Virus Survival Research Articles

TRAF6 autophagic degradation by avibirnavirus VP3 inhibits antiviral innate immunity via blocking NFKB/NF-κB activation

ABSTRACT Ubiquitination is an important reversible post-translational modification. Many viruses hijack the host ubiquitin system to enhance self-replication. In the present study, we found that Avibirnavirus VP3 protein was ubiquitinated during infection and supported virus replication by ubiquitination. Mass spectrometry and mutation analysis showed that VP3 was ubiquitinated at residues K73, K135, K158, K193, and K219. Virus rescue showed that ubiquitination at sites K73, K193, and K219 on VP3 could enhance the replication abilities of infectious bursal disease virus (IBDV), and that K135 was essential for virus survival. Binding of the zinc finger domain of TRAF6 (TNF receptor associated factor 6) to VP3 mediated K11- and K33-linked ubiquitination of VP3, which promoted its nuclear accumulation to facilitate virus replication. Additionally, VP3 could inhibit TRAF6-mediated NFKB/NF-κB (nuclear factor kappa B) activation and IFNB/IFN-β (interferon beta) production to evade host innate immunity by inducing TRAF6 autophagic degradation in an SQSTM1/p62 (sequestosome 1)-dependent manner. Our findings demonstrated a macroautophagic/autophagic mechanism by which Avibirnavirus protein VP3 blocked NFKB-mediated IFNB production by targeting TRAF6 during virus infection, and provided a potential drug target for virus infection control. Abbreviations: ATG: autophagy related; BafA1: bafilomycin A1; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; Cas9: CRISPR-associated protein 9; CHX: cycloheximide; Co-IP: co-immunoprecipitation; CRISPR: clustered regularly interspaced short palindromic repeats; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GST: glutathione S-transferase; IBDV: infectious bursal disease virus; IF: indirect immunofluorescence; IFNB/IFN-β: interferon beta; mAb: monoclonal antibody; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; MS: mass spectrometry; NFKB/NF-κB: nuclear factor kappa B; NBR1: NBR1 autophagy cargo receptor; OPTN: optineurin; pAb: polyclonal antibody; PRRs: pattern recognition receptors; RNF125: ring finger protein 125; RNF135/Riplet: ring finger protein 135; SQSTM1/p62: sequestosome 1; TAX1BP1: tax1 binding protein1; TCID50: 50% tissue culture infective dose; TRAF3: TNF receptor associated factor 3; TRAF6: TNF receptor associated factor 6; TRIM25: tripartite motif containing 25; Ub: ubiquitin; Wort: wortmannin; WT: wild type.

Novel Evidence Showing the Possible Effect of Environmental Variables on COVID-19 Spread.

Coronavirus disease (COVID‐19) remains a serious issue, and the role played by meteorological indicators in the process of virus spread has been a topic of academic discussion. Previous studies reached different conclusions due to inconsistent methods, disparate meteorological indicators, and specific time periods or regions. This manuscript is based on seven daily meteorological indicators in the NCEP reanalysis data set and COVID‐19 data repository of Johns Hopkins University from 22 January 2020 to 1 June 2021. Results showed that worldwide average temperature and precipitable water (PW) had the strongest correlation (ρ > 0.9, p < 0.001) with the confirmed COVID‐19 cases per day from 22 January to 31 August 2020. From 22 January to 31 August 2020, positive correlations were observed between the temperature/PW and confirmed COVID‐19 cases/deaths in the northern hemisphere, whereas negative correlations were recorded in the southern hemisphere. From 1 September to 31 December 2020, the opposite results were observed. Correlations were weak throughout the near full year, and weak negative correlations were detected worldwide (|ρ| < 0.4, p ≤ 0.05); the lag time had no obvious effect. As the latitude increased, the temperature and PW of the maximum confirmed COVID‐19 cases/deaths per day generally showed a decreasing trend; the 2020‐year fitting functions of the response latitude pattern were verified by the 2021 data. Meteorological indicators, although not a decisive factor, may influence the virus spread by affecting the virus survival rates and enthusiasm of human activities. The temperature or PW threshold suitable for the spread of COVID‐19 may increase as the latitude decreases.

Risks and new challenges in the food chain: Viral contamination and decontamination from a global perspective, guidelines, and cleaning.

Even during the continuing world pandemic of severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), consumers remain exposed to the risk of getting infected by existing, emerging, or re-emerging foodborne and waterborne viruses. SARS-CoV-2 is different in that it is transmitted directly via the airborne route (droplets and aerosols) or indirect contact (surfaces contaminated with SARS-CoV-2). International food and health organizations and national regulatory bodies have provided guidance to protect individuals active in food premises from potential occupational exposure to SARS-CoV-2, and have recommended chemicals effective in controlling the virus. Additionally, to exclude transmission of foodborne and waterborne viruses, hygiene practices to remove viral contaminants from surfaces are applied in different stages of the food chain (e.g., food plants, food distribution, storage, retail sector, etc.), while new and enhanced measures effective in the control of all types of viruses are under development. This comprehensive review aims to analyze and compare efficacies of existing cleaning practices currently used in the food industry to remove pathogenic viruses from air, nonfood, and food contact surfaces, as well as from food surfaces. In addition, the classification, modes of transmission, and survival of food and waterborne viruses, as well as SARS-CoV-2 will be presented. The international guidelines and national regulations are summarized in terms of virucidal chemical agents and their applications.

Experimental models of ecological niches for African swine fever virus

In this study, we investigated the possible biological factors affecting the survival of the African swine fever virus (ASFV) in the environment and their potential to influence the ecology of the ASFV. Specifically, we tested the survival and replication of ASFV in four phylogenetically distinct organisms: Paramecium caudatum, Dendrobaena alpine, Aedes aegypti andXeropicta derbentina using qReal-Time PCR and hemadsorbtion analysis. Levels of ASFV in earthworms (Dendrobaena alpina) and soil declined at similar rates, suggesting that earthworms likely have no influence on the ecology of the ASFV. Ciliates (Paramecium caudatum) significantly increase the rate of ASFV disappearance from the aquatic environment, probably using the virus as a food source. Mosquitoes (Aedes aegypti) do not provide significant support for the persistence of ASF virus in the environment, with no evidence for transmission to their offspring or pigs that ingested mosquitoes. ASFV persisted for much longer in air-breathing land snails (Xeropicta derbentina) than in the soil. Moreover, transcription of viral genes was maintained within the snail, although the question of full-fledged viral replication is still open. In addition, the active movements of snails suggests that they could play a role in the spread of the virus. The virus is likely to be localized in the intestines of snails as it is regularly excreted from their feces. These results highlight the importance of investigating invertebrates for understanding ASFV surviving, spreading and transmission in natural populations with zoonotic transmission potential.

CD33 is downregulated by influenza virus H1N1pdm09 and induces ROS and the TNF-α, IL-1β, and IL-6 cytokines in human mononuclear cells.

The influenza A virus (IAV) H1N1pdm09 induces exacerbated inflammation, contributing to disease complications. Inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), favor an inflammatory response that aids viral replication and survival. A pathway by which spontaneous TNF-α production occurs involves either the reduction of Siglec-3 (CD33) levels or the absence of its ligand, sialic acid. Influenza virus uses sialic acid to enter cells by reducing their expression; however, the role of CD33 in IAV H1N1pdm09 stimulation and its relationship with inflammation have not yet been studied. To evaluate the role of CD33 in proinflammatory cytokine production in IAV H1N1pdm09 stimulation, peripheral blood mononuclear cells from healthy subjects were incubated with IAV H1N1pdm09. We observed that the infection caused an increase in the mRNA expression of proinflammatory cytokines such as TNF-α, interleukin (IL)-1β, and IL-6 and a significant reduction in CD33 expression by monocytes at an early stage of infection. Additionally, suppressor of cytokine signaling 3 (SOCS-3) mRNA expression was upregulated at 6h, and reactive oxygen species (ROS) production increased at 1.5h. Moreover, a significant reduction in CD33 expression on the cell surface of monocytes from influenza patients or of IAV H1N1pdm09-stimulated monocytes incubated in vitro was observed by flow cytometry. The results suggest that the decrease in CD33 and increase of SOCS-3 expression induced by IAV H1N1pdm09 triggered TNF-α secretion and ROS production, suggesting an additional way to exacerbate inflammation during viral infection.

Transfer of MS2 bacteriophage from surfaces to raspberry and pitanga fruits and virus survival in response to sanitization, frozen storage and preservation technologies.

This study assessed the norovirus (NoV) surrogate bacteriophage MS2 transfer from stainless steel, glass and low-density polypropylene surfaces to raspberry and pitanga fruits. The effect of sodium hypochlorite (100 ppm, 1 min) on MS2 survival on whole fruits, the MS2 survival in sanitized fruits and derived pulps during frozen storage, and in response to preservation technologies (heat, organic acids and salts) was also assessed. The highest (p < 0.05) viral transfer (%) was observed from glass and stainless steel (∼90%) to raspberry, and from glass and polypropylene (∼75%) to pitanga, after 60 min of contact. Sodium hypochlorite reduced (p < 0.05) MS2 titer by 3.5 and 3.8 log PFU/g in raspberry and pitanga, respectively. MS2 decreased (p < 0.05) up to 1.4 log PFU/g in frozen stored sanitized fruits (whole fruits and pulps) after 15 days, with no further changes after 30 days. Thermal treatments reduced MS2 titer (p < 0.05) in both fruit pulps. MS2 inactivation was higher in pitanga pulp. The addition of ascorbic acid, citric acid, sodium benzoate, or sodium metabisulfite had little effect (<1 log PFU/g) on MS2 concentration in either fruit. These results may inform NoV risk management practice in processing and handling of fruits.

Evidence for a semisolid phase state of aerosols and droplets relevant to the airborne and surface survival of pathogens

The phase state of respiratory aerosols and droplets has been linked to the humidity-dependent survival of pathogens such as SARS-CoV-2. To inform strategies to mitigate the spread of infectious disease, it is thus necessary to understand the humidity-dependent phase changes associated with the particles in which pathogens are suspended. Here, we study phase changes of levitated aerosols and droplets composed of model respiratory compounds (salt and protein) and growth media (organic-inorganic mixtures commonly used in studies of pathogen survival) with decreasing relative humidity (RH). Efflorescence was suppressed in many particle compositions and thus unlikely to fully account for the humidity-dependent survival of viruses. Rather, we identify organic-based, semisolid phase states that form under equilibrium conditions at intermediate RH (45 to 80%). A higher-protein content causes particles to exist in a semisolid state under a wider range of RH conditions. Diffusion and, thus, disinfection kinetics are expected to be inhibited in these semisolid states. These observations suggest that organic-based, semisolid states are an important consideration to account for the recovery of virus viability at low RH observed in previous studies. We propose a mechanism in which the semisolid phase shields pathogens from inactivation by hindering the diffusion of solutes. This suggests that the exogenous lifetime of pathogens will depend, in part, on the organic composition of the carrier respiratory particle and thus its origin in the respiratory tract. Furthermore, this work highlights the importance of accounting for spatial heterogeneities and time-dependent changes in the properties of aerosols and droplets undergoing evaporation in studies of pathogen viability.

RNAi-mediated siRNA sequences to combat the COVID-19 pandemic with the inhibition of SARS-CoV2

The outbreak of the COVID-19 pandemic has cost five million lives to date, and was caused by a positive-sense RNA virus named SARS-CoV2. The lack of drugs specific to SARS-CoV2, leads us to search for an effective and specific therapeutic approach. Small interfering RNA (siRNA) is able to activate the RNA interference (RNAi) pathway to silence the specific targeted gene and inhibit the viral replication, and it has not yet attracted enough attention as a SARS-CoV2 antiviral agent. It could be a potential weapon to combat this pandemic until the completion of full scale, effective mass vaccination. For this study, specific siRNAs were designed using a web-based bioinformatics tool (siDirect2.0) against 14 target sequences. These might have a high probability of silencing the essential proteins of SARS-CoV2. such as: 3CLpro/Mpro/nsp5, nsp7, Rd-Rp/nsp12, ZD, NTPase/HEL or nsp13, PLpro/nsp3, envelope protein (E), spike glycoprotein (S), nucleocapsid phosphoprotein (N), membrane glycoprotein (M), ORF8, ORF3a, nsp2, and its respective 5′ and 3′-UTR. Among these potential drug targets, the majority of them contain highly conserved sequences; the rest are chosen on the basis of their role in viral replication and survival. The traditional vaccine development technology using SARS-CoV2 protein takes 6–8 months; meanwhile the virus undergoes several mutations in the candidate protein chosen for vaccine development. By the time the protein-based vaccine reaches the market, the virus would have undergone several mutations, such that the antibodies against the viral sequence may not be effective in restricting the newly mutated viruses. However, siRNA technology can make sequences based on real time viral mutation status. This has the potential for suppressing SARS-CoV2 viral replication, through RNAi technology.

Investigation of mouse hepatitis virus strain A59 inactivation under both ambient and cold environments reveals the mechanisms of infectivity reduction following UVC exposure

The surface contamination of SARS-CoV-2 is becoming a potential source of virus transmission during the pandemic of COVID-19. Under the cold environment, the infection incidents would be more severe with the increase of virus survival time. Thus, the disinfection of contaminated surfaces in both ambient and cold environments is a critical measure to restrain the spread of the virus. In our study, it was demonstrated that the 254 nm ultraviolet-C (UVC) is an efficient method to inactivate a coronavirus, mouse hepatitis virus strain A59 (MHV-A59). The inactivation rate to MHV-A59 coronavirus was up to 99.99% when UVC doses were 2.90 and 14.0 mJ/cm2 at room temperature (23 °C) and in cold environment (−20 °C), respectively. Further mechanistic study demonstrated that UVC could induce spike protein damage to partly impede virus attachment and genome penetration processes, which contributes to 12% loss of viral infectivity. Additionally, it can induce genome damage to significantly interrupt genome replication, protein synthesis, virus assembly and release processes, which takes up 88% contribution to viral inactivation. With these mechanistic understandings, it will greatly contribute to the prevention and control of the current SARS-CoV-2 transmissions in cold chains (low temperature-controlled product supply chains), public area such as airport, school, and warehouse.

Sublethal enteroviral infection exacerbates disease progression in an ALS mouse model

BackgroundAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of the motor neuron system associated with both genetic and environmental risk factors. Infection with enteroviruses, including poliovirus and coxsackievirus, such as coxsackievirus B3 (CVB3), has been proposed as a possible causal/risk factor for ALS due to the evidence that enteroviruses can target motor neurons and establish a persistent infection in the central nervous system (CNS), and recent findings that enteroviral infection-induced molecular and pathological phenotypes closely resemble ALS. However, a causal relationship has not yet been affirmed.MethodsWild-type C57BL/6J and G85R mutant superoxide dismutase 1 (SOD1G85R) ALS mice were intracerebroventricularly infected with a sublethal dose of CVB3 or sham-infected. For a subset of mice, ribavirin (a broad-spectrum anti-RNA viral drug) was given subcutaneously during the acute or chronic stage of infection. Following viral infection, general activity and survival were monitored daily for up to week 60. Starting at week 20 post-infection (PI), motor functions were measured weekly. Mouse brains and/or spinal cords were harvested at day 10, week 20 and week 60 PI for histopathological evaluation of neurotoxicity, immunohistochemical staining of viral protein, neuroinflammatory/immune and ALS pathology markers, and NanoString and RT-qPCR analysis of inflammatory gene expression.ResultsWe found that sublethal infection (mimicking chronic infection) of SOD1G85R ALS mice with CVB3 resulted in early onset and progressive motor dysfunction, and shortened lifespan, while similar viral infection in C57BL/6J, the background strain of SOD1G85R mice, did not significantly affect motor function and mortality as compared to mock infection within the timeframe of the current study (60 weeks PI). Furthermore, we showed that CVB3 infection led to a significant increase in proinflammatory gene expression and immune cell infiltration and induced ALS-related pathologies (i.e., TAR DNA-binding protein 43 (TDP-43) pathology and neuronal damage) in the CNS of both SOD1G85R and C57BL/6J mice. Finally, we discovered that early (day 1) but not late (day 15) administration of ribavirin could rescue ALS-like neuropathology and symptoms induced by CVB3 infection.ConclusionsOur study identifies a new risk factor that contributes to early onset and accelerated progression of ALS and offers opportunities for the development of novel targeted therapies.

Nervous necrosis virus viability modulation by water salinity and temperature.

Nervous necrosis virus (NNV) is a hazardous aquatic pathogen, distributed worldwide and in a wide range of temperatures. Viral persistence in water has been demonstrated to be affected by different factors, such as temperature, UV, or biological load. In this study, we have investigated the viability of NNV strains in low- and high-salinity seawater (LS and HS, respectively) both in laboratory and aquarium conditions, at different storage temperatures, and for comparative purposes, in culture medium. Our results showed the highest NNV viability in seawater at 15°C and as temperature increased, a drop in viral persistence was observed. Additionally, survival at 15 and 30°C was strongly affected by increasing salt content, while no differences were observed between LS and HS groups at 20 and 25°C. The results of the incubation under aquarium conditions indicated that the effect of UV light and oxygen exposure accelerate the inactivation of infective particles. According to previous studies, NNV persistence in cell culture medium was higher than in seawater, and as observed in the latter, increasing incubation temperatures led to a decrease in viral survival.

Environmental Factors Influencing COVID-19 Incidence and Severity.

Emerging evidence supports a link between environmental factors-including air pollution and chemical exposures, climate, and the built environment-and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission and coronavirus disease 2019 (COVID-19) susceptibility and severity. Climate, air pollution, and the built environment have long been recognized to influence viral respiratory infections, and studies have established similar associations with COVID-19 outcomes. More limited evidence links chemical exposures to COVID-19. Environmental factors were found to influence COVID-19 through four major interlinking mechanisms: increased risk of preexisting conditions associated with disease severity; immune system impairment; viral survival and transport; and behaviors that increase viral exposure. Both data and methodologic issues complicate the investigation of these relationships, including reliance on coarse COVID-19 surveillance data; gaps in mechanistic studies; and the predominance of ecological designs. We evaluate the strength of evidence for environment-COVID-19 relationships and discuss environmental actions that might simultaneously address the COVID-19 pandemic, environmental determinants of health, and health disparities.

Evaluation of knowledge and attitude of dental students regarding COVID-19: A multicentric study.

Despite the life-threatening risk that corona virus disease (COVID-19) poses to dentists, their role in suppressing its transmission and the need to deliver dental care to the patients is inevitable. In this regard, it is essential to determine the awareness and knowledge among Indian dental students regarding COVID-19 and to assess their preparedness to handle the current situation. This cross-sectional study enrolled 408 dental undergraduate (UG) and postgraduate (PG) students from private and government dental teaching hospitals. A closed-ended online questionnaire containing crucial elements of COVID management during dental procedures was given to the participants assessing their knowledge and perception pertaining to COVID-19 regarding dental practice. Chi-square, Mann-Whitney U, and Kruskal-Wallis tests were employed for statistical analysis. P ≤ 0.05 was considered statistically significant. The study consisted of 64.71% UG and 35.29% PG dental students, with a mean age of 25.75 ± 3.69 years and M: F ratio of 0.5:1. COVID-19 knowledge increased significantly with age (P = 0.0055) and educational level (P < 0.01). Awareness was also significantly greater in students from government institutions in comparison with private colleges (P < 0.001), with 91.67% of participants being aware of the transmission routes. However, familiarity with survival of virus in aerosols, considerations regarding the use of masks, and strategies for confirmed/suspected COVID-19 health-care workers to return to work after recovery were seen in only 35%, 53.9%, and 38.9% of the students, respectively. COVID-19 awareness among Indian dental students is significantly related to their education level. The lacunae in this knowledge need to be filled by adequate training by incorporating it as a part of their education.

Potential Role of Functional Foods in the Management of SARS-CoV-2 Omicron Variant

Abstract Omicron is a more contagious variant of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), commonly known as Coronavirus disease 2019 (COVID- 19). Dietary management of COVID-19 with functional foods plays a significant role in reducing cytokine storm and the viral replication and survival. A thorough literature search was performed in electronic databases such as PubMed and EMBASE using the keywords Coronavirus, COVID-19, Omicron, functional foods, and phytonutrient. In total, 77 research articles were identified; of those, 10 papers were included after title, abstract and full text review. Most commonly used phytonutrients for the management of COVID-19 include bromelain, curcumin, quercetin, gingerol and epigallocatechin gallate (EGCG). These phytonutrients possessed anti-inflammatory properties, and were effective in targeting the spike proteins and docking sites of the SARS-CoV-2 virus, thereby preventing viral replication and survival. To the best of our knowledge, this is the first published review indicating the significance of functional foods in reducing the severity of the Omicron variant.

Human Responses in Public Health Emergencies for Infectious Disease Control: An Overview of Controlled Topologies for Biomedical Applications.

COVID-19 originated in Wuhan city of Hubei Province in China in December three years ago. Since then, it has spread to more than 210 countries and territories. This disease is caused by Severe Acute Respiratory Syndrome Coronavirus 2. The virus has a size of one to two nanometers and a single-stranded positive RNA. Droplets spread the virus from coughing and sneezing. This condition causes coughing, fever, acute respiratory problems, and even death. According to the WHO, the virus can survive outside the body for several hours. This research aimed to determine how environmental factors influenced the COVID-19 virus's survival and behavior, as well as its transmission, in a complex environment. Based on the results, virus transmissions are influenced by various human and environmental factors such as population distribution, travel, social behavior, and climate change. Environmental factors have not been adequately examined concerning the transmission of this epidemic. Thus, it is necessary to examine various aspects of prevention and control of this disease, including its effects on climate and other environmental factors.

Can Free Living Acanthamoeba Act as a Trojan Horse for SARS-Cov-2 on Viral Survival and Transmission in the Environment? A Narrative Review.

Acanthamoeba is a free-living amoeba that has been found on surfaces, air, water and various environmental sources around the world. It enters the human body through the respiratory tract via the nose. Of note, amoebae are well known to act as a reservoir for various pathogenic microorganisms including bacteria and viruses such as Adenoviruses and Mimivirus. Given that SARS-CoV-2 of the Coronaviridae family is transmitted through the respiratory tract, and the Trojan horse nature of Acanthamoeba, it has been suggested that amoebae act as a vector in the transmission of SARS-CoV-2. The aim of this study was to provide an opinion of the possibility of the coexistence of Acanthamoeba with SARS-CoV-2.

Persistence of African Swine Fever Virus in Feed and Feed Mill Environment over Time after Manufacture of Experimentally Inoculated Feed

To reduce the risk of disease from harmful feed-based pathogens, some feed manufacturers quarantine high-risk ingredients prior to their inclusion in feed. Data exist that confirms this practice is effective, but to our knowledge there is no information about porcine pathogen survival in mill environments. The objective of this study was to determine survival of African swine fever virus (ASFV) in swine feed and on mill surfaces after manufacture of experimentally inoculated swine feed. A pilot-scale feed mill was placed within a biosecurity level (BSL) 3 facility to manufacture batches of feed. The priming batch, Batch 1, was ASFV-free feed and was followed with Batch 2 which was experimentally inoculated with ASFV (5.6 × 104 TCID50/gram). Four subsequent ASFV-free batches were then manufactured (Batch 3-6). After each batch of feed, 10 feed samples were aseptically collected in a double ‘X’ pattern. During feed manufacturing, 24 steel coupons were placed on the floor of the manufacturing area and feed dust was allowed to settle onto them overnight. Once feed manufacturing was completed, feed samples and steel coupons were stored at room temperature. On the day of (day 0) and d 3, 7, 14, 28, 60, 90, and 180 after feed manufacturing, feed samples and 3 steel coupons were randomly selected, taken out of storage, and analyzed for ASFV DNA. For feed samples there was a statistically significant (P = 0.023) batch × day interaction for log10 genomic copies per gram of feed, and a marginal statistical significance (P = 0.072) for batch × day interaction for cycle threshold (Ct) values. This indicates that the batch of feed and days held at room temperature impacted the amount of the detectable ASFV DNA in feed samples. There was no evidence (P = 0.433) of ASFV degradation on environmental coupons over the 180-d storage period. This study found that quarantine time can help reduce, but not eliminate ASFV DNA in feed over time. Surprisingly, ASFV DNA is detectable on feed manufacturing surfaces for at least 180 days.

PD-L1 and EBV LMP1 expressions in classic Hodgkin lymphomas and its correlation with clinicopathological parameters and prognosis.

Programmed death pathway leads to T cell anergy. Wide range of malignancies take advantage of this pathway by programmed death-ligand 1 (PD-L1) expression either on neoplastic cells or on the nonneoplastic cells of tumour microenvironment. New therapeutic approaches have been directed against this pathway. We studied PD-L1 expression on both neoplastic Hodgkin and Reed-Sternberg (HRS) cells and cells of tumour microenvironment in classic Hodgkin lymphoma (CHL) patients and compared it with Ebstein-Barr virus (EBV) positivity, clinical data, and survival rates. Lymph node excision materials of 56 CHL patients diagnosed between 2007 and 2017 were included in this retrospective study. PD-L1 expression of HRS cells and tumour microenvironment cells were evaluated by immunohistochemical assay. Staining intensity and rate of the PD-L1 expressions were estimated. EBV was examined by immunohistochemistry for latent membrane protein 1 (LMP1) antibody. Clinical data of 39 patients and survival data of 34 patients were compared with PD-L1 expressions on tumour cells. PD-L1 expression was present in HRS cells in 89.2% of the cases. There was more than 20% of PD-L1 expression in cells of tumour microenvironment in all the cases. PD-L1 positivity did not show statistically significant difference according to EBV expression, clinical parameters, and prognosis. Previous studies showed inconsistent rates for PD-L1 prevalence (20%-95.7%) in CHL patients due to differences in the study methods. Although high prevalence of PD-L1 positivity was found in majority of them, there was no statistically significant difference between PD-L1 positivity on HRS cells and EBV expression, clinical parameters, and prognosis. This high prevalence in patients with various clinical properties makes PD-L1 a potential target for new emerging immunotherapies for CHL.

A master autoantigen-ome links alternative splicing, female predilection, and COVID-19 to autoimmune diseases

Chronic and debilitating autoimmune sequelae pose a grave concern for the post-COVID-19 pandemic era. Based on our discovery that the glycosaminoglycan dermatan sulfate (DS) displays peculiar affinity to apoptotic cells and autoantigens (autoAgs) and that DS-autoAg complexes cooperatively stimulate autoreactive B1 cell responses, we compiled a database of 751 candidate autoAgs from six human cell types. At least 657 of these have been found to be affected by SARS-CoV-2 infection based on currently available multi-omic COVID data, and at least 400 are confirmed targets of autoantibodies in a wide array of autoimmune diseases and cancer. The autoantigen-ome is significantly associated with various processes in viral infections, such as translation, protein processing, and vesicle transport. Interestingly, the coding genes of autoAgs predominantly contain multiple exons with many possible alternative splicing variants, short transcripts, and short UTR lengths. These observations and the finding that numerous autoAgs involved in RNA-splicing showed altered expression in viral infections suggest that viruses exploit alternative splicing to reprogram host cell machinery to ensure viral replication and survival. While each cell type gives rise to a unique pool of autoAgs, 39 common autoAgs associated with cell stress and apoptosis were identified from all six cell types, with several being known markers of systemic autoimmune diseases. In particular, the common autoAg UBA1 that catalyzes the first step in ubiquitination is encoded by an X-chromosome escape gene. Given its essential function in apoptotic cell clearance and that X-inactivation escape tends to increase with aging, UBA1 dysfunction can therefore predispose aging women to autoimmune disorders. In summary, we propose a model of how viral infections lead to extensive molecular alterations and host cell death, autoimmune responses facilitated by autoAg-DS complexes, and ultimately autoimmune diseases. Overall, this master autoantigen-ome provides a molecular guide for investigating the myriad of autoimmune sequalae to COVID-19 and clues to the rare adverse effects of the currently available mRNA and viral vector-based COVID vaccines.

Site-directed mutagenesis of the C-terminal of the Newcastle disease virus V protein.

Newcastle disease virus (NDV) is aparamyxovirus that is highly pathogenic to poultry causing severe economic loss worldwide. The non-structural Vprotein is one of the virulence factors of the virus. It antagonises the interferon of the host innate immunity in order to allow successful virus replication in the host cells. However, detailed investigation of recombinant NDV expressing mutated Vprotein is scarce. In this study, amesogenic recombinant NDV expressing GFP (rAF-GFP) was used to investigate the relation of Vprotein mutation on virus pathogenicity. Site-directed mutagenesis was performed using overlapping PCR to introduce four premature stop codons 456G>T, 537G>T, 624C>T and 642G>T in the Vgene reading frame. The virus was then rescued and propagated in embryonated chicken eggs. However, instead of the substituted thymine, this nucleotide was mutated into cytosine in three rescued mutants, while 537G>T mutant could not be rescued. As aresult, the premature stop codon was substituted with other amino acid and the Vprotein was expressed in full length. The pathogenicity type of the rAF (456G>T>C), rAF (624C>T>C), and rAF (642G>T>C) mutants remained to be as in mesogenic strains, suggesting that substituted amino acids were functionally interchangeable with the original amino acids present in Vprotein. It appears that an intact Vprotein is important for the virus survival. This study explored the possibility of Vprotein mutation in NDV through exploiting genetic engineering and warrants afurther investigation on modifying mutations on aconserved protein in NDV or other paramyxoviruses. Keywords: Paramyxoviridae; Newcastle disease virus; Vprotein; C terminal; virulence factor.