Designing the fusion protein of rotavirus VP8 and hepatitis A virus VP1 and evaluating the immunological response in BALB/c mice.

Background and Objectives: Rotavirus and Hepatitis A virus are responsible for causing gastroenteritis and jaundice. The current vaccination approaches have proven insufficient, especially in low-income countries. In this study, we presented a novel dual-vaccine candidate that combines the rotavirus VP8 protein and the hepatitis A virus VP1. Materials and Methods: The VP8*-rotavirus+AAY+HAV-VP1 fusion protein was produced using an Escherichia coli expression system. The recombinant protein had a molecular weight of approximately 45.5 kDa and was purified through affinity chromatography. BALB/c mice were injected subcutaneously with the recombinant protein, VP1, VP8 and vaccines for rotavirus and hepatitis A virus, both with and without ALUM and M720 adjuvants. ELISA assays were used to measure total IgG, IgG1, IgG2, and short-term and long-term IL-5 and IFN-γ responses. Results: The fusion protein, when combined with adjuvants, elicited significantly higher total IgG, IgG1, and IgG2 responses compared to VP1 and VP8 alone, as well as the rotavirus and hepatitis A vaccines. Furthermore, it induced a higher short-term IL-5 and IFN-γ response while demonstrating a higher long-term IL-5 response compared to the rotavirus and hepatitis A vaccines. Conclusion: This study demonstrates that the VP8*-rotavirus+AAY+HAV-VP1 fusion protein is a promising dual vaccine candidate for immunization against hepatitis A and rotaviruses.

Exploring the impression of TRIM25 gene expression on COVID-19 severity and SARS-CoV-2 viral replication.

BACKGROUND: There are numerous human genes associated with viral infections, and their identification in specific populations can provide suitable therapeutic targets for modulating the host immune system response and better understanding the viral pathogenic mechanisms. Many antiviral signaling pathways, including Type I interferon and NF-κB, are regulated by TRIM proteins. Therefore, the identification of TRIM proteins involved in COVID-19 infection can play a significant role in understanding the innate immune response to this virus. METHODS: In this study, the expression of TRIM25 gene was evaluated in a blood sample of 330 patients admitted to the hospital (142 patients with severe disease and 188 patients with mild disease) as well as in 160 healthy individuals. The relationship between its expression and the severity of COVID-19 disease was assessed and compared among the study groups by quantitative Real-time PCR technique. The statistical analysis of the results demonstrated a significant reduction in the expression of TRIM25 in the group of patients with severe infection compared to those with mild infection. Furthermore, the impact of increased expression of TRIM25 gene in HEK-293 T cell culture was investigated on the replication of attenuated SARS-CoV-2 virus. RESULTS: The results of Real-time PCR, Western blot for the viral nucleocapsid gene of virus, and CCID50 test indicated a decrease in virus replication in these cells. The findings of this research indicated that the reduced expression of the TRIM25 gene was associated with increased disease severity of COVID-19 in individuals. Additionally, the results suggested the overexpression of TRIM25 gene can impress the replication of attenuated SARS-CoV-2 and the induction of beta-interferon. CONCLUSION: TRIM25 plays a critical role in controlling viral replication through its direct interaction with the virus and its involvement in inducing interferon during the early stages of infection. This makes TRIM25 a promising target for potential therapeutic interventions.

Effect of high-dose Spirulina supplementation on hospitalized adults with COVID-19: a randomized controlled trial.

Objective: Spirulina (arthrospira platensis) is a cyanobacterium proven to have anti-inflammatory, antiviral, and antioxidant effects. However, the effect of high-dose Spirulina supplementation on hospitalized adults with COVID-19 is currently unclear. This study aimed to evaluate the efficacy and safety of high-dose Spirulina platensis for SARS-CoV-2 infection. Study Design: We conducted a randomized, controlled, open-label trial involving 189 patients with COVID-19 who were randomly assigned in a 1:1 ratio to an experimental group that received 15.2g of Spirulina supplement plus standard treatment (44 non-intensive care unit (non-ICU) and 47 ICU), or to a control group that received standard treatment alone (46 non-ICU and 52 ICU). The study was conducted over six days. Immune mediators were monitored on days 1, 3, 5, and 7. The primary outcome of this study was mortality or hospital discharge within seven days, while the overall discharge or mortality was considered the secondary outcome. Results: Within seven days, there were no deaths in the Spirulina group, while 15 deaths (15.3%) occurred in the control group. Moreover, within seven days, there was a greater number of patients discharged in the Spirulina group (97.7%) in non-ICU compared to the control group (39.1%) (HR, 6.52; 95% CI, 3.50 to 12.17). Overall mortality was higher in the control group (8.7% non-ICU, 28.8% ICU) compared to the Spirulina group (non-ICU HR, 0.13; 95% CI, 0.02 to 0.97; ICU, HR, 0.16; 95% CI, 0.05 to 0.48). In non-ICU, patients who received Spirulina showed a significant reduction in the levels of IL-6, TNF-α, IL-10, and IP-10 as intervention time increased. Furthermore, in ICU, patients who received Spirulina showed a significant decrease in the levels of MIP-1α and IL-6. IFN-γ levels were significantly higher in the intervention group in both ICU and non-ICU subgroups as intervention time increased. No side effects related to Spirulina supplements were observed during the trial. Conclusion: High-dose Spirulina supplements coupled with the standard treatment of COVID-19 may improve recovery and remarkably reduce mortality in hospitalized patients with COVID-19. Clinical Trial Registration: https://irct.ir/trial/54375, Iranian Registry of Clinical Trials number (IRCT20210216050373N1).

Expression of TRIM56 gene in SARS-CoV-2 variants and its relationship with progression of COVID-19.

Aim: The present study aimed to determine a correlation between differential TRIM56 expression levels and severe infections of COVID-19 between the Alpha, Delta and Omicron BA.5 variants. Materials & methods: This study was performed on 330 COVID-19 patients, including 142 with severe and 188 with mild infections, as well as 160 healthy controls. The levels of TRIM56 gene expression were determined using a qPCR. Results: TRIM56 gene showed significantly lower mRNA expression in the severe and mild groups compared with healthy individuals. Our finding indicated the high and low reduction of TRIM56 mRNA expression in Delta and Omicron BA.5 variant, respectively. Conclusion: Further research is needed to characterize the impact of TRIM proteins on the severity of COVID-19.

Scientists looked at a protein called TRIM that helps fight viruses to see if a specific TRIM protein, TRIM56, was linked to how poorly people became with COVID-19. The study looked at the blood samples of 330 patients and found that COVID-19 patients had less TRIM56 than healthy people, especially those who were particularly ill.

Reverse Vaccinology and Immunoinformatic Approach for Designing a Bivalent Vaccine Candidate Against Hepatitis A and Hepatitis B Viruses.

Hepatitis A and B are two crucial viral infections that still dramatically affect public health worldwide. Hepatitis A Virus (HAV) is the main cause of acute hepatitis, whereas Hepatitis B Virus (HBV) leads to the chronic form of the disease, possibly cirrhosis or liver failure. Therefore, vaccination has always been considered the most effective preventive method against pathogens. At this moment, we aimed at the immunoinformatic analysis of HAV-Viral Protein 1 (VP1) as the major capsid protein to come up with the most conserved immunogenic truncated protein to be fused by HBV surface antigen (HBs Ag) to achieve a bivalent vaccine against HAV and HBV using an AAY linker. Various computational approaches were employed to predict highly conserved regions and the most immunogenic B-cell and T-cell epitopes of HAV-VP1 capsid protein in both humans and BALB/c. Moreover, the predicted fusion protein was analyzed regarding primary and secondary structures and also homology validation. Afterward, the three-dimensional structure of vaccine constructs docked with various toll-like receptors (TLR) 2, 4 and 7. According to the bioinformatics tools, the region of 99-259 amino acids of VP1 was selected with high immunogenicity and conserved epitopes. T-cell epitope prediction showed that this region contains 32 antigenic peptides for Human leukocyte antigen (HLA) class I and 20 antigenic peptides in terms of HLA class II which are almost fully conserved in the Iranian population. The vaccine design includes 5 linear and 4 conformational B-cell lymphocyte (BCL) epitopes to induce humoral immune responses. The designed VP1-AAY-HBsAg fusion protein has the potency to be constructed and expressed to achieve a bivalent vaccine candidate, especially in the Iranian population. These findings led us to claim that the designed vaccine candidate provides potential pathways for creating an exploratory vaccine against Hepatitis A and Hepatitis B Viruses with high confidence for the identified strains.

Impact of TRIM5α and TRIM22 Genes Expression on the Clinical Course of Coronavirus Disease 2019.

OBJECTIVE: The innate immune response in humans involves a wide variety of factors, including the tripartite motif-containing 5α (TRIM5α) and 22 (TRIM22) as a cluster of genes on chromosome 11 that have exhibited antiviral activity in several viral infections. We analyzed the correlation of the expression of TRIM5α and TRIM22 with the severity of Coronavirus Disease 2019 (COVID-19) in blood samples of 330 patients, divided into two groups of severe and mild disease, versus the healthy individuals who never had contact with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). METHODS: The transcription level of TRIM5α and TRIM22 was determined by quantitative real-time polymerase chain reaction (qPCR). The laboratory values were collected from the patients' records. RESULTS: The expression of both genes was significantly lower in the severe group containing the hospitalized patients than in both the mild group and the control group. However, in the mild group, TRIM22 expression was significantly higher (p <0.0001) than in the control group while TRIM5α expression was not significantly different between these two groups. We found a relationship between the cycle threshold (Ct) value of patients and the expression of the aforementioned genes. CONCLUSION: The results of our study indicated that lower Ct values or higher RNA viral load might be associated with the downregulation of TRIM5α and TRIM22 and the severity of COVID-19. Additional studies are needed to confirm the results of this study.

A quartz crystal microbalance biosensor based on polyethylenimine-modified gold electrode to detect hepatitis B biomarker.

Biomarkers-based QCM-biosensors are suitable tools for the label-free detection of infectious diseases. In the current study, a QCM-biosensor was developed for the detection of HBsAg. Briefly, anti-HBsAg antibodies were covalently bound to the primary amines after PEI and thiolated-PEI surface modifications of gold-electrode. After RSM optimization, the statistical analysis revealed no significant difference between the immobilization yields of modified layers. Therefore, the PEI-modified QCM-biosensor was selected for further analysis. The PEI-surface was evaluated by FESEM, AFM, ATR-FTIR, and CA measurement. The surface hydrophilicity and its roughness were increased after PEI-coating. Also, FTIR confirmed the PEI-layering on the gold-surface. RSM optimization increased the antibody immobilization yield up to 80%. The QCM-biosensor showed noteworthy results with a wide dynamic range of 1-1 × 103 ng/mL, LOD of 3.14 ng/mL, LOQ of 9.52 ng/mL, and detection capability in human-sera, which were comparable with the ELISA. The mean accuracy of the QCM-biosensor was obtained at 91% when measured by the spike recovery test using human-sera. The biosensor was completely regenerated using 50 mM NaOH and 1% SDS. The benefits provided by the developed biosensor such as broad dynamic range, sensitivity, selectivity, stability, regenerate ability, and low cost suggest its potential application for the non-invasive and timely monitoring of HBV-biomarker.

Comparing the expression levels of tripartite motif containing 28 in mild and severe COVID-19 infection.

BACKGROUND: Tripartite motif-containing 28 (TRIM28) is an impressive regulator of the epigenetic control of the antiviral immune response. This study evaluated if the differential expression of TRIM28 correlates with the severity of coronavirus disease 2019 (COVID-19) infection. METHODS: A total of 330 COVID-19 patients, including 188 mild and 142 severe infections, and 160 healthy controls were enrolled in this study. Quantitative real-time polymerase chain reaction (qPCR) was used to determine the expression levels of TRIM28 in the studied patients. RESULTS: TRIM28 mRNA levels were significantly lower in both groups of patients versus the control group and in the severe group indicated further reduction in comparison to mild infection. The multivariate logistic regression analysis showed the mean age, lower levels of low-density lipoprotein (LDL), high-density lipoprotein (HDL), cholesterol, lower 25-hydroxyvitamin D, and PCR cycle threshold (Ct) value and higher levels of erythrocyte sedimentation rate (ESR) and differential expression of TRIM28 were linked to the severity of COVID-19 infection. CONCLUSION: The results of this study proved that the downregulation of TRIM28 might be associated with the severity of COVID-19 infection. Further studies are required to determine the association between the COVID-19 infection severity and TRIM family proteins.

Label-Free Real-Time Detection of HBsAg Using a QCM Immunosensor.

BACKGROUND: Hepatitis B virus surface antigen (HBsAg) is an important protein in both diagnosis and prevention of hepatitis B infection. In the current study, a piezoelectric immunosensor based on antibody-antigen interaction was designed to detect HBsAg. A quartz crystal microbalance system was employed to detect antibody-antigen interaction. METHODS: At first, an oscillator was designed to measure the resonant frequency affected by the reactants using IC 74LVC1GX04. Antibody against HBsAg was immobilized on 10 MHz AT-cut quartz crystal. The surface modifications were monitored by atomic force microscopy (AFM) and contact angle measurements. Different concentrations of antibody were used for surface immobilization and the frequency shifts were assessed. The system stability was studied by evaluating the stability of the crystal and the immobilized antibody. The adsorption of antibody onto the crystal was analyzed using AFM and changes in the resonance frequency. Further, a direct immunoassay was performed with this immobilized antibody to identify HBsAg solutions at different concentrations. Finally, specific and non-specific responses were investigated using hepatitis B (HBsAg) and hepatitis C (HCV Ag) antigens, respectively. RESULTS: Antibodies against HBsAg were successfully immobilized on 10 MHz AT-cut quartz crystal. The stability tests of crystal immobilized with antibody and unimmobilized crystal revealed that both forms of crystals were stable. Theoretical and experimental frequency assays were compared. A decrease in the contact angle indicated the hydrophilicity of surface after modifications. AFM images illustrated a more uniform surface after antibody adsorption and the surface roughness (RMS) reduced from 1.13 to 0.99 nm. Changes in the frequency were detected after the physical adsorption of HBsAb on the designed chip. The standard curve of antigen revealed the frequency changes depend on concentration of antigen. Finally, the specificity test confirmed the specificity of the designed biosensor for the detection of HBsAg from HCV Ag. The quantization of immobilized antibody was characterized by the frequency shift of the QCM. The obtained results were compared with ELISA assay. The correlation coefficients of HBsAg dilution between QCM and ELISA was 0.9821. CONCLUSIONS: This study is a new step to meet the challenges regarding HBsAg detection. Physical adsorption used in this study was effective as the simplest immobilization method to design a QCM-based immunosensor for HBsAg detection. Facilitated, fast, and simple detection of HBsAg by an antibody-based QCM biosensor is our main objective.

Changes in the Distribution Pattern of Hepatitis C Virus Genotypes Among Chronic Hepatitis Patients: a Cross-Sectional Study.

BACKGROUND: Hepatitis C virus (HCV) is a major cause of liver diseases. It has been determined that HCV genotypes have a distinct geographical distribution, clinical outcome, and response to antiviral therapy. Over the past years, many studies have reported that HCV genotype 1a is the dominant genotype in Ahvaz city. In recent years, changes in the distribution pattern of HCV genotypes of different geographical regions have attracted a great deal of attention; hence, the aim of this study was to accurately evaluate such probable changes in Ahvaz in southwestern Iran. METHODS: This is a cross-sectional study conducted from September 2017 to August 2020, including 262 patients suffering from chronic hepatitis C. HCV-RNA was extracted, and HCV genotyping was performed by PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method. To evaluate the association between HCV genotype, age, gender, and viral load, statistical analyses were done by SPSS software. RESULTS: HCV genotyping was done on 260 patients where genotype 3a had the highest prevalence over the period of 4 years with an average of 48.1%, followed by genotype 1a (46.5%). HCV genotype of two patients was not typeable. Although the difference between the two genotypes is currently small, the main result was finding an increasing trend in the prevalence of genotype 3a in recent years. CONCLUSIONS: The present study showed that the distribution pattern of HCV genotypes is gradually changing among chronic hepatitis C patients in Ahvaz city. The most important cause of such changes could be the alteration in HCV transmission routes and the increase in migration from areas where genotype 3 is dominant.

Full-length Sequencing and Genotyping of Hepatitis A Virus among Acute Hepatic Patients in Iran.

BACKGROUND: Given the lack of access to a full-length sequence of hepatitis A virus (HAV) and scarce information about its circulating genotype, sub-type and strain in Iran, two specimens were isolated from two patients with clinical symptoms of acute HAV to determine the full-length sequence of HAV. Following the phylogenetic and molecular study, we determined HAV genotype, sub-genotype, and strain of circulating virus in Iran. METHODS: According to real-time PCR results, 16 pairs of overlapped specific primers were used to determine the full-length sequence of HAV by whole-genome amplification (WGA) and using the Sanger method. Moreover, the results were assessed using Chromas, CLC Genomics Workbench, Mega 6, and RDP software. RESULTS: The full-length genome of HAV was amplified and sequenced with a length of 7,182 nucleotides. According to the obtained sequences, the phylogenetic tree of the mentioned viruses was drawn using MEGA 6 software and 44 full-genome viruses registered in the GenBank worldwide. Afterwards, the same process was repeated based on the protein sequence of VP1-P2A fragment in Iranian samples along with the other 22 registered protein sequences of GenBank to confirm the results of the full-genome phylogenetic tree. CONCLUSIONS: In this study, complete sequencing of two HAV specimens was carried out using the overlapping amplification and Sanger methods. According to the results of the phylogenetic tree, the circulating HAV in Iran had Genotype I and sub-genotype B and strain HM-175. In the present study, the full sequences of HAV of the two specimens were registered with accession numbers of BankIt 2277890/MN746031 and BankIt 2287607/MN746032.

The Effects of Human Reoviruses on Cancer Cells Derived from Hepatocellular Carcinoma Biopsies.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common type of liver cancer around the world. Since this cancer is highly resistant to the existing treatments, we used a novel method, which selectively targets HCC cancer cells to improve the treatment process. As normal cells are resistant to reovirus replication, we used oncolytic reoviruses, which can infect, replicate in, and destroy cancer cells. In this study, the effects of oncolytic human reoviruses on cancer cells, derived from HCC biopsies, were investigated. METHODS: First, reoviruses were purified. Then a plaque assay was performed to estimate the number of viruses and determine the multiplicity of infection (MOI). To evaluate the effects of reoviruses on cancer cells derived from HCC biopsies, replication of reovirus RNA, viral protein production, cytopathic effects (CPE), and cancer cell viability were assessed at different intervals post-infection. RESULTS: Replication of reovirus RNA and viral protein production were detected in cancer cells. Also, different levels of viral protein production, CPE, cytotoxicity, and cancer cell viability were observed at different intervals post-infection with human reoviruses. In contrast, normal human fibroblasts, which were used as negative control, remained unchanged. CONCLUSIONS: For the first time, the effects of human reoviruses on HCC biopsies were investigated. The results showed that human reoviruses could replicate in and destroy cancer cells derived from HCC biopsies. Overall, human reoviruses can be potentially used for the treatment of HCC.

HIV-1 Vpr reactivates latent HIV-1 provirus by inducing depletion of class I HDACs on chromatin.

HIV-1 Vpr is an accessory protein that induces proteasomal degradation of multiple proteins. We recently showed that Vpr targets class I HDACs on chromatin for proteasomal degradation. Here we show that Vpr induces degradation of HDAC1 and HDAC3 in HIV-1 latently infected J-Lat cells. Degradation of HDAC1 and HDAC3 was also observed on the HIV-1 LTR and as a result, markers of active transcription were recruited to the viral promoter and induced viral activation. Knockdown of HDAC1 and HDAC3 activated the latent HIV-1 provirus and complementation with HDAC3 inhibited Vpr-induced HIV-1 reactivation. Viral reactivation and degradation of HDAC1 and HDAC3 was conserved among Vpr proteins of HV-1 group M. Serum Vpr isolated from patients or the release of virion-incorporated Vpr from viral lysates also activated HIV-1 in latently infected cell lines and PBMCs from HIV-1 infected patients. Our results indicate that Vpr counteracts HIV-1 latency by inducing proteasomal degradation of HDAC1 and 3 leading to reactivation of the viral promoter.

HIV-1 Vpr Protein Induces Proteasomal Degradation of Chromatin-associated Class I HDACs to Overcome Latent Infection of Macrophages.

Mechanisms underlying HIV-1 latency remain among the most crucial questions that need to be answered to adopt strategies for purging the latent viral reservoirs. Here we show that HIV-1 accessory protein Vpr induces depletion of class I HDACs, including HDAC1, 2, 3, and 8, to overcome latency in macrophages. We found that Vpr binds and depletes chromatin-associated class I HDACs through a VprBP-dependent mechanism, with HDAC3 as the most affected class I HDAC. De novo expression of Vpr in infected macrophages induced depletion of HDAC1 and 3 on the HIV-1 LTR that was associated with hyperacetylation of histones on the HIV-1 LTR. As a result of hyperacetylation of histones on HIV-1 promotor, the virus established an active promotor and this contributed to the acute infection of macrophages. Collectively, HIV-1 Vpr down-regulates class I HDACs on chromatin to counteract latent infections of macrophages.

Prevalence and Distribution of BK virus Subtypes in Renal Transplant Recipients Referred to Golestan Hospital in Ahvaz, Iran.

BACKGROUND: BK virus (BKV) belongs to the human Polyomaviridae and the primary BKV infection is occurred during childhood then the virus could be latent through life, especially in the kidneys and urinary system. It became reactive after an immunocompromised status, such as pregnancy or transplantation. Isolated BKV from different locations of the world is grouped into four subtypes using serological and genotyping methods. The BKV subtype I is the dominant one and has worldwide distribution. OBJECTIVES: According to our knowledge, there are no data about the BKV prevalence and its genotypes in southwest part of Iran. Considering the high prevalence of renal failure and kidney transplant patients in this part, and the role of BKV in graft rejection, this study aimed to determine the prevalence of BKV infection in renal transplant recipients referred to Golestan Hospital in Ahvaz City, Iran. PATIENTS AND METHODS: Urine samples were collected from 122 kidney transplant recipients referred to Golestan Hospital in Ahvaz, southwest of Iran. The extracted DNA was amplified by Polymerase Chain Reaction, and subtype of each positive sample was determined using Restriction Fragment Length Polymorphism (RFLP) and sequencing methods. RESULTS: From all study population, 51/122 (41.8%) urine samples were positive for BKV DNA and the other samples were negative (71/122). Forty-eight cases (94.11%) were subtype I and 3 others (5.89%) were subtype IV using the RFLP method. None of the patient's urine samples were positive for subtypes II and III. CONCLUSIONS: Our work is the second study in Iran and considering huge numbers of transplantation in Iran and Khuzestan Province, south western of Iran, in addition to the role of this virus in kidney transplant rejection, routine evaluation of BKV positivity is recommended both for graft recipient and donors. This helps better transplantation result and may prevent graft rejection.