Examining the Environment to Identify Viruses in Livestock

 With the increased  reliance on livestock and animal products in today’s world, humans and animals are in closer proximity than ever. This closeness however, also makes this connection susceptible to be corrupted by the spread of viral disease. This spread from animals to humans is called zoonotic infection and is how many of the most serious viral outbreaks began such as influenza and coronavirus. Livestock are particularly vulnerable to the spreading of these viruses as they are often clustered close together, promoting viral transmission that can eventually make its way to human hosts. Resources for monitoring viruses within livestock populations have, however, been a challenge as traditional monitoring has had to be done directly on the animals which can pose risks to both the animals and the researchers. Thus, scientists are looking to develop new ways to surveil virus transmission in non-invasive ways.

One way researchers have looked to solve this problem is that rather than testing the animals themselves, they could look at samples of aggregated materials from many animals such as manure, air samples, and wastewater to track viruses within animal populations. Methods of monitoring pathogens from aggregated samples have already proven effective in some cases and researchers wanted to further test this theory to see if aggregate samples could appropriately serve as reliable indicators of virus transmission in  populations of cattle and pigs.

The researchers began by setting up sample collection sites at cow and pig farms in Barcelona as well as at several slaughterhouses. They collected 105 samples across eleven months between slurry, manure, wastewater, and air samples from the locations. The samples were separated into different pools based on whether they were from pigs or cows and by the season they were collected. Viruses were then extracted from each sample by slowly removing organic and biological particles from the sample until viral DNA could be isolated.

In the swine samples, the researchers found evidence of 56 total viral species and saw that the viruses seemed to have seasonal variation with each virus seeming to have a preferred peak season of high abundance that matches with known viral seasonality indicating a success of using aggregated samples to test for viruses.

The researchers then compared their findings with the samples taken at cattle farms and from cattle wastewater samples. They found that since cows are housed at a lower density than pigs, there was a sharp decrease in pathogen detection since viruses could not be passed as easily when the animals are farther apart. The researchers did however notice the same pattern of seasonality for the 63 viral species that appeared in their screens, many of which appeared in a specific sample type alone showing the best ways to monitor those specific viruses. 

Based on their results, the researchers conclude that an aggregated sample approach is an effective way to monitor animal viruses in livestock populations. Based on their results and their alignment with previous direct studies on viral presence in livestock populations, the authors believe that aggregate sampling could prove to be of significant use, especially in the prevention of zoonotic infections that could infect humans. While this method can make it difficult to determine which individuals in a population are infected due to the collection methods, the overall results from this study indicate that there are some new approaches coming to virus monitoring and the prevention of virus spread.

In order to be even more effective with these new techniques for virus monitoring, we could complement them through making advances in viral detection technology that might allow us to track viruses and their mutations so we can be even more prepared to face viral threats as they develop before they infect the larger population. We could also look into applying this technique to non-livestock animals which would help to prevent the spread of infection globally. This paper demonstrates a major shift coming for viral monitoring and detection which could ultimately lead help us to be safer and more prepared for emerging viral infections

Literature Cited:

Rusiñol, M., Martínez-Puchol, S., Ribeiro, D., Verdaguer, J., Torrejón-Llorens, O., Itarte, M., Estarlich-Landajo, I., Mejías-Molina, C., Juliachs-Torroella, G., Girones, R., Ramírez, G. A., Baliellas, J., Bofill-Mas, S., & Fernández-Cassi, X. (2026). Livestock aggregated samples for monitoring viruses infecting animals and potentially zoonotic viral pathogens. One health (Amsterdam, Netherlands), 22, 101340. https://doi.org/10.1016/j.onehlt.2026.101340

A Rabies-Based Vaccine? The Emerging Vaccination Landscape.

 Over the last couple of years, many scientists and epidemiologists have been watching the spread of a new virus, the H5N1 avian influenza. While initially developed in birds, there has just been a recent development where it has now begun to spread within populations of cattle, and even humans have now been infected. This new flu is extremely worrying as though the infected humans only seem to have mild symptoms, previous H5N1 epidemics have had severe mortality rates. Thus, scientists are currently looking for some possible vaccinations that work for humans and animals, should this virus become a major public threat.

One possible vaccine avenue that scientists are interested in studying is through the use of dual rabies virus vaccines or RABV that have been known to protect against other viral pathogens. To test this theory, a lab at Thomas Jefferson University developed a RABV-based H5 dual vaccine or RABV-H5 that has antigens which would protect against both the avian influenza as well as rabies virus. 

In order to develop this vaccine, the researchers inserted a synthesized version of the avian flu into the genetic sequence of rabies virus. The proper functioning of both parts of the new vaccine sequence were then analyzed and confirmed that the vaccine sequence had effective functioning of both viruses and that the influenza sequence had successfully integrated into the rabies virus sequence.

Figure 1A.

Now that the vaccine had been made, the researchers wanted to determine how effective this vaccine was. The researchers tested the vaccine on mice using different methods to vaccinate them. They used both an inactivated, or killed, form of the RABV-H5 as the vaccine as well as a live version of RABV-H5 and vaccinated the mice either with an injection in the muscle or by applying droplets of vaccine to the nose. 

Figure 1E.

After vaccination, the researchers looked for antibodies to the H5 which would serve as a sign that the vaccine worked and led to immunity of the virus. They found that in all cases, the mice had antibodies to H5 and found that vaccinating with a muscle injection at both the primary and secondary vaccination steps, the mice had higher antibody levels which indicates that these mice would have the strongest viral immunity.

After the success of the initial vaccination test, and following the same vaccination procedure paired with non-vaccinated control mice, mice were given a lethal dose of a mouse adapted version of the avian influenza virus. The researchers found that mice given the vaccine showed fewer signs of illness including weight loss and less virus seen on their lungs and had a much higher survival rate, including a one hundred percent chance of survival when given the vaccine intramuscularly both times or intramuscularly the first time and then a live version of the RABV-H5 vaccine delivered intranasally as a booster. This result stands in stark contrast to the mice that were unvaccinated and succumbed to the virus within six days, and even the mice that were vaccinated in alternate ways that only had a forty to sixty percent survival rate.

Seeing this success, the researchers did the exact same experiment, but this time with the authentic version of the avian influenza. Similar results followed from this experiment as vaccinated mice had a high survival rate and saw limited effects from the virus while unvaccinated mice succumbed to the virus at six to seven days.

With the success of the vaccine against both the mouse adapted version of the virus and the authentic virus itself, the researchers also wanted to look at how the vaccine would perform against a similar virus strain that is currently in circulation.  The researchers found that the vaccine did seem to have some effect as it reduced weight loss in the vaccinated mice and led to high survival rates against a reduced form of the virus, but as the researchers expected, the vaccine had no effect on the lethal dose of the virus.

This paper illustrates the wide world of vaccines and the unlikely places that they can come from. The use of vaccine platforms such as RABV to build new vaccines in quick order so as to prevent disease spread should not be underestimated as these platforms most likely hold the key to preventing the spreading of emerging viruses and combatting viruses before they become a major human concern. The virus created and tested in this paper also offers a glimpse at emerging vaccine technology such as the ability to vaccinate for multiple viruses with a single vaccine and also shows the increasing need to develop vaccines with both humans and animals in mind to prevent a virus from spreading from animals to humans.

While this study shows a lot of exciting potential for vaccines against new and emerging infectious diseases, this study leaves open the future possibilities of this vaccine as the next steps would be evaluating its effectiveness in humans after the successes seen in mice. While there may be future viral threats that could begin a new epidemic, this emerging vaccine technology could help us to prevent that from happening.

Literature Cited:

Paran, N., Wirblich, C., Albrecht, R., Zabihi-Diba, L., Tarquinio, A., Kurup, D., Solomides, C. C., García-Sastre, A., & Schnell, M. J. (2026). Immunogenicity and efficacy of a rabies-based vaccine against highly pathogenic influenza H5N1 virus. Emerging microbes & infections, 15(1), 2620221. https://doi.org/10.1080/22221751.2026.2620221

Combatting Against Cancer Using Virus: Oncolytic Virus M1 Reinvigorates T-cell immunity against glioblastoma

Glioblastoma multiforme (GBM), usually arising from glial precursor cells or neural stem cells, is the most common and lethal primary brain cancer in adults. It has very limited treatment options and a median life expectancy less than 2 years after diagnosis. Systemic and local immunosuppression induced by GBMs largely contributes to malignancy aggressiveness and resistance to multiple immunotherapies, such as immune checkpoint blockade (ICB) therapy, which is powerful for primary care of other types of solid tumors. 

Oncolytic virotherapy has attracted growing awareness as a potential transformative cancer therapy in recent years. It involves delivery of a genetically modified version of virus into the host and it would selectively destroy tumor cells while leaving neighboring healthy cells undamaged, overcoming a common limitation of chemotherapy. This targeted destruction is either achieved through direct oncolysis ("onco-" meaning cancer-related, "-lysis" meaning burst) or immune-mediated attack. Oncolytic therapy using alphacirus M1(OVM), which was originally isolated from Culex mosquitoes, recently emerged as a potential glioma therapeutic approach. The virus is delivered through intravenous administration and the main advantages are OVM's ability to selectively replicate in tumor cells overexpressing matrix remodeling-associated 8 (the marker allowing them to recognize and attach) and efficiency in penetrating through the blood brain barrier, which is a dense layer of blood vessels and cells that protect our brain from harmful substances but makes drug delivery difficult at the same time. Even though we might be worried that using virus as medium can be dangerous by itself at the first glance, the toxic genes are being taken out in advance and it's previously shown to be very safe: GBM patients showed high tolerance towards OVM in previous clinical trials and repeated intravenous administration of OVM is non-pathogenic for nonhuman primates (Zhang et al., 2016).

Despite of its huge therapeutic potential, the knowledge regarding how OVM initiates adaptive immunity against gliomas and how the interactions between OVM and our immune system would influence the therapeutic effects of ICB is still lacking. Hence the researchers are seeking to address these two knowledge gaps.

First, they confirmed the efficacy of OVM in reducing glioma progression. The immunocompromised mice were injected intracranially with GBM cell lines (GL261-Luciferase, GL261 and CT2A) to induce a glioma in the brain on day 0 and followed by daily tail intravenous injection of OVM or vehicle from day 5 to day 9. Vehicle includes the buffer solutions that researchers use to deliver OVM but containing no OVM, acting as the control condition. GL261-Luciferase is a cell line where it's infected with lentivirus whose genome was engineered to contain the luciferase gene so that wherever the tumor develops, it would glow. A stronger luminescence intensity (red) indicates the progression of glioma while bluish color indicates little tumor growth. It was found that OVM significantly inhibited the growth of glioma and prolonged the survival of glioma-bearing mice. 

Moving on to investigate the immune response, the researchers found that OVM successfully induced immunogenic cell death and reverse the GBM-induced immunosuppression both locally and systemically. Immunogenic cell death is characterized by the release of damage-associated molecular patterns (DAMPs) by tumor cells, which are molecules that normally stay inside the cell and becomes 'danger signals' recognized by immune cells when being released outside, driving adaptive-immunity elimination in tumor microenvironment. They examined the release of DAMPs, revealing significant CRT (externalized calreticulin, a multifunctional protein) exposure and elevated extracellular ATP (adenosine triphosphate, energy source for our cells) in the supernatant collected from glioma cell lines with OVM treatment. This indicates the occurrence of immunogenic cell death induced by OVM. 

Additionally, the number of CD4+ and CD8+ T cells increased significantly in peripheral blood of glioma-bearing mice following intravenous OVM treatment. Spleen atrophy and loss of splenic T-cell population were also recovered. Similarly, in the tumor local environment, they also found rapid increase in tumor-specific CD8+ T cells in the spleen. The measurement is achieved by orthotopical implantation of GL261 glioma cells expressing OVA (full-length ovalbumin, processed into SIINFEKL) and H-2Kb-SIINFEKL tetramer that binds to T cell receptors where H-2Kb is the mouse version of MHC class I protein. The recognition of glioma-derived-antigen-MHC complex by T cells allows the accurate tracking of tumor-specific T cells (Hsu et al., 2025). Hence, with OVM infection, both local and systemic immunity are boosted and recovered.

  

Before diving deeper into the mechanism, the researchers digressed a bit and addressed a main concern about the efficacy of OVM: Spleen is the major body 'filter' consisting of large number of immune cells that's found previously able to eliminate delivered oncolytic viruses and OVM is often 'trapped' inside the spleen of various host animals. Therefore, the researchers removed the spleen (splenectomy) from mice to find out the role of spleen in OVM treatment. To their surprise, opposite to the greater reduction in glioma progression that they have expected, the antitumor activity of OVM treatment was completely abrogated after removal. Splenectomy-OVM mice almost have the same low survival probability as vehicle control and no expansion of T-cell population in the peripheral blood and tumor microenvironment.

Consequently, the researchers tried to find out the specific cell population in the spleen that's mediating the reversal of immunosuppression and antitumor activity. They first employed a broad RNA sequencing and computational screening and found that B cells have the highest abundance and showed strongest predicted interactions with T cells after OVM treatment. To move beyond correlation, a series of validation experiment were conducted: In vitro co-culture experiments demonstrated that B cells from OVM-treated mice significantly enhanced activation of T cells but not other splenic immune populations and this activation is dose-dependent. Further, after blocking the B cells with anti-CD19 antibody, OVM administration failed to prolong the survival of glioma-bearing mice and increase the infiltration of T cell populations in the blood and tumor microenvironment. 

To elucidate the mechanism by which splenic B cells reverse immunosuppression, the researchers compared the interactions of B cells with CD8+ T cells in the OVM-treated group and the vehicle control group. They found that OVM treatment leads to much more frequent physical contacts between B and T cells, visualized through live cell imaging.

To determine the functional significance of this cell-to-cell contact, they created two conditions where the cells can directly interact with each other and where they are forced to be separated from each other by a membrane (transwell chamber). CD8+ T cell proliferation is measured by CFSE dilution (carboxyfluorescein succinimidyl ester,  a fluorescence dye that's split between new-born cells for every division) and CD8+ T cell activation is measured by GZMB+ (granzyme B, the 'weapon' T cells produce to kill target cancer cells). Using these two tracking tools, they discovered that T cell proliferation and activation is induced only when B cells and T cells that can freely contact with each other but not in transwell-separated groups. These results together suggests that the direct contact between B and T cells is essential for T-cell expansion and activation required for OVM-mediated immune enhancement.

In order to validate this finding in vivo, researchers targeted the Major Histocompatibility Complex class I (MHC-I), a critical component of the structural interface between B and T cells. B cells communicate and 'educate' T cells by presenting a fragment of tumor on MHC-I to activate them, which process is known as antigen presentation. Blocking MHC-I with a specific antibody completely abolished B-cell-induced CD8+ T cell proliferation and activation. Collectively, these results confirm that the direct physical interface is crucial for immunity restoration by OVM treatment. Later the researchers identified the specific subset of B cells that's responsible for this interaction to be Bst2+ B cells by computational screening and in-vivo evidence: Bst+B cells significantly prolonged the survival of B-cell-deficient, GL261-OVA-bearing mice after OVM stimulation while Bst2-B cells and B cells from vehicle control group did not.

The interface between B and T cells involves B cells 'presenting' a peptide derived from cancer cells or other sick cells on MHC-I protein, which is then recognized by T cell receptors (TCR). 

Finally, to bridge the above mechanistic findings with clinical application, the researchers tested if OVM treatment can help overcome immunosuppression that previously has prevent immune checkpoint blockade (ICB) therapy from functioning in glioblastoma patients. ICB therapy works by removing the "brake" on the T cells. Our bodies have these natural immune checkpoint proteins that's naturally used to prevent excessive inflammation from happening that would otherwise harm other healthy cells. But at the same time, this also grant tumor cells a tool to escape from immune responses. ICB drugs block these inhibitory signals and boost the working capacity of our immune system. 

PD-L1 and PD-1 are the immune checkpoint proteins that 'put a brake' on the T cell when bound. If either of them are blocked, which is what the researchers and ICB drugs do, T cells are free to attack the tumor cells. 

To test the synergy between ICB and OVM therapy, immunocompromised mice was injected intracranially on day 0 and from day 5 to day 9, the mice is divided into different groups and treated daily intravenously with different combinations of immune checkpoint protein antibodies, including isotype antibody as control, PD-1 and PD-L1 antibodies, with or without OVM. The researchers revealed that, comparing with other 5 combinations, OVM treatment and PD-1 inhibitor together yielded the greatest recruitment of CD8+ T cells into tumor microenvironment, suppression of intracranial glioma growth as well as restoration of splenic size and weight. Systematically, massive expansions of effector (activated) CD8+ and CD4+ T cells in peripheral bloodstream are also observed. Therefore, OVM synergizes with PD-1 inhibitors for glioma treatment by simultaneously reshaping systemic immunity and the local immune environment in the brain.

isotype= control isotype antibody; PD-1 and PD-L1 Ab= PD-1 and PD-L1 antibodies (ICB therapy); Vehicle= control for OVM treatment; OVM= oncolytic viral therapy. 

This study provides profound mechanistic details for how emerging OVM treatment reverses the immunosuppressive environment of GBM, which is incurable and has grim prognosis. Most significantly, it establishes OVM as a transformative catalyst for ICB therapy, which is historically ineffective for GBM. The findings also illuminate a remarkable capability of OVM bypassing the 'toxic', immunosuppressed tumor microenvironment in the brain, and re-directing the site of immune cell communication to the 'clean sanctuary', the spleen. Within this protected environment, the recovery of B- and T-cell physical interface structures allows for robust antigen presentation, subsequent T cell activation, and eventually elimination of the tumor cells. The study has a strong advantage in its comprehensive validation of each step. For instance, when assessing the efficacy of the therapies, they would use several measures like the size of the spleen, tumor growth and immune cells activation level. Also, when confirming the determining role of splenic B cells, they combined both computational tools and biological checks, including knockout and correlation experiments. Moving forward, these discoveries open a new frontier for translational medicine. While mouse models provide an essential foundation, future research should evaluate OVM in non-human primates and eventually human clinical trials to characterize its safety profile in more complex in vivo environment. Long-term studies are also preferable because so far only the decrease in size of tumor is. reported but future relapse is still possible, especially given the ability of tumors to develop resistance mutations. Furthermore, the mechanism behind OVM-directed B-cell migration to the spleen and T-cell migration to the brain can be elucidated to identify potential signaling molecule targets.

Citations:

For images:

https://www.researchgate.net/figure/The-origin-of-glioblastoma-During-normal-embryonic-development-and-in-the-adult-brain_fig2_371833392

https://vyriad.com/science/oncolytic-virus-platforms/
https://www.cancerresearch.org/immunotherapy-by-treatment-types/oncolytic-virus-therapy
https://www.nature.com/articles/s41592-020-01031-0
https://www.cancer.gov/about-cancer/treatment/types/immunotherapy/checkpoint-inhibitors

https://rcastoragev2.blob.core.windows.net/13974d8439d3384abb2302e11f2da069/PMC9563749.pdf

For academic literature:
the primary article:
‌Han, Yu, et al. “Oncolytic Virus M1 Reinvigorates CD8+ T-Cell Immunity against Glioblastoma through B-Cell-Dependent Antigen Cross-Presentation in the Spleen.” Cellular & Molecular Immunology, 4 Mar. 2026, www.nature.com/articles/s41423-026-01396-w/figures/8, https://doi.org/10.1038/s41423-026-01396-w. Accessed 26 Mar. 2026.

Others:

‌National Cancer Institute. “Https://Www.cancer.gov/Publications/Dictionaries/Cancer-Terms/Def/Blood-Brain-Barrier.” Www.cancer.gov, 2 Feb. 2011, www.cancer.gov/publications/dictionaries/cancer-terms/def/blood-brain-barrier.

‌Hsu, Chung-Yao, et al. “Polymerised Superparamagnetic Antigen Presenting Cell Lymphocyte Capture for Enriching Tumour Reactive T-Cells and Neoantigen Identification.” Nature Communications, vol. 16, no. 1, 2 June 2025, www.nature.com/articles/s41467-025-60321-3, https://doi.org/10.1038/s41467-025-60321-3. Accessed 26 Mar. 2026.

“Up Close: How Immune Checkpoint Inhibitors Revolutionize Cancer Treatment | What’s up at Upstate | SUNY Upstate.” Www.upstate.edu, www.upstate.edu/whatsup/2023/100523-up-close-how-immune.php.

Zhang, Haipeng, et al. Naturally Existing Oncolytic Virus M1 Is Nonpathogenic for the Nonhuman Primates after Multiple Rounds of Repeated Intravenous Injections. Vol. 27, no. 9, 1 Sept. 2016, pp. 700–711, https://doi.org/10.1089/hum.2016.038. Accessed 1 Aug. 2023.

‌“Defined Immune Response Tracking in Mice: OVA MHC Tetramers – Caltag Medsystems.” Caltagmedsystems.co.uk, Mar. 2024, www.caltagmedsystems.co.uk/information/defined-immune-response-tracking-in-mice-ova-mhc-tetramers/. Accessed 29 Apr. 2026.

‌“Oncolytic Virus Therapy and Its Side Effects.” Cancer.org, 2023, www.cancer.org/cancer/managing-cancer/treatment-types/immunotherapy/oncolytic-virus-therapy.html.

‌

‌

Hepatitis B Virus Genome Packaging and Reverse Transcription

Introduction

Hepatitis B Virus (HBV) is an infectious liver virus that contains a genome consisting of circular, double-stranded DNA. HBV is a member of a family of viruses that takes RNA genetic information generated during infection, and transcribes it into DNA to package into new viral particles (1). The process of reverse transcription is unique to viruses, as there is no need to generate DNA from RNA anywhere else in biological life. 

For liver cells infected with HBV to form infectious viral particles, the viral DNA produced during this infection must fit inside new viral particles to generate more viruses to infect more cells (1). This process is called packaging, and is required in all viral infections, regardless of the types of cells they infect, or the type of genome that the virus packages its cells with. Viral genomes are not very large compared to more complex life, such as in human cells, but the size of an average viral particle is also much smaller than an animal cell, and fitting the entire genome of a virus into its particle is a major hurdle that any virus must overcome, HBV included, and understanding the mechanisms behind this process may prove valuable in understanding viral replication (1). This paper describes their process of uncovering how HBV packages its DNA genome into the mature viral particle (2).

Viral Particle Imaging

The paper by Gibes et al. 2026 describes their process of imaging Duck HBV (DHBV) and human HBV viral particles to uncover how the DNA inside these viruses is organized in a manner that allows them to fit inside such a small particle. DHBV was generally easy to use, which is why some of their experiments started using it rather than HBV (2). They did this by purifying DHBV and later HBV viral particles and imaging them using cryo-electron microscopy, which is used to take pictures of small particles such as viruses that would be too small for normal microscopes (Figure 1).

Figure 1: Cryo-em image of HBV particle. a. Fully developed HBV protein capsid. White shapes represent the geometric points of symmetry that pattern the capsid's outer layer. b. First layer of DNA (lavender) showing 11 concentric rings of DNA. c. Second layer of DNA (purple) showing 7 concentric rings of DNA. d. The third and final layer of DNA (blue) showing less-defined concentric rings of DNA (2).

Structural Analysis

They saw that the viruses contained their DNA in layers wrapped around protein, forming concentric DNA rings inside the viral particle (Figure 1 b-d). To generate this structure, the precursor RNA associates with the capsid, or viral shell, proteins. The RNA is then reverse transcribed into the DNA, which replaces the RNA. The structures they derived from their experiments hint that the process of copying the RNA into DNA may cause the rings of DNA to slide around the proteins, and as more layers are added, the DNA would become more rigid in its position in the viral particle (2). One of the ends of the capsid protein monomers that the DNA associates with is rich in arginine, a positively charged amino acid, which can interact with the negatively charged backbone of DNA to stabilize the negative charges that would end up being so tightly packed into the viral particle (Figure 2). 

Figure 2: Structures of charged molecules relevant to capsid stability a. Structure of L-arginine b. Structure of a portion of the DNA backbone, “Base” represents A, T, C, or G (2)

They speculate that the tightly packed DNA can also act as a spring inside the viral particle, so that when it infects a new liver cell, the force from the tightly wound DNA can cause the particle to burst open and facilitate genome entry into the cell (2). 

Conclusions

To better understand how HBV packages its entire genome into its viral particle, the authors imaged mature HBV and DHBV particles to visualize the organization of the DNA inside. They revealed that the DNA of HBV and DHBV is contained in layers of circular concentric loops. The authors speculate that the process of packaging the particle takes place during reverse transcription of the RNA segments that HBV creates following infection, and that the positively charged capsid proteins facilitate stabilizing the DNA inside the virus (2). 

Works Cited

1. Gibes N, Culhane K, Liu H, Xi J, Pionek K, Nair S, Loeb DD, Hu J, Zlotnick A, Wang JC. DNA synthesis inside the hepatitis B virus creates a high-energy spool. bioRxiv [Preprint]. 2026 Jan 23:2026.01.22.701187. doi: 10.64898/2026.01.22.701187. PMID: 41648622; PMCID: PMC12871785.

2. Watson AG, Mulay AS, Gill US. Chronic hepatitis B in 2025: diagnosis, treatment and future directions. Clin Med (Lond). 2025 Nov;25(6):100527. doi: 10.1016/j.clinme.2025.100527. Epub 2025 Nov 4. PMID: 41192690; PMCID: PMC12681808.

Synthesis and Testing of PB05, a Dual Inhibitor of IAV PB2 and Cellular JAK2

 Synthesis and Testing of PB05, a Dual Inhibitor of IAV PB2 and Cellular JAK2

Influenza A virus (IAV) is a member of the influenza family, which contains Influenza A-D. Influenza A and B are the subtypes primarily responsible for the seasonal influenza cases.  Influenza subtypes have further genetic diversity due to their hemagglutinin (HA) protein, which has 18 identified variants, and the neuraminidase (NA) protein that has 11 identified variants. The variants of these proteins present on the viral surface determine the nomenclature for a specific subtype of virus; for example, the influenza A H1N1 virus (Spanish Flu) would present variant 1 for both the HA and NA proteins (1).

Although vaccinations are available each year for the emerging strains, ~5 million people each year develop severe symptoms, with 650,000 yearly deaths. In addition to its well-known symptoms of fever, tiredness, dizziness, and headache, influenza infection can cause rarer, yet more dangerous symptoms such as pneumonia and myocarditis. A major source of the symptoms and major illnesses that come from influenza infection can be attributed to the immune response. This can be an issue when unregulated, pro-inflammatory molecules are produced out of control, leading to more severe symptoms. This process is heavily regulated by the JAK-STAT pathway, which is responsible for the activation of many pro-inflammatory molecules. Common viral signals are recognized by cells, which leads to signaling events that activate JAK proteins. Influenza antiviral drugs primarily target the viral replication process, while ignoring the burden the immune response can have on a patient. This highlights the need for antivirals that can target both the viral replication and immune response elements of IAV infection (2).

By nature of the atypical genome of IAV, it requires its own special enzyme that can produce its transcripts, called an RdRp. The IAV RdRp is also capable of stealing essential portions of cellular machinery in a mechanism called “cap snatching, ” which is essential for its replication. The PB2 subunit of RdRp contains the cap-binding region required to steal caps, and thus is a suitable target for drugs that seek to inhibit cap snatching, and by extension, viral replication (2).

The authors of this paper were able to screen for drugs that either inhibit JAK proteins or drugs that inhibit the PB2 domain of IAV RdRp. They identified two drugs with similar structures: decernotinib, which inhibits JAK, and pimodivir, which inhibits PB2. Their goal was to synthesize a drug that mimics both structures and is able to inhibit both JAK and PB2. The paper describes their process of synthesizing and testing this molecule, which they call PB05, against various influenza infection conditions (2).

Synthesis

Figure 1. Visual Representation of PB05. (b) Images of pimodivir and decernotinib in the cap binding domain of PB2. (c) Visual representation of the workflow to create a pimodivir and decernotinib-like molecule, with possible modifications to the green “R” group in the pink box

Both drugs shared a main skeleton of carbon and nitrogen-based rings, which interact with portions of PB2 (Figure 1b,c). Fluorine substituents present on pimdinovir were incorporated into the final molecule, while the modified carbon chain of decernotinib was incorporated as well. They synthesized 5 drugs with this structure, with slight structural variations named PB01, PB02, PB03, PB04, and PB05. These molecules were tested for IAV inhibition, JAK inhibition, and PB2 inhibition. From these tests, the authors were able to determine that the PB05 drug performed the best (2).

Antiviral Activity of PB05

Figure 2 PB05 and decernotinib binding visualization. (c) (Left) PB05 and decernotinib in the active region of JAK2. (Right) PB05 and decernotinib in the cap binding domain of PB2.

The effectiveness of PB05 was tested against the H1N1 and H3N2 strains of IAV, which demonstrated that PB05 was capable of reducing the levels of IAV proteins and genetic material, but about 10x less efficiently than pimodivir, the PB2 inhibitor. Their imaging technology revealed that PB05 was directly binding to PB2 (Figure 2c). As for JAK-STAT inhibition, PB05 was also able to decrease the levels of transcripts responsible for producing inflammatory molecules, and was also found to be interacting with JAK (Figure 2c). In mice, inflammation-induced damage was also severely decreased. From these experiments, they were able to prove that PB05 was an effective antiviral therapy against IAV, which worked by both inhibiting the immune response to reduce inflammation-based damage, but also by inhibiting a key IAV enzyme in viral replication (2). 

Pharmacokinetics

Aside from a drug's ability to act on its target, another key aspect of drug design is making a drug that can be taken into the body without interference from common pathways of degradation and removal. This means that digestion, excretion, and distribution of the drug can also become a problem. Thankfully, they determined that PB05 was able to be taken orally with a reasonable dosage that wasn't harmful to the mice they tested on, and stayed in the body long enough to be effective when taken twice daily (2).

Conclusion

This paper demonstrated the need for drugs that both inhibit the immune response to influenza infections to reduce the injuries caused by inflammation, and inhibit viral replication to assist the immune system in successfully clearing the infection. They were able to find two drugs that performed these actions with structural similarities, and synthesize a single drug that combined the properties of these two. They claim that PB05 is an important first step in developing better IAV treatments, but more studies are needed on its interactions with the body's metabolism and its ability to circulate. It functions by blocking the JAK pathway and the cap snatching enzyme of IAV, demonstrating the dual action ability of this drug.

Works Cited

1. Kyokha Ameen Y. Seasonal Influenza: A Narrative Review of Epidemiology, Clinical Features, and Preventive Strategies. Cureus. 2025 Oct 24;17(10):e95336. doi: 10.7759/cureus.95336. PMID: 41287674; PMCID: PMC12640676.

2. Rong B, Yang Y, Lu K, Zhou X, Zheng P, Lin X, Wen Y, Lin S, Deng X, Zhou Q, Liu S. Dual Inhibition of PB2 and JAK2 for Influenza: A Strategy Combining Antiviral and Host-Directed Immune Modulation. Molecules. 2026 Feb 17;31(4):696. doi: 10.3390/molecules31040696. PMID: 41752472; PMCID: PMC12943364.