Distribution of group a Rotavirus circulating in Mashhad, Iran.

Background and Objectives: Group A Rotavirus (RVA) is the most important causative agent of acute diarrheal disease in pediatrics 5 years and below. This study aimed to determine the distribution of circulating RVA in Mashhad, Iran to develop health improvement strategies and vaccine decision making. Materials and Methods: A total of 106 fecal specimens were collected from children admitted to Akbar and Dr. Sheikh referral pediatric hospitals of Mashhad City during the December 2020 to March 2021 and December 2021 to March 2022. All specimens were tested for specific bacterial, parasitic, and amoebic infections. Negative samples were analyzed for RVA infections using the RT-PCR method. Results: RVA was detected in 31.3% of the specimens, indicating no statistical significance in gender distribution or between fall and winter positivity rates. The number of RVA-positive specimens increased following age increasing in the range of 1 to 60 months. Conclusion: Today, acute diarrheal disease (ADD) is still caused mostly by Rotavirus infections in pediatrics in Mashhad. Comprehensive studies are needed to determine the genetic diversity of circulating Rotavirus strains in this era.

Toll-like receptor 7 and RIG-I-like receptors expression in peripheral blood mononuclear cells of naïve patients with hepatitis C.

BACKGROUND: The proper function of Pattern Recognition Receptors (PRRs) as a part of the host immune system can eliminate numerous pathogens from the body. However, some viruses can manipulate PRRs to escape the innate immune system. As there is controversy in the activation of PRRs in patients infected with HCV, we decided to evaluate the gene expression changes of PRRs in HCV cases compared to the healthy control. METHODS: In this study, the relative expression of Toll-like receptor 7, RIG-I, and MAD-5 in peripheral mononuclear blood cells of twenty HCV patients and twenty healthy controls of the same gender and age were analyzed by quantitative Real-time PCR. RESULTS: Our results showed that the expression of RIG-I and MAD-5 significantly increased in HCV-infected samples compared to the controls (P value:0.01; P value:0.05), while the expression of TLR7 was similar between the case and the control group (P value:0.1). CONCLUSION: It seems in suppressing HCV, RIG-I and MAD-5 receptors are likely to be more activated than TRL7 in HCV patients. The lack of TLR7 gene expression might reflect the defect of the host in the stimulation of the innate immune system through the TLR7 pathway.

Diagnostic genosensor for detection of rotavirus based on HFGNs/MXene/PPY signal amplification.

A novel genosensor was developed for rotavirus specific cDNA sequence detection. The genosensor was comprised of hierarchical flower-like gold nanostructures, MXene, and polypyrrole (HFGNs/MXene/PPY) nanocomposite as a signal amplification tag, specific antisense ssDNA oligonucleotide as a recognition bioelement, and methylene blue (MB) as a redox marker. The morphological and electrochemical features of the biosensor were first tested and optimized and the high performance of the platform was confirmed in terms of sensitivity and reproducibility. Then, 20 rotavirus RNA isolated from clinical and cell-cultured samples (10 positive and 10 negative confirmed by RT-PCR and electrophoresis methods) were evaluated by the genosensor. The analysis results revealed that the genosensor is able to differentiate successfully between the positive and negative control groups. The developed genosensor for rotavirus RNA detection presented an excellent limit of detection of ∼ 0.8 aM and a determination  range of  10-18 and 10-7 M. In addition, the ssDNA/HFGNs/MXene/PPY/GCE showed high selectivity and long-term stability of ~ 24 days. Therefore, this novel genosensor would be of great benefit for the clinical diagnosis of rotavirus.

The protection quest is a primary key to sharing the neutralizing antibody response to cover against all emerging VOCs based on BIV1-CovIran studies.

Over time, the antigenic evolution of emerging variants of SARS-CoV-2 has demanded the development of potential protective vaccines. Administration of additional doses of current vaccines based on the WT spike protein may boost immunity, but their effectiveness has dwindled for patients with more recent variants. Here, we studied the neutralization activity of post-WT strain-based vaccination and a structural simulation in-silico based on the interactions of the RBD-hACE2 as the key to initiating infection among the VOCs of SARS-CoV-2. Our data display shows that WT sera showed a markedly greater reduction in Delta and Omicron, suggesting that the Wuhan-based vaccines may be more susceptible to breakthrough and new VOCs. According to the MD simulation, mutations of Omicron result in a significant change in the variant charge distribution throughout the binding interface that consequently alters the critical interface electrostatic potential in comparison to other variants. This observation provides new insights into immunization policy and next-generation vaccine development.

Corrigendum to "Inhibition of HIV-1 replication using the CRISPR/noNLS-Cas9 system as a prophylactic strategy" <[Heliyon 8 (9) (2022) e10483]>.

[This corrects the article DOI: 10.1016/j.heliyon.2022.e10483.].

CRISPR/Cas9 mediated knocking out of OPN gene enhances radiosensitivity in MDA-MB-231 breast cancer cell line.

PURPOSE: Although chemotherapy and radiotherapy in conjunction with surgery have been known as the standard methods for patients with breast cancer, they frequently face resistance due to the failure of cells to death. Accordingly, improving the results requires discovering novel therapeutic approaches based on the changes in the molecular biology of cancer cells. Osteopontin (OPN) is a secreted protein that previous studies have shown to be associated with progression, poor prognosis, and metastasis in breast cancer. The current study examined the synergistic effects of radiotherapy and knocking out of OPN gene, utilizing CRISPR/Cas9 technique in MDA-MB-231 breast cancer cells. METHODS: We used to knock out the OPN gene by the two different gRNAs. The cells irradiated 24 h after transfection. The mRNA expression, tumor cell proliferation, cell cycle distribution, growth, and apoptosis were measured. Moreover, activation of Chk1 and AKT were measured via western blot. RESULTS: We demonstrated the OPN knocking out along with radiation led to the promotion of apoptosis, suppression of downstream genes, reduction of cell viability, and inhibition of cell-cycle progression. The western blot analysis has indicated that the knocking out of the OPN gene along with radiotherapy changes DNA damage responses substantially. CONCLUSIONS: The OPN gene knocking out with radiotherapy might be an efficient approach to overcome the radioresistance in breast cancer.

Significant alteration of IFN stimulated genes expression in MA104 cells infected with bovine rotavirus RF strain.

The pattern recognition receptors (PRRs) trigger signaling cascades, such as nuclear factor kappa B (NF-κB) and interferon regulatory factors (IRFs). Rotavirus (RV) countermeasures against innate responses and understanding of these processes will improve our knowledge regarding immunopathogenesis of RV infection. In this study, we investigated the effect of RV RF strain on the important ISG candidate genes engaging in virus infections for which little information is known in RV RF strain. To this end, MA104 cells were mock/infected with RF followed by incubation in the presence or absence of IFN-α and the expression of MX1, OAS1, STAT1, ISG15, and ISG56 mRNA was analyzed by real-time PCR. All of ISGs' mRNAs showed higher expression levels in IFN I treated cells compared to virus-infected cells except for ISG56. Infecting the cells with RV and treatment with IFN type I led to overexpression of ISG56 compared to cells were either infected with the virus or only treated with IFN I. In conclusion, we showed that the RV RF strain efficiently blocks type I IFN-induced gene expression particularly ISG15, MX1, STAT, and OSA1 as antiviral proteins. Furthermore, viruses may use some ISGs such as ISG 56 to regulate IFN I signaling pathway, negatively.

Inhibition of HIV-1 replication using the CRISPR/cas9-no NLS system as a prophylactic strategy.

Globally, it is estimated that 43 million people are living with human immunodeficiency virus type 1 (HIV-1), and there are more than 600,000 acquired immunodeficiency syndrome (AIDS)-related deaths in 2020. The only way to increase the life expectancy of these patients right now is to use combination antiretroviral therapy (cART) for the lifetime. Due to the integration of the HIV-1 DNA in lymphocytes, the replication of the virus can only be reduced by using antiretroviral drugs. If the drug is stopped, the virus will replicate and reduce the number of lymphocytes. In recent years, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9-mediated genome editing system has been considered, preventing HIV-1 replication by causing DNA double-stranded breaks (DSBs) or disrupting the integrated virus replication by targeting the provirus. In this study, we utilized the CRISPR/Cas9 without the nuclear localization signal sequence (w/o NLS) system to inhibit the VSV-G-pseudotyped HIV-1 replication by targeting the HIV-1 DNA as a prophylactic method. To this end, we designed a multiplex gRNA (guide RNA) cassette to target the pol, env, and nef/long terminal repeat (nef/LTR) regions of the HIV-1 genome and then cloned it in plasmid expressing no-NLS-Cas9 protein as an all-in-one CRISPR/Cas9 vector. Using HIV-1 pseudovirus transduction into HEK-293T cell lines, our results showed that the CRISPR/Cas9-no NLS system disrupts the pseudotyped HIV-1 DNA and significantly (P-value < 0.0001) decreases the p24 antigen shedding and viral RNA load in cell culture supernatants harvested 48h after virus transduction. Although these results revealed the potential of the CRISPR/Cas9-no NLS nuclease system as a prophylactic strategy against HIV-1 infections, due to inefficient impairments of HIV-1 DNA, further studies are required to enhance its effectiveness and application in clinical practice.

Curcumin inhibits the replication of rotavirus in vitro.

Rotavirus is the most important etiological agent of infectious diarrhea in children under 5 years of age with more than 125,000 deaths occurring annually worldwide. The present study aims to determine the effect of curcumin, a natural polyphenol compound, on rotavirus in a cell culture model. The anti-viral activity of curcumin was evaluated by reverse-transcriptase quantitative PCR (RT-qPCR), TCID50, and western blot techniques to assess CC50 in curcumin-treated MA104 cells as well as EC50 and SI within the infected MA104 cell line. Our findings supported that curcumin exerted an inhibitory influence against rotavirus in a dose-dependent manner and decreased the viral titer and VP6 expression by ~99% at a concentration of 30 µM (p Keywords: curcumin; rotavirus; RT-qPCR; in vitro; anti-rotavirus agent.

Frequency of Hepatitis B Markers in Systemic Lupus Erythematosus Patients in Iran.

BACKGROUND: Systemic lupus erythematosus (SLE) is a chronic autoimmune disease. Hepatitis B virus is the causative agent for chronic, acute, cirrhosis, and hepatocellular carcinoma.  SLE patients with chronic or occult hepatitis B infection undergoing immunosuppressive drugs may become reactive and develop fatal hepatitis. Therefore, this study was conducted to determine HBV markers in SLE patients before the administration of immunosuppressive drugs in Ahvaz city, Iran. MATERIALS AND METHODS: The sera of 92 SLE patients were  tested for HBs Ag and anti-HBc using ELISA, HBV DNA (by Nested PCR) testes. Real-time PCR was performed for the patients with positive anti-HBc and negative HBsAg. The positive HBV DNA samples were checked for HBV genotype and HBV subtypes. RESULTS: Among the 92 SLE patients, three (3.3%) were males and 89 (96.7%) females . The patients' ages ranged from 14 to 70 years [mean age of 38.9±10.1]. Three of 92 (3.26%) subjects [2/3 males and 1/89 female] were positive for HBsAg, anti-HBc Ab, and HBV DNA detected with PCR (p=0.000003)].  Five of 89 (5.61%) subjects [1 male and 4/88 females were only positive for anti-HBc and negative for HBs Ag, HBV DNA(PCR) using Real-time PCR (p=0.05).  The results of the nucleotide data and phylogenetic tree showed all three HBV patients were genotype D1. The results of amino acid sequencing revealed all three HBV patients were HBV subtype ayw2. CONCLUSION: This study proved that 3.26% of SLE patients were positive for overt HBV infection (positive for anti-HBc, HBsAg and HBV-DNA using PCR). All the three isolated HBV were genotype D1 and subtype ayw2. The fact that 5.61% of  the patients were only positive for anti-HBc characterized the occult hepatitis B infection (OBI) although further investigation is needed. To prevent HBV or OBI reactivation for SLE patients before immunosuppression treatment, HBV markers including anti-HBc, HBsAg, HBV-DNA should be implemented using PCR and Real-time PCR .

The role of microbiota in colorectal cancer.

Cancer is one of the most important causes of death throughout the world, and the mortality rate is increasing significantly due to the aging of the population. One of the most common types of cancer is colorectal cancer (CRC). Human microbial ecosystems use metabolism to make important impacts on the body physiology. An intensive literature review was made to investigate the correlations between human gut microbiota and the incidence of CRC. The results of these studies show that there are differences in the composition of microbiota between CRC patients and normal people and the microorganisms in CRC patients are very different from healthy individuals. Therefore, changes in the microbiome can be used as a biomarker for the early detection of CRC. On the other hand, the intestinal flora is may be act as a powerful weapon against CRC in the future.

Principles and Applications of CRISPR Toolkit in Virus Manipulation, Diagnosis, and Virus-Host Interactions.

Viruses are one of the most important concerns for human health, and overcoming viral infections is a worldwide challenge. However, researchers have been trying to manipulate viral genomes to overcome various disorders, including cancer, for vaccine development purposes. CRISPR (clustered regularly interspaced short palindromic repeats) is becoming one of the most functional and widely used tools for RNA and DNA manipulation in multiple organisms. This approach has provided an unprecedented opportunity for creating simple, inexpensive, specific, targeted, accurate, and practical manipulations of viruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human immunodeficiency virus-1 (HIV-1), and vaccinia virus. Furthermore, this method can be used to make an effective and precise diagnosis of viral infections. Nevertheless, a valid and scientifically designed CRISPR system is critical to make more effective and accurate changes in viruses. In this review, we have focused on the best and the most effective ways to design sgRNA, gene knock-in(s), and gene knock-out(s) for virus-targeted manipulation. Furthermore, we have emphasized the application of CRISPR technology in virus diagnosis and in finding significant genes involved in virus-host interactions.

Is Immuno-PCR Better than ELISA Test for Detection of Toxoplasma gondii IgG Antibody?

INTRODUCTION: IgG antibodies against T. gondii persist for years, and can act as a reliable serological biomarker for the diagnosis of previous exposure to this parasite. Hence, the current investigation was designed to compare diagnostic power of immuno-polymerase chain reaction (iPCR) and enzyme-linked immunosorbent assay (ELISA) methods for detection of T. gondii IgG antibody. METHODS: Immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies against T. gondii were measured by the ELISA method in 81 participants. In addition, detection of acute and chronic toxoplasmosis was performed via the ELISA IgG avidity. The set-up of iPCR was carried out and then, serum IgG of subjects were detected using the iPCR method. RESULTS: Of 81 samples, 4 (4.9%) and 30 (37%) cases were be found positive for IgM and IgG against T. gondii in the ELISA method, respectively. Moreover, of 81 specimens, 42 (51.9%) and 39 (48.1%) samples had low-avidity IgG and high-avidity IgG by the IgG avidity kit, respectively. While, 59 (72.8%) of 81 samples were detected positive using the iPCR technique. Kappa (κ) value coefficient, between the iPCR and ELISA (for IgG) showed a strong agreement (0.360, p value < 0.001). A value of 0.25 I.U./ml for serum IgG [area under curve (AUC) = 0.720 (CI = 0.613-0.827); p = 0.002] was the cut-off value to differentiating between positive and negative toxoplasmosis (with sensitivity 66.0% and specificity 60.0%). CONCLUSION: Our findings indicated despite a strong agreement shown between iPCR and ELISA methods, the diagnostic power of iPCR technique was more sensitive than ELISA test for detection of T. gondii IgG antibody. However, more complementary investigations are widely needed in this regard.

Evaluation of SARS-CoV-2 in Indoor Air of Sina and Shahid Beheshti Hospitals and Patients' Houses.

Side by side air sampling was conducted using a PTFE filter membrane as dry sampler and an impinger containing a suitable culture medium as a wet sampler. Most of the samples were collected from two hospitals and few air samples were collected from private houses of non-hospitalized confirmed COVID-19 patients. The collected air samples were analyzed using RT-PCR. The results indicated that all air samples collected from the hospitals were PCR negative for SARS-CoV-2. While two of four air samples collected from the house of non-hospitalized patients were PCR positive. In this study, most of the hospitalized patients had oxygen mask and face mask, and hence this may be a reason for our negative results regarding the presence of SARS-CoV-2 in indoor air of the hospitals, while non-hospitalized patients did not wear oxygen and protective face masks in their houses. Moreover, a very high concentration of particles in the size range of droplet nuclei (< 5 µm) was identified compared to particles in the size range of respiratory droplets (> 5-10 µm) in the areas where patients were hospitalized. It can be concluded that using face mask by patients can prevent the release of viruses into the indoor air, even in hospitals with a high density of patients.

The evaluation of the overexpression of the ERG-11, MDR-1, CDR-1, and CDR-2 genes in fluconazole-resistant Candida albicans isolated from Ahvazian cancer patients with oral candidiasis.

INTRODUCTION: Resistance to azole drugs has been observed in candidiasis due to their long-term use and poor response to treatment. Resistance to azole drugs in Candida albicans isolates is controlled by several genes including ERG11, CDR1, CDR2, and MDR1. In this study, the expression of the mentioned genes was evaluated in C. albicans isolates susceptible and resistant to fluconazole. METHODS: After identifying the Candida isolates using morphological and molecular methods, the minimum inhibitory concentration (MIC) and drug susceptibility were determined using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) method. RNA was then extracted and cDNA was synthesized from 24 C. albicans isolates from patients with cancer. Then, the mean expressions of these genes were compared in two groups using real-time polymerase chain reaction (RT-PCR). RESULTS: A total of 74 Candida isolates were obtained from the oral cavity of 61 cancer patients with oral candidiasis. After 24 h, 21.6% of the isolates were fluconazole-resistant, 10.8% were identified as dose-dependent, and the rest of the isolates (67.6%) were fluconazole-sensitive. The mean expressions of the CDR1 and MDR1 genes were significantly higher in the resistant isolates than in the sensitive ones. However, the ERG11 and CDR2 genes were not significantly increased in the resistant isolates. CONCLUSION: The increased mean expressions of the CDR1 and MDR1 genes had a greater effect on fluconazole resistance among the drug-resistant strains of C. albicans in chemotherapy patients. It seemed that the accumulation of chemotherapeutic drugs in this organism stimulated some regulatory factors and increased the expression of these two genes and ultimately helped to further increase their expression and resistance to fluconazole.

Household Secondary Attack Rate of COVID-19 among Healthcare Workers and Related Factors.

Background: The risk for transmission of COVID-19 to people in close contact with infected people, especially healthcare workers, has not been well estimated. Therefore the present study was conducted to assess the household secondary attack rate (SAR) of COVID-19 among healthcare workers and related factors. Materials and Methods: The present prospective case-ascertained study was conducted on 202 healthcare workers with confirmed COVID-19 in Hamadan, diagnosed from March 1, 2020, to August 20, 2020. For households with close contact with the index case, RT-PCR was performed regardless of symptoms. We defined SAR as the proportion of secondary cases from the total contacts that live in the index case household. SAR was reported as a percentage and 95% confidence interval (CI). Multiple logistic regression was performed to explore the predictors of COVID-19 transmission of index cases to their households. Results: We found 36 secondary cases out of 391 household contacts with laboratory confirmation (RT-PCR), representing a household SAR of 9.2% (95% CI: 6.3, 12.1). Among factors related to the family member, female gender (OR: 2.9, 95% CI: 1.2, 6.9), being the patient's spouse (OR: 2.2, 95% CI: 1.0, 4.6), and living in the apartment (OR: 2.78, 95% CI: 1.24, 6.23), and among factors related to index cases, hospitalization (OR: 5.9, 95% CI: 1.3, 26.9) and caught (OR: 2.4, 95% CI: 1.1, 5.2) were the significant predictors of disease transmission to other family members (P<0.05). Conclusion: The findings of this study suggest that the SAR is remarkable in household contacts of infected healthcare workers. Some characteristics of family members of the index case, including female gender, being the patient's spouse, and living in the apartment, and some characteristics of the index case, including hospitalization and caught, were associated with the increased SAR.

Dual role of microbiota-derived short-chain fatty acids on host and pathogen.

A growing body of documents shows microbiota produce metabolites such as short-chain fatty acids (SCFAs) as crucial executors of diet-based microbial influence the host and bacterial pathogens. The production of SCFAs depends on the metabolic activity of intestinal microflora and is also affected by dietary changes. SCFAs play important roles in maintaining colonic health as an energy source, as a regulator of gene expression and cell differentiation, and as an anti-inflammatory agent. Additionally, the regulated expression of virulence genes is critical for successful infection by an intestinal pathogen. Bacteria rely on sensing environmental signals to find preferable niches and reach the infectious state. This review will present data supporting the diverse functional roles of microbiota-derived butyrate, propionate, and acetate on host cellular activities such as immune modulation, energy metabolism, nervous system, inflammation, cellular differentiation, and anti-tumor effects, among others. On the other hand, we will discuss and summarize data about the role of these SCFAs on the virulence factor of bacterial pathogens. In this regard, receptors and signaling routes for SCFAs metabolites in host and pathogens will be introduced.

Enhanced High Mutation Rate and Natural Selection to Produce Attenuated Viral Vaccine with CRISPR Toolkit in RNA Viruses especially SARS-CoV-2.

The best and most effective way to combat pandemics is to use effective vaccines and live attenuated vaccines are among the most effective vaccines. However, one of the major problems is the length of time it takes to get the attenuated vaccines. Today, the CRISPR toolkit (Clustered Regularly Inerspaced Short Palindromic Repeats) has made it possible to make changes with high efficiency and speed. Using this toolkit to make point mutations on the RNA virus's genome in a coculture of permissive and nonpermissive cells and under controlled conditions can accelerate changes in the genome and accelerate natural selection to obtain live attenuated vaccines.

Safety and potency of BIV1-CovIran inactivated vaccine candidate for SARS-CoV-2: A preclinical study.

The development of effective and safe COVID-19 vaccines is a major move forward in our global effort to control the SARS-CoV-2 pandemic. The aims of this study were (1) to develop an inactivated whole-virus SARS-CoV-2 candidate vaccine named BIV1-CovIran and (2) to determine the safety and potency of BIV1-CovIran inactivated vaccine candidate against SARS-CoV-2. Infectious virus was isolated from nasopharyngeal swab specimen and propagated in Vero cells with clear cytopathic effects in a biosafety level-3 facility using the World Health Organization's laboratory biosafety guidance related to COVID-19. After characterisation of viral seed stocks, the virus working seed was scaled-up in Vero cells. After chemical inactivation and purification, it was formulated with alum adjuvant. Finally, different animal species were used to determine the toxicity and immunogenicity of the vaccine candidate. The study showed the safety profile in studied animals including guinea pig, rabbit, mice and monkeys. Immunisation at two different doses (3 or 5 µg per dose) elicited a high level of SARS-CoV-2 specific and neutralising antibodies in mice, rabbits and nonhuman primates. Rhesus macaques were immunised with the two-dose schedule of 5 or 3 µg of the BIV1-CovIran vaccine and showed highly efficient protection against 104 TCID50 of SARS-CoV-2 intratracheal challenge compared with the control group. These results highlight the BIV1-CovIran vaccine as a potential candidate to induce a strong and potent immune response that may be a promising and feasible vaccine to protect against SARS-CoV-2 infection.