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Protein OCLN found to play crucial role in SARS-CoV-2 cell-to-cell transmission

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In a recent study published in the journal PNAS, researchers demonstrated that the tight junction (TJ) protein occludin (OCLN) is critical for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S)-mediated, direct cell-to-cell transmission inside a host.

Study: Tight junction protein occludin is an internalization factor for SARS-CoV-2 infection and mediates virus cell-to-cell transmission. Image Credit: Naeblys / Shutterstock

Background

Viral transmission via tight cell-to-cell contact diminishes the effectiveness of antiviral drugs and helps viruses evade neutralizing antibodies (nAbs). Studies have documented that many human viruses, including SARS-CoV-2, influenza A, and respiratory syncytial virus (RSV), use this mechanism, cell-free particles, or both to establish infection and survive inside a host.

Previous studies have also associated the formation of syncytia in human lung tissues with SARS-CoV-2. In SARS-CoV-2 and other coronaviruses (CoVs), e.g., Middle Eastern Respiratory Syndrome (MERS-CoV), syncytia formation contributes to the augmented direct cell-to-cell transmission. However, the host factors involved in this process and the underlying mechanisms of S glycoprotein–induced cell-to-cell dissemination of SARS-CoV-2 remain unclear.

Epithelial cells in human airways have intercellular structures called TJs that regulate the passage of ions and small solutes. They also serve as the first barrier to pathogens. TJs contain the protein OCLN, acknowledged as a vital host factor for the entry of several viruses inside a human host, e.g., rotavirus, and hepatitis C virus.

Since the virus-induced fusion of virus-infected cells with neighboring cells and the creation of syncytia requires breaking the intercellular barrier, the researchers speculated that TJ proteins might be involved in the spread of SARS-CoV-2 via cell-to-cell transmission.

About the study

In the present study, researchers examined the effect of SARS-CoV-2 infection on TJ proteins, which helped them uncover a previously unknown role of OCLN as a host-origin internalization factor for SARS-CoV-2 entry and subsequent cell-to-cell transmission within the host cells. They used Vero-E6 cells infected with replication-competent vesicular stomatitis virus (rVSV) expressing enhanced green fluorescent protein (eGFP) and SARS-CoV-2 S (rVSV-eGFP-S), and alternatively, a SARS-CoV-2 recombinant expressing the mNeonGreen gene to determine the distribution of OCLN in infected Vero-E6 cells.

Western blot confirmed that infections by both recombinants altered OCLN expression. Accordingly, OCLN levels became undetectable 48 hour postinfection (pi) in infected Vero-E6 cells. The team also confirmed these results in human-derived A549-hACE2 cells and a hamster SARS-CoV-2 infection model.

Next, the team determined whether permissive cells, e.g., Vero-E6 and A549-hACE2 cells, also need OCLN for the establishment of SARS-CoV-2 infection. So, they transfected Vero-E6 cells with two OCLN-targeted small interfering RNAs (siRNAs), which helped them confirm OCLN expression using the immunofluorescence assay (IFA) and quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR). Furthermore, the researchers performed the coimmunoprecipitation (co-IP) assay to visualize the OCLN’s interaction with the SARS-CoV-2 S protein.

OCLN is a transmembrane protein with four-helical domains, viz., an intracellular N-terminal domain, a long cytoplasmic tail, also known as C-terminal, two extracellular loops, EL1 & EL2 linked via a short intracellular loop. So, finally, the researchers determined which OCLN domain was critical for SARS-CoV-2 internalization and cell-to-cell transmission.

To this end, the team constructed four OCLN deletion constructs, viz., hOCLN/ΔC, hOCLN/ΔE1, hOCLN/ΔE2, and hOCLN/ΔN, and transfected them into the OCLN KO Vero-E6 cell line. Next, they confirmed the expression of each using Western blot and IFA.

Results

The study pointed out that OCLN, a host factor, mediated SARS-CoV-2 internalization and subsequent cell-to-cell transmission. Thus, its knockdown decreased SARS-CoV-2 spread, while its overexpression promoted it in experiments with rVSVs expressing S proteins of multiple SARS-CoV-2 variants. In vitro and in vivo SARS-CoV-2 infection markedly decreased and eventually completely destructed OCLN.

All rVSVs expressing S proteins of the SARS-CoV-2 Delta, Beta, and Kappa variants exhibited enhanced cell-to-cell transmission than the WA-1 strain. The Gamma & Alpha variants exhibited comparable abilities to drive syncytium formation as the WA-1 strain. Intriguingly, Omicron exhibited a markedly decimated capacity to form syncytia in A549-hACE2 cells relative to other SARS-CoV-2 variants and WA-1, indicating that Omicron has limited ability to spread cell-cell using the endocytic pathway. This observation also justifies why Omicron BA.1/BA.2 sublineages trigger trivial clinical symptoms than WA-1 and other SARS-CoV-2 variants.

Further, the study experiments suggested that SARS-CoV-2 infection only degraded OCLN but had minimal impact on other TJ proteins, e.g., Claudin-1. Future studies should investigate the mechanisms underlying the mechanisms of SARS-CoV-2-induced down-regulation of the synthesis and stability of the OCLN protein in TJs. In SARS-CoV-2 permissive cells, the knockdown of OCLN did not impact angiotensin-converting enzyme 2 (ACE2) expression but markedly reduced viral infection, especially during the internalization step and subsequent viral replication. Conversely, its overexpression significantly enhanced both SARS-CoV-2’s internalization and replication but not its binding. Thus, OCLN could not be considered a receptor or coreceptor for SARS-CoV-2 entry.

The study results also revealed that the endosomal entry pathway was engaged in OCLN-mediated SARS-CoV-2 cell-to-cell transmission. Finally, treatment with trypsin markedly promoted SARS-CoV-2 cell-to-cell transmission and augmented virus yield in host cells, suggesting that this phenomenon helps preserve viral infectivity and transmissibility more than cell-free transmission.

Conclusions

The current study provided mechanistic insights into SARS-CoV-2 entry and subsequent spread inside a human host. The researchers presented robust substantiation of OCLN-driven cell-to-cell transmission of WA-1, and all other SARS-CoV-2 variants examined in this study. Indeed, OCLN is a crucial host factor involved in SARS-CoV-2 internalization and subsequent cell-to-cell viral transmission.

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Health Canada approves updated Moderna COVID-19 vaccine

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TORONTO – Health Canada has authorized Moderna’s updated COVID-19 vaccine that protects against currently circulating variants of the virus.

The mRNA vaccine, called Spikevax, has been reformulated to target the KP.2 subvariant of Omicron.

It will replace the previous version of the vaccine that was released a year ago, which targeted the XBB.1.5 subvariant of Omicron.

Health Canada recently asked provinces and territories to get rid of their older COVID-19 vaccines to ensure the most current vaccine will be used during this fall’s respiratory virus season.

Health Canada is also reviewing two other updated COVID-19 vaccines but has not yet authorized them.

They are Pfizer’s Comirnaty, which is also an mRNA vaccine, as well as Novavax’s protein-based vaccine.

This report by The Canadian Press was first published Sept. 17, 2024.

Canadian Press health coverage receives support through a partnership with the Canadian Medical Association. CP is solely responsible for this content.

The Canadian Press. All rights reserved.

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These people say they got listeria after drinking recalled plant-based milks

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TORONTO – Sanniah Jabeen holds a sonogram of the unborn baby she lost after contracting listeria last December. Beneath, it says “love at first sight.”

Jabeen says she believes she and her baby were poisoned by a listeria outbreak linked to some plant-based milks and wants answers. An investigation continues into the recall declared July 8 of several Silk and Great Value plant-based beverages.

“I don’t even have the words. I’m still processing that,” Jabeen says of her loss. She was 18 weeks pregnant when she went into preterm labour.

The first infection linked to the recall was traced back to August 2023. One year later on Aug. 12, 2024, the Public Health Agency of Canada said three people had died and 20 were infected.

The number of cases is likely much higher, says Lawrence Goodridge, Canada Research Chair in foodborne pathogen dynamics at the University of Guelph: “For every person known, generally speaking, there’s typically 20 to 25 or maybe 30 people that are unknown.”

The case count has remained unchanged over the last month, but the Public Health Agency of Canada says it won’t declare the outbreak over until early October because of listeria’s 70-day incubation period and the reporting delays that accompany it.

Danone Canada’s head of communications said in an email Wednesday that the company is still investigating the “root cause” of the outbreak, which has been linked to a production line at a Pickering, Ont., packaging facility.

Pregnant people, adults over 60, and those with weakened immune systems are most at risk of becoming sick with severe listeriosis. If the infection spreads to an unborn baby, Health Canada says it can cause miscarriage, stillbirth, premature birth or life-threatening illness in a newborn.

The Canadian Press spoke to 10 people, from the parents of a toddler to an 89-year-old senior, who say they became sick with listeria after drinking from cartons of plant-based milk stamped with the recalled product code. Here’s a look at some of their experiences.

Sanniah Jabeen, 32, Toronto

Jabeen says she regularly drank Silk oat and almond milk in smoothies while pregnant, and began vomiting seven times a day and shivering at night in December 2023. She had “the worst headache of (her) life” when she went to the emergency room on Dec. 15.

“I just wasn’t functioning like a normal human being,” Jabeen says.

Told she was dehydrated, Jabeen was given fluids and a blood test and sent home. Four days later, she returned to hospital.

“They told me that since you’re 18 weeks, there’s nothing you can do to save your baby,” says Jabeen, who moved to Toronto from Pakistan five years ago.

Jabeen later learned she had listeriosis and an autopsy revealed her baby was infected, too.

“It broke my heart to read that report because I was just imagining my baby drinking poisoned amniotic fluid inside of me. The womb is a place where your baby is supposed to be the safest,” Jabeen said.

Jabeen’s case is likely not included in PHAC’s count. Jabeen says she was called by Health Canada and asked what dairy and fresh produce she ate – foods more commonly associated with listeria – but not asked about plant-based beverages.

She’s pregnant again, and is due in several months. At first, she was scared to eat, not knowing what caused the infection during her last pregnancy.

“Ever since I learned about the almond, oat milk situation, I’ve been feeling a bit better knowing that it wasn’t something that I did. It was something else that caused it. It wasn’t my fault,” Jabeen said.

She’s since joined a proposed class action lawsuit launched by LPC Avocates against the manufacturers and sellers of Silk and Great Value plant-based beverages. The lawsuit has not yet been certified by a judge.

Natalie Grant and her seven year-old daughter, Bowmanville, Ont.

Natalie Grant says she was in a hospital waiting room when she saw a television news report about the recall. She wondered if the dark chocolate almond milk her daughter drank daily was contaminated.

She had brought the girl to hospital because she was vomiting every half hour, constantly on the toilet with diarrhea, and had severe pain in her abdomen.

“I’m definitely thinking that this is a pretty solid chance that she’s got listeria at this point because I knew she had all the symptoms,” Grant says of seeing the news report.

Once her daughter could hold fluids, they went home and Grant cross-checked the recalled product code – 7825 – with the one on her carton. They matched.

“I called the emerg and I said I’m pretty confident she’s been exposed,” Grant said. She was told to return to the hospital if her daughter’s symptoms worsened. An hour and a half later, her fever spiked, the vomiting returned, her face flushed and her energy plummeted.

Grant says they were sent to a hospital in Ajax, Ont. and stayed two weeks while her daughter received antibiotics four times a day until she was discharged July 23.

“Knowing that my little one was just so affected and how it affected us as a family alone, there’s a bitterness left behind,” Grant said. She’s also joined the proposed class action.

Thelma Feldman, 89, Toronto

Thelma Feldman says she regularly taught yoga to friends in her condo building before getting sickened by listeria on July 2. Now, she has a walker and her body aches. She has headaches and digestive problems.

“I’m kind of depressed,” she says.

“It’s caused me a lot of physical and emotional pain.”

Much of the early days of her illness are a blur. She knows she boarded an ambulance with profuse diarrhea on July 2 and spent five days at North York General Hospital. Afterwards, she remembers Health Canada officials entering her apartment and removing Silk almond milk from her fridge, and volunteers from a community organization giving her sponge baths.

“At my age, 89, I’m not a kid anymore and healing takes longer,” Feldman says.

“I don’t even feel like being with people. I just sit at home.”

Jasmine Jiles and three-year-old Max, Kahnawake Mohawk Territory, Que.

Jasmine Jiles says her three-year-old son Max came down with flu-like symptoms and cradled his ears in what she interpreted as a sign of pain, like the one pounding in her own head, around early July.

When Jiles heard about the recall soon after, she called Danone Canada, the plant-based milk manufacturer, to find out if their Silk coconut milk was in the contaminated batch. It was, she says.

“My son is very small, he’s very young, so I asked what we do in terms of overall monitoring and she said someone from the company would get in touch within 24 to 48 hours,” Jiles says from a First Nations reserve near Montreal.

“I never got a call back. I never got an email”

At home, her son’s fever broke after three days, but gas pains stuck with him, she says. It took a couple weeks for him to get back to normal.

“In hindsight, I should have taken him (to the hospital) but we just tried to see if we could nurse him at home because wait times are pretty extreme,” Jiles says, “and I don’t have child care at the moment.”

Joseph Desmond, 50, Sydney, N.S.

Joseph Desmond says he suffered a seizure and fell off his sofa on July 9. He went to the emergency room, where they ran an electroencephalogram (EEG) test, and then returned home. Within hours, he had a second seizure and went back to hospital.

His third seizure happened the next morning while walking to the nurse’s station.

In severe cases of listeriosis, bacteria can spread to the central nervous system and cause seizures, according to Health Canada.

“The last two months have really been a nightmare,” says Desmond, who has joined the proposed lawsuit.

When he returned home from the hospital, his daughter took a carton of Silk dark chocolate almond milk out of the fridge and asked if he had heard about the recall. By that point, Desmond says he was on his second two-litre carton after finishing the first in June.

“It was pretty scary. Terrifying. I honestly thought I was going to die.”

Cheryl McCombe, 63, Haliburton, Ont.

The morning after suffering a second episode of vomiting, feverish sweats and diarrhea in the middle of the night in early July, Cheryl McCombe scrolled through the news on her phone and came across the recall.

A few years earlier, McCombe says she started drinking plant-based milks because it seemed like a healthier choice to splash in her morning coffee. On June 30, she bought two cartons of Silk cashew almond milk.

“It was on the (recall) list. I thought, ‘Oh my God, I got listeria,’” McCombe says. She called her doctor’s office and visited an urgent care clinic hoping to get tested and confirm her suspicion, but she says, “I was basically shut down at the door.”

Public Health Ontario does not recommend listeria testing for infected individuals with mild symptoms unless they are at risk of developing severe illness, such as people who are immunocompromised, elderly, pregnant or newborn.

“No wonder they couldn’t connect the dots,” she adds, referencing that it took close to a year for public health officials to find the source of the outbreak.

“I am a woman in my 60s and sometimes these signs are of, you know, when you’re vomiting and things like that, it can be a sign in women of a bigger issue,” McCombe says. She was seeking confirmation that wasn’t the case.

Disappointed, with her stomach still feeling off, she says she decided to boost her gut health with probiotics. After a couple weeks she started to feel like herself.

But since then, McCombe says, “I’m back on Kawartha Dairy cream in my coffee.”

This report by The Canadian Press was first published Sept. 16, 2024.

Canadian Press health coverage receives support through a partnership with the Canadian Medical Association. CP is solely responsible for this content.

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B.C. mayors seek ‘immediate action’ from federal government on mental health crisis

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VANCOUVER – Mayors and other leaders from several British Columbia communities say the provincial and federal governments need to take “immediate action” to tackle mental health and public safety issues that have reached crisis levels.

Vancouver Mayor Ken Sim says it’s become “abundantly clear” that mental health and addiction issues and public safety have caused crises that are “gripping” Vancouver, and he and other politicians, First Nations leaders and law enforcement officials are pleading for federal and provincial help.

In a letter to Prime Minister Justin Trudeau and Premier David Eby, mayors say there are “three critical fronts” that require action including “mandatory care” for people with severe mental health and addiction issues.

The letter says senior governments also need to bring in “meaningful bail reform” for repeat offenders, and the federal government must improve policing at Metro Vancouver ports to stop illicit drugs from coming in and stolen vehicles from being exported.

Sim says the “current system” has failed British Columbians, and the number of people dealing with severe mental health and addiction issues due to lack of proper care has “reached a critical point.”

Vancouver Police Chief Adam Palmer says repeat violent offenders are too often released on bail due to a “revolving door of justice,” and a new approach is needed to deal with mentally ill people who “pose a serious and immediate danger to themselves and others.”

This report by The Canadian Press was first published Sept. 16, 2024

The Canadian Press. All rights reserved.

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