronavirus

    • Thus, results may differ in real-life context (e.g., virus on dry surfaces, severely contaminated face masks)

      • Scientists believe that this is unlikely though

  • These tests were not performed on SARS-CoV-2

    • Results would most likely be similar since SARS-CoV and SARS-CoV-2 are genetically similar

Take home message

• • If we ever completely run out of face masks or other PPEs, thermal disinfection could potentially be used to recycle old PPEs

What is the purpose of this research letter?

• • To inform the public that SARS-CoV-2 (or COVID-19) can be transmitted through blood via blood donations

What did they find in this study?

• • Using real-time reverse transcription PCR, scientists in Wuhan, China screened 2,430 blood donations by March 4 and perform retrospective testing of blood samples from 4,995 donations between December 21, 2019 and January 22, 2020

  • Of the 7425 patients, 4 plasma samples from asymptomatic patients were positive for SARS-CoV-2 RNA

    • Not much information on the 4 asymptomatic patients were obtained

    • All blood donations from these patients were removed from circulation

Take home message

• • Although only 0.05% of blood donations were positive for SARS-CoV-2, there should be a slightly better screening approach to detect SARS-CoV-2 RNA in blood donors

There are reports of COVID-19 cases with mild upper respiratory tract symptoms, suggesting the potential for pre- or oligosymptomatic transmission. There is an urgent need for information on body site-specific virus replication, immunity, and infectivity. This study sampled 9 patients for active virus replication.

• No discernible differences in viral loads or detection rates were found when comparing naso- vs. oropharyngeal swabs.

• Pharyngeal virus shedding was very high during the first week of symptoms (peak = day 4). Findings suggest a more efficient transmission of SARS-CoV-2 than SARS-CoV through active pharyngeal viral shedding at a time when symptoms are still mild and typical of an upper respiratory tract infection.

• Tissue preference of SARS-CoV-2 in the throat is supported, particularly during the first 5 days of symptoms. The disturbance of gustatory and olfactory sense points at upper respiratory tract tissue infection. Sequence-distinct virus populations were consistently detected in throat and lung samples from the same patient, proving independent replication.

• Shedding of viral RNA from sputum outlasted the end of symptoms. The last positive-testing swab sample was taken on day 28 post-onset. Therefore, early discharge with ensuing home isolation could be chosen for patients who are beyond day 10 of symptoms with less than 100,000 viral RNA copies per mL of sputum.

• Infectious virus was not readily isolated from stool samples in spite of high virus RNA concentration. None of 27 urine samples and none of 31 blood serum samples tested positive for SARS-CoV2 RNA.This suggests that measures to contain viral spread should aim at droplet-, rather than fomite-based transmission.

• Seroconversion occurred after 7 days in 50% of patients (14 days in all), but was not followed by a rapid decline in viral load. Hence, vaccine approaches targeting mainly the induction of antibody responses should aim to induce particularly strong antibody responses in order to be effective.

What is the purpose of this study?

• • To review the endocytic pathway’s and autophagy’s roles in SARS-CoV-2 (or COVID-19) infection and the potential of developing antivirals against these pathways

What is the endocytic pathway and autophagy?

• • The endocytic pathway is where a cell endocytoses extracellular components and these endosomes later fuse with lysosomes for degradation

  • Similarly, in autophagy, intracellular components are engulfed by autophagosomes, which then fuse with lysosomes to form autolysosomes (its contents are then degraded)

  • Figure 1. Involvement of the endocytic pathway and autophagy (non-endosomal pathway) in the entry and replication of CoVs in host cells

    • Two potential lysosomotropic agents, chloroquine (CQ) and chlorpromazine, targets this pathway, which could show potential therapeutic effect against SARS-CoV-2

How does SARS-CoV-2 utilize these pathways?

• • Actually, it has not been reported that SARS-CoV-2 utilize these pathways; however, there has been evidence that SARS-CoV, a genetically similar virus, does

  • SARS-CoV-2 and SARS-CoV use the same receptor (angiotensin converting enzyme II) for viral entry

  • Using this knowledge, three main classes of inhibitors could be used to block viral entry and infection:

    • Lysosomotropic agents, which neutralize the acidic pH of endosome-lysosomes

      • This is why chloroquine has been thought of a potential therapy for SARS-CoV-2

    • Direct endosomal-lysosomal protease inhibitors

    • Clathrin-mediated endocytosis inhibitors

Take home message

• • By better understanding SARS-CoV-2’s life cycle, scientists can create new and novel therapies that can target various stages of the virus’ life cycle

  • Although it is not completely clear whether SARS-CoV-2 utilizes the endocytic pathway and autophagy in its life cycle, it most likely does due to its genetic similarity to SARS-CoV

  • Since there are multiple FDA-approved drugs that target these pathways, more clinical trials should be initiated to test these compounds on SARS-CoV-2-infected patients

Review by Chris Ma on 4/4/20.

Significance of this study

• In the United States, 34.2 million (10.5% of the total population) have diabetes mellitus (DM).

• This population is at a higher risk for hospitalization and death from COVID-19.

• Ironically, plasma glucose levels and DM are independent predictors for morbidity and mortality in patients with SARS.

• This paper discusses potential mechanisms by which diabetes modulates the host-viral interactions and host-immune response, including: a higher affinity of cellular binding and efficient virus entry, decreased viral clearance, diminished T cell function, increased susceptibility to hyperinflammation and cytokine storm syndrome, and presence of cardiovascular disease.

Potential Mechanisms that increase COVID-19 risk in Diabetics:

• • Higher affinity of cellular binding and efficient virus entry: Increased expression of ACE2 receptors are found in diabetic rodent models. DM independently and causally increases lung ACE2 expression. Insulin administration attenuates ACE2 expression, while other agents such as GLP-1 agonists, thiazolidinediones, ACEI, and statins upregulate ACE2. Levels of cellular protease furin are also elevated in patients with DM.

  • Decreased viral clearance and diminished T cell function: DM inhibits neutrophil chemotaxis, phagocytosis, and intracellular killing of microbes. This initial delay in adaptive immunity also delays activation of Th1 cell-mediated immunity and a late hyper-inflammatory response is observed in DM.

  • Increased susceptibility to hyperinflammation and cytokine storm syndrome: Peripheral counts of CD4+ and CD8+ T cells are low but a higher proportion of Th17 proinflammatory CD4+ cells may lead to accentuated inflammatory cytokines.

  • Presence of cardiovascular disease: A humanized mouse model for MERS-CoV demonstrated a more severe and prolonged disease in diabetic mice with high-fat diets.

Review by Karisma Gupta on 4/3/20.

Significant findings:

• Both severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and SARS-CoV-2 interact with the RAAS through angiotensin-converting enzyme 2 (ACE2) and is theorized to the the cause of the virus’ infectivity

• With the wide spread use of ACE inhibitors and angiotensin II receptor blockers (ARBs), there are concerns with continued use of of these medications.

• Raised concern that withdrawal of RAAS inhibitors may be harmful in certain high-risk patients with Covid-19.

• SARS-CoV-2 appears not only to gain initial entry through ACE2 but also to subsequently down-regulate ACE2 expression. Down-regulation of ACE2 activity in the lungs may result in unopposed angiotensin II accumulation and local RAAS activation. Unabated angiotensin II activity may be in part responsible for organ injury in Covid-19.

• In a small case study, patients with Covid-19 appeared to have elevated levels of plasma angiotensin II, which correlated with total viral load and degree of lung injury.

• In preclinical models it was found that administration of recombinant ACE2 appeared to reverse this lung-injury process in other viral infections

Major Conclusions and Future clinical implications

• Among patients with unstable clinical status, myocardial injury associated with Covid-19 may pose even higher early risks after withdrawal of RAAS inhibitors.

• RAAS inhibitors should be continued in patients in otherwise stable condition who are at risk for, are being evaluated for, or have Covid-19.

• Current trials to test whether administering recombinant ACE2 protein may be beneficial in restoring balance to the RAAS network and potentially preventing organ injury.

• Paired trials of losartan and other high blood pressure medication as a concurrent treatment for Covid-19 are being conducted among patients who have not previously received treatment with a RAAS inhibitor

Review by Jeeva Jacob on 4/2/20.

What is the significance of this study?

• • To introduce that SARS-CoV-2 (also known as COVID-19) may also be able to target and infect the kidneys

What proof do we have that SARS-CoV-2 can target the kidneys?

• • In patients infected with SARS-CoV (not SARS-CoV-2), SARS-CoV antigens, RNA, and virions were found via immunohistochemical examination in the kidneys

  • The proximal tubular epithelial cells in nephrons express ACE2, which may explain why SARS-CoV viruses were able to infect those cells

  • Due to SARS-CoV’s and SARS-CoV-2’s similar genetic make-up, it is likely that SARS-CoV-2 can target and infect nephrons with a similar mechanism to SARS-CoV

  • Acute kidney injury has already been reported in some COVID-19 infected patients with and without preexisting kidney disease

    • A few other studies reported that among their COVID-19 patient cohort, 4% to 7% of these patients eventually had acute kidney injury

What is its potential mechanism of action of nephron injury?

• • Unclear but it is most likely multifactorial

  • A few possible mechanisms could be related to direct infection of the virus, immune and inflammatory response of the infection, and systemic toxicity caused by respiratory failure

Take home message

• • Special care for renal function should be given to infected COVID-19 patients

  • Future studies should look into whether COVID-19 can be transmitted through urine

Review by Chris Ma on 4/1/20.

What is the significance of this study?

• • To further understand COVID-19’s origins using its sequenced genome

What is SARS-CoV-2’s (also known as COVID-19) origins?

• • Initially, many thought that SARS-CoV-2’s sole origin was zoonotic in a Wuhan city, China seafood and wildlife market

  • However, through genomic sequencing and phylogenetic analysis of “environmental samples” (e.g., surfaces) from the market, these samples showed significant similarity to the virus’ genome in the earliest infected Wuhan patients

  • It is impossible to confirm whether the virus’ origins were environmental or zoonotic since direct animal sampling was not completed during the start of the outbreak

  • The most closely related viruses to SARS-CoV-2 were from bat coronaviruses, such as RaTG13 and RmYN02

    • Bats are probably extremely important as reservoir species; however, there has not been clear evidence of SARS-CoV-2 originating from bats

  • It is possible that other mammalian species may have acted as amplifying or intermediate hosts, allowing the virus to mutate to infect humans

    • E.g., camels and civets were intermediate hosts for MERS and SARS, respectively

  • It is possible that humans were infected with a less virulent, asymptomatic strain of SARS-CoV-2 much earlier than envisioned and it gradually acquired mutations, including the furin cleavage site, that made it to what it is today

  • It is possible that recombination events occurred; however, it is difficult to determine its exact pattern and genetic ancestry

How was SARS-CoV-2’s viral genome sequenced?

• • Next-generation meta-transcriptomic sequencing was used to obtain a complete viral genome on January 5, 2020 from an infected patient admitted to the Central Hospital of Wuhan on December 26, 2019

  • The virus’ sequence was released on the open access website, http://virological.org/, on January 11, 2020

What are the main differences between SARS-CoV-2 and SARS-CoV?

• • Approximately 79% of SARS-CoV-2’s genome was similar to SARS-CoV’s

    • Approximately 72% of SARS’CoV-2’s spike (S) protein, a host cell receptor binding glycoprotein, was similar to SARS-CoV’s

  • The most important difference in SARS-CoV-2’s genome is the insertion of a furin cleavage site in the S1/S2 junction of the S protein

    • Cleavage of this furin site significantly enhances viral entry

What happens next?

• • SARS-CoV-2 has an error prone RNA polymerase so it is likely for more mutations to arise as the pandemic progresses

    • In comparison to other RNA viruses, SARS-CoV-2 has a lower mutation rate though

    • We have no evidence that these mutations will cause changes in phenotypes, such as virulence and transmissibility, but it is important to monitor any phenotypic changes as time progresses

  • In general, limiting our exposure to animal pathogens may be an effective and the simplest method to prevent future outbreaks

Review by Chris Ma on 3/31/20.

What is the significance of this study?

• • To highlight the importance of elevated plasmin(ogen) levels in explaining why patients with preexisting conditions, such as diabetes, hypertension, COPD, coronary heart disease, cerebrovascular disease, and kidney dysfunction, have more severe clinical outcomes when infected with COVID-19 (also known as SARS-CoV-2)

What major findings did they discover?

• • Severe SARS-CoV-2 patients generally present with significantly increased fibrin-degraded products and decreased platelets, and hemorrhage in multiple organs

    • This indicates that these patients may also have hyper-fibrinolysis

  • In general, the virulence of viruses containing a furin site in their envelope proteins increase when cleaved by plasmin and other proteases

    • SARS-CoV-2 has a furin site in its envelope proteins, which may explain why patients with other comorbidities may have more severe outcomes when infected by this virus

What is so special about this “furin site?”

• • Furin is a cellular endoprotease and it is hypothesized that the cleavage of a furin site increases viral virulence, as seen in other viruses containing this site such as HIV, herpes, human influenza, and Ebola

  • SARS-CoV-2’s receptor-binding protein, Spike (S) protein, binds to human ACE2 receptors with higher affinity than SARS-CoV virus

    • This higher affinity may be due to the insertion of this furin-cleavage site into the spike proteins of SARS-CoV-2, increasing its ability to enter host cells

  • Furin is highly expressed in human alveolar type II cells found in the respiratory system which may explain why most SARS-CoV-2-infected patients present with respiratory ailments

Are there any non-furin proteases that we should worry about?

• • Plasmin(ogen), trypsin and kallikrein, which are expressed in alveolar type I and II epithelial cells, also show some protease activity against SARS-CoV-2’s S proteins

    • Plasminogen is also expressed in endothelial cells and demonstrates that its active form, plasmin, can cleave furin sites on human epithelial sodium channels (ENaC)

      • Decreased ENaC function causes fluid to accumulate in body cavities (e.g., lung edema, increased blood volume) and may lead to greater susceptibility to bacteremia and sepsis

  • Plasmin(ogen) is elevated in patients with hypertension, cardiovascular disease, diabetes, and other comorbidities

  • Plasmin(ogen) is also elevated in ARDS

Why is this study important?

• • Plasmin(ogen) levels are elevated in patients with preexisting medical conditions, making them more susceptible to SARS-CoV-2 infection and fatality

  • Elevated plasmin(ogen) levels may be used as a biomarker for disease severity

  • Elevated plasmin(ogen) levels may be a critical factor for risk stratification

Review by Chris Ma on 3/30/20.

What is the significance of this study?

• The authors were able to elucidate the dynamic mechanism of viral entry of mouse hepatitis coronavirus (MHV) in DBT cells with CEACAM1a, a cell adhesion protein

• Although the authors did not elucidate the entry mechanism for COVID-19 in a human model, this study provided a better mechanistic insight of how coronavirus enters cells as a family

What is the CoV spike protein?

• CoV spike protein has a critical role in viral entry into host cells

• The CoV spike protein has two forms: pre-fusion (homo-trimer with 3 receptor-binding S1 heads on top of a trimeric membrane-fusion S2 stalk) and post-fusion (coiled-coil structure containing only S2)

  • During viral entry, the spike protein is cleaved via host proteases at two sites: first at the S1/S2 site and second within the S2 site

  • After these cleavages occur, the pre-fusion form transitions into the post-fusion form (irreversible process)

• The S1 subunit of spike contains an N-terminal domain (S1-NTD; recognizes sugar receptors) and a C-terminal domain (S1-CTD)

  • Receptor binding stabilizes the S1-CTD conformation, weakening the S1/S2 interaction and facilitating the dissociation of S1/S2

  • The S1-CTD has two dynamic conformations: “lying down” (to evade immune surveillance) and “standing up” (for receptor binding)

What did they discover in this study?

• MHV entry is the only coronavirus that uses its spike S1-NTD to date

Why is there a lot of focus on the entry stage of the CoV’s life cycle?

• Cov’s entry mechanism is a unique, complicated process

• Targeting these specific entry proteins allow us to create antivirals and potentially vaccines that can prevent infection from occurring and to have fewer off-target effects

Review by Chris Ma (3/27/20)

What is the pathophysiology of COVID-19?

• Coronavirus is an enveloped, positive-stranded RNA virus with a nucleocapsid

• An atypical coronavirus genome can have at least 6 open reading frames (ORFs) where ORF1a and ORF1b encode 16 non-structural proteins

  • The non-structural proteins (e.g., proteases, polyprotein 1a/1ab) can block the host innate immune response

• All the other ORFs encode structural proteins (e.g., spike, membrane, envelope, and nucleocapsid proteins)

  • Homotrimers of spike glycoproteins (subunits S1 and S2) are found on the viral surface and allows host receptor attachment

    • The S2 subunit in the spike protein is highly conserved and is a promising target for antivirals

    • A mutation in this region from Sars-CoV is what most likely caused the COVID-19 (Sars-CoV-2) outbreak

Pneumonia mechanism

• COVID-19 infection causes a cytokine storm after tissue damage, releasing significant amounts of interleukin 6 (IL-6)

• IL-6 promotes B lymphocyte differentiation, stimulating and inhibiting the growth of certain cells

  • IL-6 also stimulates acute phase proteins and has an important role in thermoregulation in bone maintenance and central nervous system functionality

Histological findings in two patients

• Lungs showed edema and proteinaceous exudates in the form of large protein globules

• Inflammatory clusters of fibrinoid material and multinucleated giant cells and pneumocyte hyperplasia were also observed

Why is this important?

• It is important to discover and understand the mechanism of action of COVID-19 for scientists to create novel antivirals that can target different steps of the virus’ life cycle

  • Increases the cure rate

  • Increases the barrier to resistance

Review by Chris Ma (3/27/20)