What impact the SARS-CoV-2 virus had inside our lung cells within the first few hours of being infected?

What if scientists knew exactly what impact the SARS-CoV-2 virus had inside our lung cells, within the first few hours of being infected?

Could they use that information to find drugs that would disrupt the virus’ replication process before it ever gets fully underway?

The discovery that several existing FDA-approved drugs—including some originally designed to fight cancer—can stop coronavirus in its tracks indicates the answer is a resounding yes.

A team of Boston University researchers—hailing from BU’s National Emerging Infectious Diseases Laboratories (NEIDL), the Center for Regenerative Medicine (CReM) at BU’s Medical Campus, and BU’s Center for Network Systems Biology (CNSB)—embarked on a months-long, collaborative and interdisciplinary quest, combining multiple areas of expertise in virology, stem cell-derived lung tissue engineering, and deep molecular sequencing to begin answering those questions.

They simultaneously infected tens of thousands of human lung cells with the SARS-CoV-2 virus, and then tracked precisely what happens in all of those cells during the first few moments after infection. As if that was not complicated enough, the team had to cool their entire high-containment research facility inside the NEIDL to a brisk 61 degrees Fahrenheit.

The result of that challenging and massive undertaking?

The BU team has revealed the most comprehensive map to date of all the molecular activities that are triggered inside lung cells at the onset of coronavirus infection. They also discovered there are at least 18 existing, FDA-approved drugs that could potentially be repurposed to combat COVID-19 infections shortly after a person becomes infected.

Experimentally, five of those drugs reduced coronavirus spread in human lung cells by more than 90 percent. Their findings were recently published in Molecular Cell.

Now, academic and industry collaborators from around the world are in contact with the team about next steps to move their findings from bench to bedside, the researchers say. (Although COVID-19 vaccines are starting to be rolled out, it’s expected to take the better part of a year for enough people to be vaccinated to create herd immunity. And there are no guarantees that the current vaccine formulations will be as effective against future SARS-CoV-2 strains that could emerge over time.)

More effective and well-timed therapeutic interventions could help reduce the overall number of deaths related to COVID-19 infections.

“What makes this research unusual is that we looked at very early time points [of infection], at just one hour after the virus infects lung cells. It was scary to see that the virus already starts to damage the cells so early during infection,” says Elke Mühlberger, one of the study’s senior investigators and a virologist at BU’s NEIDL. She typically works with some of the world’s most lethal viruses like Ebola and Marburg.

“The most striking aspect is how many molecular pathways are impacted by the virus,” says Andrew Emili, another of the study’s senior investigators, and the director of BU’s CNSB, which specializes in proteomics and deep sequencing of molecular interactions.

“The virus does wholesale remodeling of the lung cells – it’s amazing the degree to which the virus commandeers the cells it infects.”

Viruses can’t replicate themselves because they lack the molecular machinery for manufacturing proteins – that’s why they rely on infecting cells to hijack the cells’ internal machinery and use it to spread their own genetic material.

When SARS-CoV-2 takes over, it completely changes the cells’ metabolic processes, Emili says, and even damages the cells’ nuclear membranes within three to six hours after infection, which the team found surprising.

In contrast, “cells infected with the deadly Ebola virus don’t show any obvious structural changes at these early time points of infection, and even at late stages of infection, the nuclear membrane is still intact,” Mühlberger says.

The nuclear membrane surrounds the nucleus, which holds the majority of a cell’s genetic information and controls and regulates normal cellular functions. With the cell nucleus compromised by SARS-CoV-2, things rapidly take a bad turn for the entire cell.

Under siege, the cells – which normally play a role in maintaining the essential gas exchange of oxygen and carbon dioxide that occurs when we breathe – die. As the cells die, they also emit distress signals that boost inflammation, triggering a cascade of biological activity that speeds up cell death and can eventually lead to pneumonia, acute respiratory distress, and lung failure.

“I couldn’t have predicted a lot of these pathways, most of them were news to me,” says Andrew Wilson, one of the study’s senior authors, a CReM scientist, and a pulmonologist at Boston Medical Center (BMC), BU’s teaching hospital.

At BMC, Boston’s safety net hospital, Wilson has been on the front lines of the COVID-19 pandemic since March 2020, trying to treat and save the sickest patients in the hospital’s ICU. “That’s why our [experimental] model is so valuable.”

The team leveraged the CReM’s organoid expertise to grow human lung air sac cells, the type of cell that lines the inside of lungs. Air sac cells are usually difficult to grow and maintain in traditional culture and difficult to extract directly from patients for research purposes.

That’s why much coronavirus research to date by other labs has relied on the use of more readily available cell types, like kidney cells from monkeys. The problem with that is kidney cells from monkeys don’t react the same way to coronavirus infection as lung cells from humans do, making them a poor model for studying the virus – whatever is learned from them doesn’t easily translate into clinically relevant findings for treating human patients.

“Our organoids, developed by our CReM faculty, are engineered from stem cells—they’re not identical to the living, breathing cells inside our bodies, but they are the closest thing to it,” says Darrell Kotton, one of the study’s senior authors.

He is a director of the CReM and a pulmonologist at BMC, where he has worked alongside Wilson in the ICU treating COVID-19 patients. The two of them often collaborated with Mühlberger, Emili, and other members of their research team via Zoom calls that they managed to join during brief moments of calm in the ICU.

In another recent study using the CReM’s engineered human lung cells, the research team confirmed that existing drugs remdesivir and camostat are effective in combating the virus, though neither is a perfect fix for controlling the inflammation that COVID-19 causes.

Remdesivir, a broad-use antiviral, has already been used clinically in coronavirus patients. But based on the new study’s findings that the virus does serious damage to cells within hours, setting off inflammation, the researchers say there’s likely not much that antiviral drugs like remdesivir can do once an infection has advanced to the point where someone would need to be put on a ventilator in the ICU. “[Giving remdesivir] can’t save lives if the disease has already progressed,” Emili says.

Seeing how masterfully SARS-CoV-2 commandeers human cells and subverts them to do the manufacturing work of replicating the viral genome, it reminded the researchers of another deadly invader.

“I was surprised that there are so many similarities between cancer cells and SARS-CoV-2-infected cells,” Mühlberger says. The team screened a number of cancer drugs as part of their study and found that several of them are able to block SARS-CoV-2 from multiplying.

Like viruses, cancer cells want to replicate their own genomes, dividing over and over again.

To do that, they need to produce a lot of pyrimidine, a basic building block for genetic material. Interrupting the production of pyrimidine – using a cancer drug designed for that purpose – also blocks the SARS-CoV-2 genome from being built.

But Mühlberger cautions that cancer drugs typically have a lot of side effects. “Do we really want to use that heavy stuff against a virus?” she says. More studies will be needed to weigh the pros and cons of such an approach.

The findings of their latest study took the four senior investigators and scientists, postdoctoral fellows, and graduate students from their laboratories almost four months, working nearly around the clock, to complete the research. Of critical importance to the team’s leaders was making sure that the experimental setup had rock-solid foundations in mimicking what’s actually happening when the SARS-CoV-2 virus infects people.

“Science is the answer – if we use science to ask the lung cells what goes wrong when they are infected with coronavirus, the cells will tell us,” Kotton says. “Objective scientific data gives us hints at what to do and has lessons to teach us. It can reveal a path out of this pandemic.”

He’s particularly excited about the outreach the team has received from collaborators around the world. “People with expertise in supercomputers and machine learning are excited about using those tools and the datasets from our publication to identify the most promising drug targets [for treating COVID-19],” he says.

Kotton says the theme that’s become obvious among COVID-19 clinicians and scientists is understanding that timing is key. “Once a patient is on a ventilator in the ICU, we feel limited in what we can do for their body,” he says. “Timing is everything, it’s crucial to identify early windows of opportunity for intervention. You can keep guessing and hope we get lucky—or you [do the research] to actually understand the infection from its inception, and take the guesswork out of drug development.”

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Mechanism of SARS-CoV-2 infection

Based on SARS and MERS research and the latest SARS-CoV-2 sequence, these three coronaviruses capable of infecting humans share the same receptor (ACE2). The infection mechanism is shown in Fig. 1 . ACE2, also known as ACEH, is a member of the angiotensin-converting enzyme (ACE) family of dipeptidyl-carboxydipeptidase and is highly homologous to ACE1. ACE1 and ACE2 convert angiotensin 1 into angiotensin (Ang) 1–9 and angiotensin 2 into Ang 1−7.

ACE2 has high affinity to Ang II type 1 and type 2 receptors and plays an important role in many physiological functions, such as cell proliferation and hypertrophy, inflammatory response, blood pressure, and fluid balance. ACE2 is specifically expressed in certain organs and tissues, suggesting that it plays an important role in regulating cardiovascular, renal, and reproductive functions [^9,10].

The S-glycoprotein functional receptors for SARS-CoV and HCoV-NL63 are encoded by the ACE2 gene. RNA sequencing has been used to analyze 27 different types of tissues and 95 human tissue samples, showing that the expression of ACE2 is high in duodenum and small intestine and low in lung. Moreover, ACE2 expression is detected in placental choriocarcinoma cells (BEWO), immortalized human epidermal cells (HaCaT), liver cancer cells (HepG2), acute promyelocytic leukemia cells (NB-4), multiple myeloma cells (RPMI 8226), bladder cancer cells (RT4), and glioblastoma cells (SH-SY5Y).

In another study, the ACE2 protein level was determined in human organs and tissues, including respiratory mucosa, lung, stomach, small intestine, colon, skin, lymph nodes, thymus, bone marrow, spleen, liver, kidney, and brain. The results showed that ACE2 is abundantly expressed in the lungs and small intestine and is highly expressed in endothelial cells and smooth muscle cells of virtually all organs.

Therefore, once in the circulatory system, SARS-CoV-2 is likely to spread via blood flow [¹¹]. These data suggest that SARS-CoV-2 not only affects the respiratory system but is also a potential threat to the digestive system, urogenital system, central nervous system, and circulatory system.

SARS-CoV-2 and the digestive system
Based on current clinical and epidemiological data, the clinical symptoms of SARS-CoV-2 infection vary a great deal from patient to patient. The virus first affects the respiratory epithelial cells and alveolar cells, followed by the digestive system.

Evidence from previous studies about SARS proved that coronavirus has a tropism to the gastrointestinal tract. The viral nucleic acid could be readily detected in stool specimens of patients who have been infected with SARS. In addition, the replication process of this virus in the intestinal tissue is very active according to the observation of vivisection and autopsy under the electron microscope.

The kind of tropism above may explain the frequent occurrence of diarrhoea in coronavirus infection. Wong et al’s research showed that SARS-CoV-2 has the ability of encoding and expressing the spike (S) glycoproteins that could bind to the entry receptor ACE2. It is worth noting that in cytoplasm of gastric, duodenal and rectal epithelium, the expression of viral nucleocapsid protein is visualized [12,13].

Some SARS-CoV-2 cases present with diarrhea as the initial symptom. SARS-CoV-2 enters cells via the cell-surface receptor ACE2, and ACE2 regulates intestinal inflammatory response, which can be used to track SARS-CoV-2-mediated routes of transmission. Single-cell RNA sequencing showed that ACE2 mRNA is abundantly expressed in intestinal cells of healthy adults; in contrast, the expression level is relatively low in the lungs. Moreover, ACE2 expression is high in epithelial cells of the proximal and distal intestines.

The intestinal epithelium is in direct contact with exogenous pathogens, and we surmise that after consumption of SARS-CoV-2–infected wild animals, small intestinal epithelial cells are the first affected by the virus, and diarrhea may be an important sign of infection and clinical manifestation. This suggests that clinicians should pay attention to suspected patients who have diarrhea [14]. Lan and Cai performed RNA sequencing and found high, specific ACE2 expression in bile duct cells, suggesting that it is important to monitor liver function of SARS-CoV-2 patients, especially liver indicators involving bile duct function. In case of liver dysfunction, targeted treatment and care should be given in a timely manner [15].

ACE2 expression in the lungs reduces SARS-CoV-2 spike protein–induced lung injury via the renin-angiotensin system. In the intestines, ACE2 plays an important role in maintaining amino acid balance and regulating the expression of antimicrobial peptides and the equilibrium of the intestinal flora.

A recent RNA sequencing analysis in patients with inflammatory bowel disease (IBD) or colitis showed that ACE2 expression in colon cells was positively correlated with the regulation of viral infection and congenital cellular immunity and was negatively correlated with viral transcription, protein translation, phagocytosis, and complement activation [16]. Therefore, ACE2-mediated SARS-CoV-2 infection may be a double-edged sword with respect to susceptibility and immunity.

In summary, during clinical diagnosis and treatment of patients with SARS-CoV-2 infection, clinicians should pay attention to patients who present digestive symptoms (especially diarrhea) as the initial symptoms. Moreover, should diarrhea or other related symptoms of intestinal infection arise during treatment, the patient should receive prompt integrative treatment as needed, including anti-diarrhea therapy, hydration, correction of electrolyte disturbance, and antiviral therapy. In addition, because the virus and antiviral therapy may cause liver damage, patients should be closely monitored for liver function and receive liver-protective therapy as needed.

SARS-CoV-2 and the urogenital system
SARS-CoV-2 shares the same receptor as SARS-CoV. A retrospective analysis found that among SARS patients, the proportion of patients with acute renal insufficiency (ARI) was low but the mortality rate was more than 90 %. As a control, the researchers conducted a clinical study to assess kidney function in 59 patients with SARS-CoV-2 infection, including 28 severe cases and three deaths.

The results showed that 19 % of the patients had elevated serum creatinine, 27 % had elevated urea nitrogen, and 63 % had urine protein (+ to ++). Kidney CT was abnormal in all the patients [17]. In addition, three separate clinical studies in six, 41, and 99 patients with SARS-CoV-2 infection, respectively, showed that besides severe respiratory dysfunction, 3 %–10 % of the patients had renal insufficiency, and 7 % had acute kidney injury [18]. Zhong et al. showed that viral nucleic acid was isolated from the urine samples of SARS-CoV-2 patients. These data indicate that the incidence of kidney dysfunction is high after SARS-CoV-2 infection. The bladder may also be affected and may ultimately lead to multiple-organ failure and death.

Fan et al. further investigated the effect of SARS-CoV-2 infection on the urinary system and the male reproductive system. The researchers analyzed the online database and plotted ACE2 expression in various organs. The results showed that ACE2 is highly expressed in renal tubular cells, mesenchymal cells, and testicular and vas deferens cells. Surprisingly, ACE2 mRNA and protein levels are higher in testis than in any other organ.

The researchers surmise that SARS-CoV-2 binds to ACE2 to affect the kidneys and testis and subsequently causes their dysfunction [18]. Wang et al. found that ACE2 was predominantly enriched in spermatogonia and Leydig and Sertoli cells. Gene Set Enrichment Analysis (GSEA) indicated that Gene Ontology (GO) categories associated with viral reproduction and transmission were highly expressed in ACE2-positive spermatogonia, but terms which related to male gamete generation showed significantly low expression [[19]].

Both viral infection and antiviral therapy have potential nephrotoxicity and may cause kidney injury. Therefore, SARS-CoV-2 patients should be closely monitored for fluid and electrolyte disturbance and kidney function and receive specific targeted treatment and care as needed. Patients with chronic renal insufficiency should undergo hemodialysis or renal replacement therapy if necessary to facilitate metabolic waste removal. In addition, the virus may affect testicular tissue, and clinicians should assess the risk of testicular lesions in younger patients during the hospital treatment and follow-up and provide prompt prevention and treatment for potential SARS-CoV-2–related reproductive injury.

SARS-CoV-2 and the central nervous system [20]
SARS-CoV-2 may infect the central nervous system(CNS), as the viral nucleic acid has been detected in patient’s cerebrospinal fluid and brain tissue from autopsy. As for the route of virus entering the CNS, the blood route still needs to be further verified due to the existence of blood-brain barrier.

But on the other hand, neuronal pathway is important vehicles for neurotropic viruses to enter the CNS. Viruses can migrate after infecting sensory or motor nerve endings. Under the action of motor proteins, dynein and kinesins, the viruses can achieve neuronal transport in a way of retrograde or anterograde.

Here’s a classic example. Based on the unique anatomical structure of olfactory nerves and olfactory bulb, it becomes a channel between the nasal epithelium and the CNS. In the early stages of SARS-CoV-2 infection of the respiratory system, Olfactory tract becomes an important channel for virus transmission to brain. In addition to the above research, an Gu et al. study of gene sequences in neurons in the brain showed that coronavirus can invade the CNS from the periphery through neural pathways [21,22,24].

In addition, as the life center of human body, the brainstem controls vital functions such as heart beating, blood pressure maintenance and respiration. Studies have shown that some coronaviruses can invade brainstem via a synapse-connected route from the lungs and airways. The potential invasion to CNS of SARS-CoV-2 may be one reason for the acute respiratory failure. Therefore, it is of great significance for the treatment of severe patients to clarify the disease mechanism of patients with respiratory failure clinically whether it is caused by pulmonary lesions or viral infection of the brainstem [23].

Some confirmed cases present with specific headache, epilepsy, and confusion, which are similar to symptoms of intracranial infection. In some cases, intracranial infection–related symptoms have been the initial symptoms, coming before the symptoms of pulmonary infection, such as cough, fever, and dyspnea. Therefore, for suspected SARS-CoV-2 cases with intracranial symptoms, plain and enhanced brain magnetic resonance imaging and lumbar puncture (SARS-CoV-2 nucleic acid test on cerebral spinal fluid) are essential. For SARS-CoV-2 patients with intracranial symptoms, early treatment should be given to in addition to routine anti-infective therapy, including rehydration (reduce intracranial pressure), nerve nourishment, epilepsy prevention, and acid suppression.

Since SARS-CoV-2 binds to ACE2, some patients with underlying hypertension may have unusually high blood pressure and increased risk of intracranial hemorrhage after SARS-CoV-2 infection. Severely low platelets is also an important manifestation of critical SARS-CoV-2 infection, as well as an independent risk factor for acute cerebrovascular events. Angiotensin-converting enzyme inhibitors and angiotensin Ⅱ receptor blockers may increase the expression of ACE2. Therefore, it is important to adjust antihypertensive drug dosages in SARS-CoV-2 patients with underlying hypertension. Diuretics and calcium channel blockers are alternatives for management of blood pressure.

Moreover, some patients may have primary or secondary muscle injury, such as limb pain, fatigue, and elevated muscle enzymes, which may result from the inflammatory response or direct muscle damage following viral infection. Patients with SARS-CoV-2 infection and muscle injury should receive appropriate nutritional support, and γ-globulin injection in case of severe muscle injury.

Such as viral encephalitis, infectious toxic encephalopathy and some kind of acute cerebrovascular diseases, they are all related to coronavirus infections. For mechanisms by which SARS-CoV-2 infection may cause neurological damage, in addition to direct damage associated with ACE2 receptors, when SARS-CoV-2 proliferates in lung tissue cells, diffuse alveolar, interstitial inflammatory exudation and edema are all very common pathological changes.

It causes alveolar gas exchange disorders and hypoxia in the CNS. Due to anaerobic metabolism in the mitochondria in brain and excessive accumulation of acid metabolites, relevant clinical manifestations will appear such as swelling of brain cells, interstitial edema, obstruction of cerebral blood flow, headache due to ischemia and congestion and even a coma. Additionally, previous studies showed that when coronavirus attacked primary glial cells, a large amount of inflammatory factors such as IL-6, IL-12, IL-15, and TNF-α after being infected were released. This is also one of the pathophysiological processes of CNS damage caused by inflammatory factors [21,23].

SARS-CoV-2 and the circulatory system

Huang et al. showed that five of the 41 patients first diagnosed with SARS-CoV-2 infection in Wuhan, China, had viral myocarditis, mainly manifested as elevated hs-cTnl. Among them, four patients developed severe conditions, accounting for 31 % of all severe cases. The mean systolic blood pressure was significantly higher in severe cases than in nonsevere cases (145 mm Hg vs 122 mm Hg).

Clinical data show that an increasing number of SARS-CoV-2 patients present circulatory symptoms (palpitations, chest tightness, short of breath) as the initial symptoms [25]. On January 23, the National Health Commission (China) released a report on 17 deaths, which shows that two patients had no underlying cardiovascular disease but developed apparent cardiac symptoms after the diagnosis of SARS-CoV-2 infection. One patient had changes in the ST segment on electrocardiogram and persistent abnormal myocardial enzymes, and one patient had sudden progressive decline in heart rate and undetectable heart sounds.

Hou et al published an analysis of 84 cases of SARS-CoV-2 infection, which showed that elevated creatine kinase and creatine kinase isoenzyme MB during treatment is a sign of severe condition and disease progression. After SARS-CoV-2 infection in humans, immune disorders exacerbate the inflammatory response, which directly or indirectly leads to high risk of cardiovascular symptoms and diseases.

At present, researchers believe that three mechanisms may be involved in how SARS-CoV-2 infection induces acute myocardial injury. First, the virus infects the heart and causes myocardial injury directly. Second, Xu et al. showed that SARS-CoV-2 binds to highly expressed ACE2 receptors in the cardiovascular system to cause myocardial injury via certain signaling pathways [26].

Third, Huang et al. showed that in SARS-CoV-2 patients, Th1/Th2 imbalance triggers cytokine cascade, and the release of a large amount of cytokines causes myocardial injury [25]. In addition, hypoxaemia and respiratory dysfunction instigated by SARS-CoV-2 may also cause damage to myocardial cells. Meanwhile, plasma high-sensitivity C-reactive protein (hsCRP) is one of the most classical inflammation markers, as well as levels of cytokines linked to cardiovascular risk, are also related to adverse outcomes and could be some potential biomarkers to assess the disease progression [27,28].

SARS-CoV-2 and the inflammatory cascade

In the wake of SARS epidemic in late 2002 in Guangdong, China, many researchers investigated the pathogenesis of SARS in humans. Inflammatory cytokines and chemical factors are significantly elevated in SARS patients, but it is unclear whether these factors are the culprit or a secondary virus-induced cytopathology. Moreover, researchers are still debating whether the type 1 interferon response is involved during SARS-CoV infection. In vitro studies showed minimal interferon induction and signaling, while other studies reached different conclusions after analyzing peripheral white blood cells from patients with SARS-CoV infection.

Since the recent outbreak, cytokine cascades have been observed in many severe SARS-CoV-2 cases [25,29]. Fig. 2 shows the mechanism of cytokine cascade, also known as an inflammatory cascade. Pathogen infection triggers an intense immune response and inflammatory response and rapid release of a large amount of cytokines (such as tumor necrosis factor-α, interleukin (IL)-1, IL-6, and interferon-γ (IFN-γ)).

In this context, patients with viral infection are particularly susceptible to acute respiratory distress syndrome and multiple-organ failure. Cytokine cascades and low lymphocytes are also specific in other severe coronavirus diseases (such as SARS and MERS) and are positively related to disease progression and severity [[30], [31], [32]].

Recent studies have confirmed this conclusion, showing low lymphocytes and elevated inflammatory cytokines in most SARS-CoV-2 cases [33,34]. Once triggered, the cytokine cascade may cause rapid failure of one or more organs with extremely adverse prognosis if not treated promptly. It is considered an important risk factor for mortality in critical SARS-CoV-2 cases. Wei et al. and Xu et al. from the University of Science and Technology of China conducted immunological analyses of blood samples from 33 patients with SARS-CoV-2 infection to investigate how the cytokine cascade occurs in severe SARS-CoV-2 cases.

The results showed that SARS-CoV-2 rapidly activates pathogenic T cells and induces the release of a large amount of inflammatory cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1, IL-6, and IFN-γ. GM-CSF activates CD14+ cells, CD16+ cells, and monocytes, resulting in further release of inflammatory cytokines such as IL-6. This process continues to strengthen the inflammatory cascade.

The intense immune response causes damage to the lungs and other vital organs. Li et.al also found that the direct attack on other organs by disseminated SARS-CoV-2, the immune pathogenesis caused by the systemic cytokine storm, and the microcirculation dysfunctions together lead to viral sepsis. Therefore, effective antiviral treatment and measures to modulate the innate immune response and restore the adaptive immune response are important for breaking the vicious cycle and improving the treatment effect [6].

reference link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7186209/

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More information: Ryan M. Hekman et al, Actionable Cytopathogenic Host Responses of Human Alveolar Type 2 Cells to SARS-CoV-2, Molecular Cell (2020). DOI: 10.1016/j.molcel.2020.11.028