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.
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.
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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.
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 government says it will donate up to 200,000 vaccine doses to fight the mpox outbreak in Congo and other African countries.
It says the donated doses of Imvamune will come from Canada’s existing supply and will not affect the country’s preparedness for mpox cases in this country.
Minister of Health Mark Holland says the donation “will help to protect those in the most affected regions of Africa and will help prevent further spread of the virus.”
Dr. Madhukar Pai, Canada research chair in epidemiology and global health, says although the donation is welcome, it is a very small portion of the estimated 10 million vaccine doses needed to control the outbreak.
Vaccine donations from wealthier countries have only recently started arriving in Africa, almost a month after the World Health Organization declared the mpox outbreak a public health emergency of international concern.
A few days after the declaration in August, Global Affairs Canada announced a contribution of $1 million for mpox surveillance, diagnostic tools, research and community awareness in Africa.
On Thursday, the Africa Centres for Disease Control and Prevention said mpox is still on the rise and that testing rates are “insufficient” across the continent.
Jason Kindrachuk, Canada research chair in emerging viruses at the University of Manitoba, said donating vaccines, in addition to supporting surveillance and diagnostic tests, is “massively important.”
But Kindrachuk, who has worked on the ground in Congo during the epidemic, also said that the international response to the mpox outbreak is “better late than never (but) better never late.”
“It would have been fantastic for us globally to not be in this position by having provided doses a much, much longer time prior than when we are,” he said, noting that the outbreak of clade I mpox in Congo started in early 2023.
Clade II mpox, endemic in regions of West Africa, came to the world’s attention even earlier — in 2022 — as that strain of virus spread to other countries, including Canada.
Two doses are recommended for mpox vaccination, so the donation may only benefit 100,000 people, Pai said.
Pai questioned whether Canada is contributing enough, as the federal government hasn’t said what percentage of its mpox vaccine stockpile it is donating.
“Small donations are simply not going to help end this crisis. We need to show greater solidarity and support,” he said in an email.
“That is the biggest lesson from the COVID-19 pandemic — our collective safety is tied with that of other nations.”
This report by The Canadian Press was first published Sept. 13, 2024.
Canadian Press health coverage receives support through a partnership with the Canadian Medical Association. CP is solely responsible for this content.
HALIFAX – The Nova Scotia government says it could be months before it reveals how many people are on the wait-list for a family doctor.
The head of the province’s health authority told reporters Wednesday that the government won’t release updated data until the 160,000 people who were on the wait-list in June are contacted to verify whether they still need primary care.
Karen Oldfield said Nova Scotia Health is working on validating the primary care wait-list data before posting new numbers, and that work may take a matter of months. The most recent public wait-list figures are from June 1, when 160,234 people, or about 16 per cent of the population, were on it.
“It’s going to take time to make 160,000 calls,” Oldfield said. “We are not talking weeks, we are talking months.”
The interim CEO and president of Nova Scotia Health said people on the list are being asked where they live, whether they still need a family doctor, and to give an update on their health.
A spokesperson with the province’s Health Department says the government and its health authority are “working hard” to turn the wait-list registry into a useful tool, adding that the data will be shared once it is validated.
Nova Scotia’s NDP are calling on Premier Tim Houston to immediately release statistics on how many people are looking for a family doctor. On Tuesday, the NDP introduced a bill that would require the health minister to make the number public every month.
“It is unacceptable for the list to be more than three months out of date,” NDP Leader Claudia Chender said Tuesday.
Chender said releasing this data regularly is vital so Nova Scotians can track the government’s progress on its main 2021 campaign promise: fixing health care.
The number of people in need of a family doctor has more than doubled between the 2021 summer election campaign and June 2024. Since September 2021 about 300 doctors have been added to the provincial health system, the Health Department said.
“We’ll know if Tim Houston is keeping his 2021 election promise to fix health care when Nova Scotians are attached to primary care,” Chender said.
This report by The Canadian Press was first published Sept. 11, 2024.
