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How Virus Detectives Trace the Origins of an Outbreak – and Why it’s So Tricky

Every time there is a major disease outbreak, one of the first questions scientists and the public ask is: “Where did this come from?”

In order to predict and prevent future pandemics like COVID-19, researchers need to find the origin of the viruses that cause them. This is not a trivial task. The origin of HIV was not clear until 20 years after it spread around the world. Scientists still don’t know the origin of Ebola, even though it has caused periodic epidemics since the 1970s.

As an expert in viral ecology, I am often asked how scientists trace the origins of a virus. In my work, I have found many new viruses and some well-known pathogens that infect wild plants without causing any disease. Plant, animal or human, the methods are largely the same. Tracking down the origins of a virus involves a combination of extensive fieldwork, thorough lab testing and quite a bit of luck.

Viruses Jump from Wild Animal Hosts to Humans

Many viruses and other disease agents that infect people originate in animals. These diseases are zoonotic, meaning they are caused by animal viruses that jumped to people and adapted to spread through the human population.

It might be tempting to start the viral origin search by testing sick animals at the site of the first known human infection, but wild hosts often don’t show any symptoms. Viruses and their hosts adapt to each other over time, so viruses often don’t cause obvious disease symptoms until they’ve jumped to a new host species. Researchers can’t just look for sick animals.

Another problem is that people, and their food animals aren’t stationary. The place where researchers find the first infected person is not necessarily close to the place where the virus first emerged.

In the case of COVID-19, bats were an obvious first place to look. They’re known hosts for many coronaviruses and are the probable source of other zoonotic diseases like SARS and MERS.

For SARS-CoV-2, the virus that causes COVID-19, the nearest relative scientists have found so far is BatCoV RaTG13. This virus is part of a collection of bat coronaviruses discovered in 2011 and 2012 by virologists from the Wuhan Virology Institute. The virologists were looking for SARS-related coronaviruses in bats after the SARS-CoV-1 pandemic in 2003. They collected fecal samples and throat swabs from bats at a site in Yunnan Province about 932 miles (1,500 kilometers) from the institute’s lab in Wuhan, where they brought samples back for further study.

To test whether the bat coronaviruses could spread into people, researchers infected monkey kidney cells and human tumor-derived cells with the Yunnan samples. They found that a number of the viruses from this collection could replicate in the human cells, meaning they could potentially be transmitted directly from bats to humans without an intermediate host. Bats and people don’t come into direct contact very often, however, so an intermediate host is still quite likely.

Finding the Nearest Relatives

The next step is to determine how closely related a suspected wildlife virus is to the one infecting humans. Scientists do this by figuring out the genetic sequence of the virus, which involves determining the order of the basic building blocks, or nucleotides, that make up the genome. The more nucleotides two genetic sequences share, the more closely related they are.

Genetic sequencing of bat coronavirus RaTG13 showed it to be over 96% identical to SARS-CoV-2. This level of similarity means that RaTG13 is a pretty close relative to SARS-CoV-2, confirming that SARS-CoV-2 probably originated in bats, but is still too distant to be a direct ancestor. There likely was another host that caught the virus from bats and passed it on to humans.

Because some of the earliest cases of COVID-19 were found in people associated with the wildlife market in Wuhan, there was speculation that a wild animal from this market was the intermediate host between bats and humans. However, researchers never found the coronavirus in animals from the market.

Likewise, when a related coronavirus was identified in pangolins confiscated in an anti-smuggling operation in southern China, many leaped to the conclusion that SARS-CoV-2 had jumped from bats to pangolins to humans. The pangolin virus was found to be only 91% identical to SARS-CoV-2, though, making it unlikely to be a direct ancestor of the human virus.

To pinpoint the origin of SARS-CoV-2, a lot more wild samples need to be collected. This is a difficult task – sampling bats is time-consuming and requires strict precautions against accidental infection. Since SARS-related coronaviruses are found in bats across Asia, including Thailand and Japan, it’s a very big haystack to search for a very small needle.

Creating a family tree for SARS-CoV-2

In order to sort out the puzzle of viral origins and movement, scientists not only have to find the missing pieces, but also figure out how they all fit together. This requires collecting viral samples from human infections and comparing those genetic sequences both to each other and to other animal-derived viruses.

To determine how these viral samples are related to each other, researchers use computer tools to construct the virus’s family tree, or phylogeny. Researchers compare the genetic sequences of each viral sample and construct relationships by aligning and ranking genetic similarities and differences.

The direct ancestor to the virus, sharing the greatest genetic similarity, could be thought of as its parent. Variants sharing that same parent sequence but with enough changes to make them distinct from each other are like siblings. In the case of SARS-CoV-2, the South African variant, B.1.351, and the U.K. variant, B.1.1.7, are siblings.

Building a family tree is complicated by the fact that different analysis parameters can give different results: The same set of genetic sequences can produce two very different family trees.

The nucleotide sequences of six fictional viruses are shown on the top. Below are two family trees of these viruses created using two different programs. The tree on the left uses only percent identity, while the tree on the right also considers whether the two sequences share similar characters. – Marilyn Roossinck, CC BY-ND

For SARS-CoV-2, phylogenetic analysis proves particularly difficult. Though tens of thousands of SARS-CoV-2 sequences are now available, they don’t differ from one another enough to form a clear picture of how they’re related to each other.

The Current Debate: Wild Host or Lab Spillover?

Could SARS-CoV-2 have been released from a research lab? Although current evidence implies that this is not the case, 18 prominent virologists recently suggested that this question should be further investigated.

Although there has been speculation about SARS-CoV-2 being engineered in a lab, this possibility seems highly unlikely. When comparing the genetic sequence of wild RaTG13 with SARS-CoV-2, differences are randomly spread across the genome. In an engineered virus, there would be clear blocks of changes that represent introduced sequences from a different viral source.

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There is one unique sequence in the SARS-CoV-2 genome that codes for a part of the spike protein that seems to play an important role in infecting people. Interestingly, a similar sequence is found in the MERS coronavirus that causes a disease similar to COVID-19.

Though it is not clear how SARS-CoV-2 acquired these sequences, viral evolution suggests they arose from natural processes. Viruses accumulate changes either by genetic exchange with other viruses and their hosts, or by random mistakes during replication. Viruses that gain a genetic change that gives them a reproductive advantage would typically continue to pass it on through replication. That MERS and SARS-CoV-2 share a similar sequence in this part of the genome suggests that it naturally evolved in both and spread because it helps them infect human cells.

Where to Go from Here?

Figuring out the origin of SARS-CoV-2 could give us clues to understand and predict future pandemics, but we may never know exactly where it came from. Regardless of how the SARS-CoV-2 jumped into humans, it’s here now, and it’s probably here to stay. Going forward, researchers need to continue monitoring its spread, and get as many people vaccinated as possible.

This article was republished with permission from The Conversation, a news site dedicated to sharing ideas from academic experts.  Written by Marilyn J. Roossinck

Professor of Plant Pathology and Environmental Microbiology, Penn State

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The Anti-Aging and Rejuvenating Properties of Stem Cells

In recent years, biomedical research on topics surrounding longevity and aging has gained a tremendous amount of traction. The biotech industry as well as major pharmaceutical companies, have also jumped onto the bandwagon of discovering ways to alter and reverse the aging process. Currently, the business of anti-aging is valued at about $110 billion and is expected to expand to more than $600 billion within the next four years¹. This new obsession with anti-aging is not without good reason; global aging is a concerning phenomenon. The United Nations estimates that by 2050, there will be more than 1.5 billion people aged 65 and over². As a result, the global incidence and burden of disease of age-related diseases will follow a similar trend. Therefore, the goal of anti-aging research is not solely to extend the human lifespan but to treat or prevent age-related diseases ranging from Alzheimer’s to Parkinson’s to diabetes and cardiovascular diseases, etc.

In order to achieve these goals, many of these biotech companies are leveraging on the regenerative prowess of stem cells
³.  This comes as no surprise since human pluripotent stem cells (hPSCs) are unlimited sources of self-renewing cells that can be differentiated into any cell type. This essentially means that large quantities of healthy and young hPSC-derived cells can easily be generated for cell replacement therapy. Also, stem cell-derived exosomes have recently come under the spotlight for their anti-aging properties. In addition, the induced pluripotent stem cells (iPSCs) technology can be leveraged to reverse aging phenotypes in cells. These will be further elaborated on in this article.

Aging and Stem Cells Share an Intimate Relationship

Aging is a complex biological process, but research has pointed to an intimate relationship between aging and stem cells⁴. Adult stem cells are found in many different organs and tissues, but they are low in numbers. They function to regenerate damaged cells and tissues. However, their regenerative capacity declines with age. This explains why injuries in older individuals take a longer time to heal as compared to a younger person. There are several mechanisms underlying the depreciation of adult stem cell functions⁵; targeting these mechanisms head-on may be a strategy to enhance the functions of adult stem cells as we age. However, there are other alternative solutions.

Firstly, therapeutics may be designed to enhance the function or proliferation of endogenous (existing) adult stem cells. Secondly, these adult stem cells can be isolated from tissues and expanded in vitro. Although difficult, researchers have been successful in isolating and expanding certain adult stem cells^6–8. These expanded cells can subsequently be used for transplantation to enhance cellular repair and tissue regeneration. In Herrera et al.’s (2006) study ⁶, they were able to isolate adult stem cells from adult human liver and after transplanting these cells into immune-deficient mice, the stem cells were able to regenerate portions of the liver. Thirdly, hPSCs can be differentiated into different types of progenitor cells using well-defined protocols. These hPSC-derived progenitors can then be used for transplantation⁹. Post-transplantation, these cells contribute to cell repair and regeneration, similar to that of adult stem cells. Recently, a group of researchers from the University of Tehran carefully differentiated hPSCs to cardiovascular progenitors before transplanting them into a rat model of myocardial infarction (heart attack). These progenitor cells significantly improved functionalities of the heart and reduced scar tissue formation in these rats¹⁰.

Although more work has to be done on improving the methods of isolating and expanding adult stems in vitro, and differentiating hPSCs to progenitor cells to ensure that the cells are safe and effective for use in transplantation therapies, there is great promise in utilizing these strategies to delay or prevent aging and age-associated diseases.

Exosomes Produced from Stem Cells

Another exciting frontier lies in the anti-aging properties of stem cell-derived exosomes. Studies have shown that exosomes derived from human embryonic stem cells, mesenchymal and stromal stem cells have regenerative capabilities^11–13. Exosomes are nano-sized, membrane-bound, extra-cellular particles secreted by cells. They contain various biological materials such as nucleic acids, proteins, metabolites, and function to facilitate cell-cell communication. While the underlying mechanisms as to how exosomes promote regeneration remains to be fully elucidated, it is likely that the amalgamation of inflammatory signals, growth factors, microRNAs, long non-coding RNAs, enzymes, and proteins found in exosomes promotes and reversal of cellular senescence, thus contributing to its anti-aging properties¹¹. Stem cell-derived exosomes are currently being explored as a therapeutic option for age-related musculoskeletal disorders¹⁴. Although research on exosomes is still in the early phases, exploitation of its anti-aging effects might potentially be a safe and effective anti-aging therapeutic strategy.

Stem Cell Technology Can Rejuvenate Old Cells

Yamanaka and colleagues’ Nobel prize-winning discovery of iPSCs revolutionized stem cell research^15,16. By simply introducing key transcription factors into adult cells, they can be reprogrammed back into a pluripotent state (‘infancy’). This process is known as cellular reprogramming, and the transcription factors are collectively referred to as ‘Yamanaka factors’. Recently, researchers from Stanford University were able to exploit this cellular reprogramming technology to rejuvenate old human cells¹⁷. By introducing several factors (including Yamanaka factors) transiently into old human cells, they were able to rewind these cells to a ‘youthful’ state, while preserving the cellular identities of cells. After further analysis, these rejuvenated cells were found to be more similar to younger cells than elderly cells.

As a proof of concept, the researchers then went on to introduce the same set of factors into aged mouse-derived skeletal muscle stem cells (MuSC) and transplanted them into the muscles of aged mice. Post-transplantation, these rejuvenated MuSCs were able to restore muscle functionality of old mice to a younger state. This remarkable study has illustrated that stem cell technology has great potential in reversing aging in cells to obtain ‘youthful’ cells and these cells could be used in cell transplantation therapies to treat age-related conditions. 

In the Coming Years

Experts agree that stem cell science holds answers to human longevity and has great potential in treating and preventing age-related diseases¹⁸. Numerous stakeholders in the biomedical industry have invested interests in tapping on stem cells to realize these goals. In the coming years, we will be expecting more research efforts dedicated towards a deeper level of understanding, as well as further optimization of stem cell-based interventions for aging. Companies involved in utilizing this science are worth researching and learning about while this segment of biotech is yet to approach potential.

About the Author:  Nicole Pek is a stem cell biologist and enthusiastic science communicator.
She has worked on using human pluripotent stem cells to study cellular
development in multiple organ systems, to model complex human diseases, and
screen for therapeutics that could treat the diseases. Outside of the lab,
Nicole plays a pro-active role in communicating to the public through her
science blog ‘Two Cells’ and her education podcast ‘The Diploid Duo’.

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References

1.         Grover, N. Healthier, longer lifespans will be a reality sooner than you think, Juvenescence promises as it closes $100M round. Endpoints News https://endpts.com/healthier-longer-lifespans-will-be-a-reality-sooner-than-you-think-juvenescence-promises-as-it-closes-100m-round/.

2.         World Population Ageing 2019 Highlights.

3.         Jefferson, R. S. ‘Extraordinary’ Breakthroughs In Anti-Aging Research ‘Will Happen Faster Than People Think’. Forbes https://www.forbes.com/sites/robinseatonjefferson/2019/08/26/how-extraordinary-breakthroughs-in-anti-aging-research-will-happen-faster-than-people-think/.

4.         Ullah, M. & Sun, Z. Stem cells and anti-aging genes: double-edged sword—do the same job of life extension. Stem Cell Res Ther 9, (2018).

5.         Ahmed, A. S. I., Sheng, M. H., Wasnik, S., Baylink, D. J. & Lau, K.-H. W. Effect of aging on stem cells. World J Exp Med 7, 1–10 (2017).

6.         Herrera, M. B. et al. Isolation and characterization of a stem cell population from adult human liver. Stem Cells 24, 2840–2850 (2006).

7.         Messina, E. et al. Isolation and Expansion of Adult Cardiac Stem Cells From Human and Murine Heart. Circulation Research 95, 911–921 (2004).

8.         Mantovani, C., Terenghi, G. & Shawcross, S. G. Isolation of adult stem cells and their differentiation to Schwann cells. Methods Mol Biol 916, 47–57 (2012).

9.         Thies, R. S. & Murry, C. E. The advancement of human pluripotent stem cell-derived therapies into the clinic. Development 142, 3077–3084 (2015).

10.       Vahdat, S. et al. Expansion of Human Pluripotent Stem Cell-derived Early Cardiovascular Progenitor Cells by a Cocktail of Signaling Factors. Sci Rep
9, 16006 (2019).

11.       Ha, D. H. et al. Mesenchymal Stem/Stromal Cell-Derived Exosomes for Immunomodulatory Therapeutics and Skin Regeneration. Cells 9, (2020).

12.       Chen, B. et al. Human embryonic stem cell-derived exosomes promote pressure ulcer healing in aged mice by rejuvenating senescent endothelial cells. Stem
Cell Research & Therapy 10, 142 (2019).

13.       Newton, W. C., Kim, J. W., Luo, J. Z. Q. & Luo, L. Stem cell-derived exosomes: a novel vector for tissue repair and diabetic therapy. Journal of Molecular
Endocrinology 59, R155–R165 (2017).

14.       Yao, X.
et al. Stem cell derived exosomes: microRNA therapy for age-related musculoskeletal disorders. Biomaterials 224, 119492 (2019).

15.       Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006).

16.       Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).

17.       Sarkar, T. J. et al. Transient non-integrative expression of nuclear reprogramming factors promotes multifaceted amelioration of aging in human cells. Nat Commun 11, (2020).

18.       Stem Cells and Aging: What’s Next? Cell Stem Cell 16, 578–581 (2015).

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PDS Biotechnology Set to Join Russell Microcap® Index

FLORHAM PARK, N.J., June 07, 2021 (GLOBE NEWSWIRE) — PDS Biotechnology Corporation (Nasdaq: PDSB), a clinical-stage immunotherapy company developing novel cancer therapies based on the Company’s proprietary Versamune^® T-cell activating technology, today announced the Company is set to join the Russell Microcap^® Index at the conclusion of the 2021 Russell indexes annual reconstitution, effective after the US market opens on June 28, according to a preliminary list of additions posted June 4.

Membership in the Russell Microcap^® Index, which remains in place for one year, means automatic inclusion in the appropriate growth and value style indexes. FTSE Russell determines membership for its Russell indexes primarily by objective, market-capitalization rankings and style attributes.

“Inclusion in the Russell Microcap Index underscores the progress we are making to increase shareholder value by driving our three (3) Phase 2 clinical programs forward for our lead HPV-cancer immunotherapy PDS0101, including an oral presentation of interim data for our National Cancer Institute-led trial at the American Society of Clinical Oncology annual meeting later today,” said Dr. Frank Bedu-Addo, President and CEO of PDS Biotech. “Inclusion in the Index significantly benefits our Company and shareholders by elevating our visibility within the global investment community. We look forward to continuing to progress the clinical development of PDS0101 while also moving additional oncology pipeline products into human testing within the next year.”

Russell indexes are widely used by investment managers and institutional investors for index funds and as benchmarks for active investment strategies. Approximately $10.6 trillion in assets are benchmarked against Russell’s US indexes. Russell indexes are part of FTSE Russell, a leading global index provider.

For more information on the Russell Microcap^® Index and the Russell indexes reconstitution, go to the “Russell Reconstitution” section on the FTSE Russell website.

About PDS Biotechnology

PDS Biotech is a clinical-stage immunotherapy company developing a growing pipeline of cancer immunotherapies based on the Company’s proprietary Versamune^® T-cell activating technology platform. Our Versamune^®-based products overcome the limitations of current immunotherapy by inducing in vivo, large quantities of high-quality, highly potent polyfunctional tumor specific CD4+ helper and CD8+ killer T-cells. PDS Biotech has developed multiple therapies, based on combinations of Versamune^® and disease-specific antigens, designed to train the immune system to better recognize diseased cells and effectively attack and destroy them. Our immuno-oncology product candidates are initially being studied in combination therapy to potentially enhance efficacy without compounding toxicity across a range of cancer types. The company’s lead investigational cancer immunotherapy product PDS0101 is currently in Phase 2 clinical studies in HPV-associated cancers. The company’s pipeline products address various cancers including breast, colon, lung, prostate and ovarian cancers. To learn more, please visit www.pdsbiotech.com or follow us on Twitter at @PDSBiotech.

About FTSE Russell

FTSE Russell is a global index leader that provides innovative benchmarking, analytics and data solutions for investors worldwide. FTSE Russell calculates thousands of indexes that measure and benchmark markets and asset classes in more than 70 countries, covering 98% of the investable market globally.

FTSE Russell index expertise and products are used extensively by institutional and retail investors globally. Approximately $17.9 trillion is currently benchmarked to FTSE Russell indexes. For over 30 years, leading asset owners, asset managers, ETF providers and investment banks have chosen FTSE Russell indexes to benchmark their investment performance and create ETFs, structured products and index-based derivatives.

A core set of universal principles guides FTSE Russell index design and management: a transparent rules-based methodology is informed by independent committees of leading market participants. FTSE Russell is focused on applying the highest industry standards in index design and governance and embraces the IOSCO Principles. FTSE Russell is also focused on index innovation and customer partnerships as it seeks to enhance the breadth, depth and reach of its offering.

FTSE Russell is wholly owned by London Stock Exchange Group. For more information, visit www.ftserussell.com.

Forward Looking Statements

This communication contains forward-looking statements (including within the meaning of Section 21E of the United States Securities Exchange Act of 1934, as amended, and Section 27A of the United States Securities Act of 1933, as amended) concerning PDS Biotechnology Corporation (the “Company”) and other matters. These statements may discuss goals, intentions and expectations as to future plans, trends, events, results of operations or financial condition, or otherwise, based on current beliefs of the Company’s management, as well as assumptions made by, and information currently available to, management. Forward-looking statements generally include statements that are predictive in nature and depend upon or refer to future events or conditions, and include words such as “may,” “will,” “should,” “would,” “expect,” “anticipate,” “plan,” “likely,” “believe,” “estimate,” “project,” “intend,” “forecast,” “guidance”, “outlook” and other similar expressions among others. Forward-looking statements are based on current beliefs and assumptions that are subject to risks and uncertainties and are not guarantees of future performance. Actual results could differ materially from those contained in any forward-looking statement as a result of various factors, including, without limitation: the Company’s ability to protect its intellectual property rights; the Company’s anticipated capital requirements, including the Company’s anticipated cash runway and the Company’s current expectations regarding its plans for future equity financings; the Company’s dependence on additional financing to fund its operations and complete the development and commercialization of its product candidates, and the risks that raising such additional capital may restrict the Company’s operations or require the Company to relinquish rights to the Company’s technologies or product candidates; the Company’s limited operating history in the Company’s current line of business, which makes it difficult to evaluate the Company’s prospects, the Company’s business plan or the likelihood of the Company’s successful implementation of such business plan; the timing for the Company or its partners to initiate the planned clinical trials for PDS0101, PDS0203 and other Versamune^® based products; the future success of such trials; the successful implementation of the Company’s research and development programs and collaborations, including any collaboration studies concerning PDS0101, PDS0203 and other Versamune^® based products and the Company’s interpretation of the results and findings of such programs and collaborations and whether such results are sufficient to support the future success of the Company’s product candidates; the success, timing and cost of the Company’s ongoing clinical trials and anticipated clinical trials for the Company’s current product candidates, including statements regarding the timing of initiation, pace of enrollment and completion of the trials (including our ability to fully fund our disclosed clinical trials, which assumes no material changes to our currently projected expenses), futility analyses, presentations at conferences and data reported in an abstract, and receipt of interim results, which are not necessarily indicative of the final results of the Company’s ongoing clinical trials; any Company statements about its understanding of product candidates mechanisms of action and interpretation of preclinical and early clinical results from its clinical development programs and any collaboration studies; the acceptance by the market of the Company’s product candidates, if approved; the timing of and the Company’s ability to obtain and maintain U.S. Food and Drug Administration or other regulatory authority approval of, or other action with respect to, the Company’s product candidates; and other factors, including legislative, regulatory, political and economic developments not within the Company’s control, including unforeseen circumstances or other disruptions to normal business operations arising from or related to COVID-19. The foregoing review of important factors that could cause actual events to differ from expectations should not be construed as exhaustive and should be read in conjunction with statements that are included herein and elsewhere, including the risk factors included in the Company’s annual and periodic reports filed with the SEC. The forward-looking statements are made only as of the date of this press release and, except as required by applicable law, the Company undertakes no obligation to revise or update any forward-looking statement, or to make any other forward-looking statements, whether as a result of new information, future events or otherwise.

Media & Investor Relations Contact:

Deanne Randolph
PDS Biotech
Phone: +1 (908) 517-3613
Email: drandolph@pdsbiotech.com

Rich Cockrell
CG Capital
Phone: +1 (404) 736-3838
Email: rich@cg.capital