Virus World

Viral variant is one known risk factor associated with post-acute sequelae of COVID-19 (PASC), yet the pathogenesis is largely unknown. Here, we studied SARS-CoV-2 Delta variant-induced PASC in K18-hACE2 mice. The virus replicated productively, induced robust inflammatory responses in lung and brain tissues, and caused weight loss and mortality during the acute infection. Longitudinal behavior studies in surviving mice up to 4 months post-acute infection revealed persistent abnormalities in neuropsychiatric state and motor behaviors, while reflex and sensory functions recovered over time. In the brain, no detectable viral RNA and minimal residential immune cell activation was observed in the surviving mice post-acute infection.

Transcriptome analysis revealed persistent activation of immune pathways, including humoral responses, complement, and phagocytosis, and gene expression levels associated with ataxia telangiectasia, impaired cognitive function and memory recall, and neuronal dysfunction and degeneration. Furthermore, surviving mice maintained potent systemic T helper 1 prone cellular immune responses and strong sera neutralizing antibodies against Delta and Omicron variants months post-acute infection. Overall, our findings suggest that infection in K18-hACE2 mice recapitulates the persistent clinical symptoms reported in long-COVID patients and provides new insights into the role of systemic and brain residential immune factors in PASC pathogenesis.

Published in Frontiers in Immunology (May 2, 2024):

https://doi.org/10.3389/fimmu.2024.1384516

Significance

Abstract

Scientists have created a first-of-its-kind mouse model of the COVID-19 disease, capable of revealing how the SARS-CoV-2 virus wreaks havoc on multiple organ systems in the animal's body. While this experimental model doesn't directly correspond to human coronavirus cases, it's a breakthrough in other ways, giving us a potential test system for exploring how the deadly respiratory pathogen extends way beyond the respiratory system in many cases of infection. "This mouse model is a really powerful tool for studying SARS-CoV-2 in a living system," explains cardiologist Arjun Deb from UCLA. Mouse models of COVID-19 have been engineered before, but none have gotten us to this point, the researchers say, by demonstrating what extra-pulmonary manifestations of COVID-19 look like. That's been a significant limitation for animal-based research into the virus's progression, and it's due to a couple of reasons. While mouse cells contain an analogue of the ACE2 receptor that the SARS-CoV-2 uses to bind to human cells, the virus doesn't seem to attach to the mouse version of the molecule. Genetically engineering mice with the human version of ACE2 provides a workaround of sorts, but before now, scientists hadn't succeeded in inducing multiple organ failure in mice, which could mimic the way human cases of extra-pulmonary infection tend to present. The shortfall may have been because prior studies used nasal inoculation on mice, infecting the animals with SARS-CoV-2 through the nose, which doesn't seem to develop into full-blown, system-wide coronavirus infections in mice.

In the new study, led by first author Shen Li, a cardiologist at UCLA, the team got around this by injecting the virus into the bloodstreams of engineered mice, where it could reach the human version of ACE2 (called hACE2) in the heart and other vital organs. Unfortunately for the subjects, the experimental tweaks worked. And while it wasn't a good outcome for the mice involved, being able to study systemically induced toxicity in SARS-CoV-2-infected mice could significantly expand our knowledge of the virus's branching capabilities in human cases. Such knowledge is sorely needed. "Among COVID-19 patients, those who have organs involved other than the lungs are most at risk of a bad outcome," Deb says. "So we felt it was really important to understand how the virus affects those other organs." In the experiment, systemic administration of the infection provoked rapid results. Within seven days, the infected mice "demonstrated profound morbidity, severely restricted activity, and were found huddled at the corner of the cage", in contrast with a control group of similarly engineered mice who were spared the infection, remaining healthy. In the same amount of time, the infected mice lost up to about 25 percent of their body weight due to sharply reduced food consumption, necessitating euthanisation. The infected mice also had damaged spleens, irregular heart activity and blood pressure, and altered levels of immune cells – all symptoms resembling human cases of COVID-19.

After the animals were euthanised, analysis of their organs revealed changes in gene expression in multiple tissues, disrupting cellular processes that generate energy in the body. "If a virus snuffs out the energy-generating pathways in multiple organs of the body, that's going to really wreak havoc," Deb explains.  Beyond these effects, the infected mice also bore numerous signs of epigenetic changes, which could explain the altered gene expression evident in multiple organs. It's not known for sure, but the impacts of this could potentially be felt long after an infection has been beaten by the immune system – and, hypothetically speaking, could be the basis of the prolonged symptoms experienced by COVID-19 'long haulers'. "Although the physiological significance of SARS-CoV-2 altered DNA methylation patterns is not clear from our study, our model provides proof of concept that such epigenetic changes do occur soon after SARS-CoV-2 infection and can potentially led to persistent transcriptional changes affecting tissue homeostasis and organ function," the authors write in their paper. "Such epigenetic changes potentially occurring in humans with COVID-19 could lead to symptoms from persistent changes in dysregulated gene expression in infected tissues even in the absence of tissue viral burden."...

Cited Research Published in JCI Insight (Dec. 7, 2020):

https://doi.org/10.1172/jci.insight.145027

The studies were run on a total of 36 rhesus macaques and 50 hamsters. The company is careful to caution that positive animal studies do not guarantee success in humans. In early July, Regeneron Pharmaceuticals, with the U.S. National Institute of Allergy and Infectious Diseases (NIAID), announced they were launching Phase III trials of REGN-COV-2, the company’s two-antibody cocktail to treat and prevent COVID-19. Although those results of those trials are still pending, the company issued a scientific article, not yet peer-reviewed, that shows the cocktail was able to “almost completely block establishment of virus infection,” in rhesus macaques and hamsters. Using its proprietary VelocImmune mice, Regeneron screened thousands of fully-human antibodies. The mice have been genetically modified to have a human immune system. They also evaluated antibodies isolated from people who have recovered from COVID-19. They then chose the two most potent, non-competing antibodies that were most effective at neutralizing SARS-CoV-2, the virus that causes COVID-19. They then scaled up the dual-antibody cocktail for clinical use with the company’s own VelociMab and manufacturing capabilities. The two antibodies bind non-competitively to the receptor binding domain of the virus’s spike (S) protein. This decreases the ability of mutant viruses to avoid treatment and protects against spike variants that have evolved in the human population. On June 11, the company announced the publication of two scientific articles describing the effectiveness of REGN-COV-2 in the journal Science. The first paper was, “Studies in humanized mice and convalescent humans yield a SARS-COV-2 antibody cocktail.”

In the latest animal study paper, the authors said the cocktail in monkeys and hamsters demonstrated “that REGEN-COV-2 can greatly reduce virus load in lower and upper airway and decrease virus-induced pathological sequelae when administered prophylactically or therapeutically. Our results provide evidence of the therapeutic potential of this antibody cocktail.” Essentially, the study took two approaches. One was to infect animals with the virus, then dose them with the antibody cocktail to see if it could halt the infection. It appeared to do this successfully. 

The second approach was to dose the animals with the antibody cocktail, then infect them with the virus. This also appeared to prevent infection. It’s important to note that the antibody cocktail is not a vaccine. Therapeutic antibodies such as these do not have long-range effects, although they may last several months. The studies were run on a total of 36 rhesus macaques and 50 hamsters. The company is careful to caution that positive animal studies do not guarantee success in humans. The human trials have already started. There are specifically two trials. A Phase III trial will study if REGN-COV-2 can prevent infection in uninfected people who have had close exposure to a COVID-19 patient. The drug has also been advanced into the Phase II/III portion of two adaptive Phase I/II/III trials evaluating the cocktail in treating hospitalized and non-hospitalized patients with COVID-19....

Preprint available at bioRxiv (August 3, 2020):

https://doi.org/10.1101/2020.08.02.233320

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of human infections and an effective vaccine is critical to mitigate coronavirus-induced disease 2019 (COVID-19). Previously, we developed a replication-competent vesicular stomatitis virus (VSV) expressing a modified form of the SARS-CoV-2 spike gene in place of the native glycoprotein gene (VSV-eGFP-SARS-CoV-2).

Here, we show that vaccination with VSV-eGFP-SARS-CoV-2 generates neutralizing immune responses and protects mice from SARS-CoV-2. Immunization of mice with VSV-eGFP-SARS-CoV-2 elicits high antibody titers that neutralize SARS-CoV-2 and target the receptor binding domain that engages human angiotensin converting enzyme-2 (ACE2). Upon challenge with a human isolate of SARS-CoV-2, mice expressing human ACE2 and immunized with VSV-eGFP-SARS-CoV-2 show profoundly reduced viral infection and inflammation in the lung, indicating protection against pneumonia. Passive transfer of sera from VSV-eGFP-SARS-CoV-2-immunized animals also protects naïve mice from SARS-CoV-2 challenge. These data support development of VSV-eGFP-SARS-CoV-2 as an attenuated, replication-competent vaccine against SARS-CoV-2.

Original study published in Cell (July 30, 2020):

https://doi.org/10.1016/j.chom.2020.07.018

Researchers at Georgia State University.develop universal flu vaccine that protects against six influenza viruses in mice. The researchers developed and showed that a novel nanoparticle vaccine that combines two major influenza proteins is effective in providing broad, long-lasting protection against the influenza virus in mice, showing promise as a universal flu vaccine. Findings from the new study—performed in mice and published recently in Advanced Healthcare Materials through an article titled “Double‐Layered M2e‐NA Protein Nanoparticle Immunization Induces Broad Cross‐Protection against Different Influenza Viruses in Mice”—suggest this unique vaccine combination has potential as a universal influenza vaccine or component of such vaccines.

The double-layered nanoparticle vaccine contains the influenza virus proteins matrix protein 2 ectodomain (M2e) and neuraminidase (NA). Mice were immunized with the nanoparticle vaccine before being exposed to the influenza virus, and they were protected against six different strains of the virus. “This nanoparticle antigen combination conferred mice with strong cross-protection,” explained lead study investigator Ye Wang, a doctoral candidate at the Institute for Biomedical Sciences. “It can protect mice from different strains of influenza virus. Each season, we have different flu strains that affect us. By using this approach, we hope this nanoparticle vaccine can protect humans from different strains of the influenza virus.”

Influenza is a leading cause of death by infection. Seasonal flu vaccines are insufficient to prevent influenza outbreaks and developing a universal influenza vaccine is the ideal strategy for eliminating public health threats of influenza epidemics and pandemics. A universal influenza vaccine would eliminate the need for vaccinations each season and offers universal protection against all influenza strains. The influenza virus protein M2e is found in all influenza virus strains, with each strain having a very similar version, and the protein has mutated very slowly over time. The protein NA is found on the surface of the influenza virus and has also mutated much slower than other influenza proteins. This double-layered nanoparticle vaccine uses M2e as its core, and NA is coated on the surface. In the current study, mice were exposed to one of six influenza virus strains after receiving the nanoparticle vaccine by intramuscular injection. The vaccine proved to have long-lasting immune protection, which was unchanged against viral challenges up to four months after immunizations. 

Published in Advanced Healthcare Materials (Dec. 15, 2019):

https://doi.org/10.1002/adhm.201901176

But doubts remain about whether cell reprogramming technique could one day help.  A decade after Kyoto University biologist Shinya Yamanaka won a share of a Nobel Prize for discovering a cocktail of proteins that reprogram adult cells into versatile stem cells, two teams argue the proteins can turn back the clock for entire organisms—perhaps one day humans. One group at a biotech used gene therapy to deliver some of the so-called Yamanaka factors into old mice, and modestly extended their life span. And a separate team followed a similar strategy to reverse aging-like changes in genetically engineered mice.  In both cases, the Yamanaka factors appear to have restored part of the animals’ epigenome, chemical modifications on DNA and proteins that help regulate gene activity, to a more youthful state. But scientists not involved in the work say suggestions of age reversal are premature. “These studies use reprogramming factors to reverse epigenetic changes that happen during aging,” says Matt Kaeberlein, a geroscientist at the University of Washington, Seattle, but that’s a far cry from making an old animal young again. Several groups had already found genetically engineered mice that begin expressing Yamanaka factors in adulthood show reversal of certain aging symptoms. To explore an approach that might lead to a more practical treatment for people, San Diego–based company Rejuvenate Bio injected elderly (124-week-old) mice with adeno-associated viruses (AAVs) carrying genes for three of the factors, collectively known as OSK.

These animals lived another 18 weeks on average, compared with 9 weeks for a control group, the company reported in a preprint on bioRxiv this month. They also partially regained patterns of DNA methylation—a type of epigenetic mark—typical of younger animals. Although some studies have suggested Yamanaka factors can promote cancer, Noah Davidsohn, Rejuvenate’s chief scientific officer and co-founder, says the company has so far found no obvious negative effects in mice given the gene therapy. “I would say it is provocative—possibly a breakthrough,” says Steven Austad of the University of Alabama, Birmingham, who studies the biology of aging. “But it will need to be replicated and the mechanism explored before we can say for sure.” The second study, published yesterday in Cell, is from a team led by Harvard Medical School geneticist David Sinclair, who has backed several controversial “antiaging” interventions over the past 2 decades. (Rejuvenate’s approach grew from an earlier collaboration between Sinclair and Davidsohn, but Sinclair isn’t involved in the company’s research, Davidsohn says.) Sinclair’s team set out to test his “information theory of aging,” which posits that our bodies get old because of the cumulative loss of epigenetic marks. Cells’ DNA repair mechanisms, operating throughout a lifetime to fix DNA cuts and other damage, are what degrade these marks, he argues.

To test the theory in mammals, the team genetically engineered a mouse strain that, when given a particular drug, makes an enzyme that cuts their DNA at 20 sites in the genome, which are then faithfully repaired. Widespread changes in cells’ DNA methylation patterns and gene expression followed, consistent with Sinclair’s theory. The mice ended up with an epigenetic signature more like that of older animals, and their health deteriorated. Within weeks, they lost hair and pigment; within months, they showed multiple signs of frailty and tissue aging. To see whether the epigenetic degradation was reversible, the researchers injected some of these elderly seeming mice with AAVs carrying OSK genes, which Sinclair’s group recently reported could reverse loss of vision in aging rodents. Analyses of the mice’s muscles, kidneys, and retinas suggest the cocktail reversed some of the epigenetic changes induced by the DNA breaks. The findings point to a way to drive an animal’s age “forwards and backwards at will,” Sinclair says, and support the idea of epigenome-targeting treatments for aging in humans. Molecular biologist Wolf Reik, director of the Altos Cambridge Institute of Science (opened last year by rejuvenation-focused company Altos Labs), praised the sophistication and thoroughness of the Harvard team’s study, but says the team’s indirect way of inducing epigenetic changes with dramatic DNA breaks that could have other effects makes it hard to prove those changes are what’s causing aging. It’s also unclear how well mice with induced DNA breaks mimic naturally aging animals, says Jan Vijg, a geneticist at the Albert Einstein College of Medicine. He and others stress that aging is a complex process with multiple contributing factors, and that in both papers, the effects of OSK treatment were moderate: a small extension of life span in one, and a partial reversal of artificially induced symptoms in the other. “The jump that now aging is a program” that can be wound backward isn’t justified by the research, Vijg says. Still, both groups want to move their work toward the clinic. Rejuvenate is examining the mechanisms underlying the treatment’s action and tweaking its delivery and composition, Davidsohn says. “OSK might not be the final set” of factors, he adds. Sinclair says his team is already testing AAV-delivered OSK in the eyes of monkeys. “If those studies in monkeys go well and everything looks safe enough for humans, the plan is to immediately apply to the FDA [Food and Drug Administration] to do a study in one or more [age-related] diseases of blindness.”

Published in Science (Jan.13, 2023): https://doi.org/10.1126/science.adg6801 

See also:

 https://doi.org/10.1101/2023.01.04.522507 

https://doi.org/10.1016/j.cell.2022.12.027 

Infected rodents pose no immediate danger to humans, but the research suggests that mutations are helping the coronavirus expand its range of potential hosts.  Bats, humans, monkeys, minks, big cats and big apes — the coronavirus can make a home in many different animals. But now the list of potential hosts has expanded to include mice, according to an unnerving new study.

Yunjeong Kim and Kyeong-Ok "KC" Chang, virologists in the College of Veterinary Medicine at Kansas State University, have published a study showing a possible therapeutic treatment for COVID-19. Pathogenic coronaviruses are a major threat to global public health, as shown by severe acute respiratory syndrome coronavirus, or SARS-CoV; Middle East respiratory syndrome coronavirus, known as MERS-CoV; and the newly emerged SARS-CoV-2, the virus that causes COVID-19 infection. The study, "3C-like protease inhibitors block coronavirus replication in vitro and improve survival in MERS-CoV-infected mice," appears in the Aug. 3 issue of the prestigious medical journal Science Translational Medicine. It reveals how small molecule protease inhibitors show potency against human coronaviruses. These coronavirus 3C-like proteases, known as 3CLpro, are strong therapeutic targets because they play vital roles in coronavirus replication. Vaccine developments and treatments are the biggest targets in COVID-19 research, and treatment is really key," said Chang, professor of diagnostic medicine and pathobiology. "This paper describes protease inhibitors targeting coronavirus 3CLpro, which is a well-known therapeutic target."

The study demonstrates that this series of optimized coronavirus 3CLpro inhibitors blocked replication of the human coronaviruses MERS-CoV and SARS-CoV-2 in cultured cells and in a mouse model for MERS. These findings suggest that this series of compounds should be investigated further as a potential therapeutic for human coronavirus infection. Chang and Kim have been using National Institutes of Health grants to develop antiviral drugs to treat MERS and human norovirus infections. Their work extends to other human viruses such as rhinoviruses and SARS-CoV-2...

Original study published in Science Translational Medicine (Aug. 3, 2020):

https://doi.org/10.1126/scitranslmed.abc5332

A team of UK scientists believe they are among the first to start animal testing of a vaccine for the new coronavirus outbreak that has killed more than 1,000 people and spread around the world. Researchers at Imperial College London said their ultimate goal was to have an effective and safe way of halting the SARS-like strain's spread by the end of the year.

"At the moment we have just put the vaccine that we've generated from these bacteria into mice," Imperial College London researcher Paul McKay told AFP in an interview on Monday. "We're hoping that over the next few weeks we'll be able to determine the response that we can see in those mice, in their blood, their antibody response to the coronavirus." Scientists across the world are racing to develop a way to stamp out the new strain of a well-known virus that has been successfully combatted in the past. Imperial College London said it cannot be sure how advanced other teams' research is at the moment.

China's Xinhua state news agency cited a local news report as saying that a Shanghai university also injected a test vaccine into mice on Sunday. But the local report cited unnamed sources and there has been no official announcement about the Chinese tests. Britain has recorded eight cases of the virus and been forced to shut down two branches of a medical centre in the southeast city of Brighton where at least two staff members tested positive. But coming up with a vaccine is a laborious process that usually involves years of animal testing and clinal trials on humans.

Clinical evidence is mounting that Zika virus can contribute to Guillain-Barré syndrome which causes temporary paralysis, yet the mechanism is unknown. We investigated the mechanism of temporary acute flaccid paralysis caused by Zika virus infection in aged interferon αβ-receptor knockout mice used for their susceptibility to infection. Twenty-five to thirty-five percent of mice infected subcutaneously with Zika virus developed motor deficits including acute flaccid paralysis that peaked 8-10 days after viral challenge. These mice recovered within a week. Despite Zika virus infection in the spinal cord, motor neurons were not destroyed.

We examined ultrastructures of motor neurons and synapses by transmission electron microscopy. The percent coverage of motor neurons by boutons was reduced by 20%; more specifically, flattened-vesicle boutons were reduced by 46%, and were normalized in recovering mice. Using electromyographic procedures employed in people to help diagnose Guillain-Barré syndrome, we determined that nerve conduction velocities between the sciatic notch and the gastrocnemius muscle were unchanged in paralyzed mice. However, F-wave latencies were increased in paralyzed mice, which suggests that neuropathy may exist between the sciatic notch to the nerve rootlets. Reversible synaptic retraction may be a previously unrecognized cofactor along with peripheral neuropathy for the development of Guillain-Barré syndrome during Zika virus outbreaks.

Published in Nature Scientific Reports (20 December 2019):

https://doi.org/10.1038/s41598-019-55717-3