Genomic analysis of SARS-CoV-2 variants: diagnosis and vaccination challenges.

SARS-CoV-2 put a heavy financial burden on the healthcare system, with millions of laboratory-confirmed cases and deaths worldwide in the last 2 years. During the seventh wave of this pandemic, the continuously evolving nature of SARS-CoV-2 resulted in the emergence of new variants that harbor different mutations. Mutations are associated with changes in the virus behavior, including increased transmissibility, increased virulence, and evasion of neutralizing antibodies. Currently, we need detailed and comprehensive genomic information on all SARS-CoV-2 variants. One of the key points in this study was the genome survey of mutation profiles across variants as a genomic data source, to determine the efficiency of RT-qPCR assays. We also used the source to calculate the binding affinity changes of neutralizing antibodies-mutant receptor binding domain (RBD) complexes and determine vaccine efficacy. Our result revealed that the number of nucleotide mismatches is variable in the WHO-recommended primer-probe sets. Mismatches located at the 3' ends of the oligonucleotide, may lead to false-negative results. Only the primer-probe sets designed by the Ministry of Public Health of Thailand were exclusive and cannot detect the omicron variant reliably. Binding affinity changes showed that E484K was more deleterious than other mutations and decreased stability between the mutant RBD protein and neutralizing antibodies. The Omicrons show the highest change in binding affinity which may lead to immune escape and increase transmissibility. Additionally, the 7D6 monoclonal antibody in the 7eam complex could neutralize all variants of SARS-CoV-2. We strongly recommend creating and improving a matrix accuracy by processing a large number of SARS-CoV-2 sequences to update RT-qPCR assays and identified immunogenic residues among conserved RBD. Also, a detail computational analysis is needed to investigate distinctive amino acid substitution patterns which may be foundational in the vaccines. Finally, designing in-vitro studies can help confirm the present study and manage COVID-19 patients.Communicated by Ramaswamy H. Sarma.

Research Note: Effect of corn silage and alfalfa meal as alternative induced molt methods to improving Salmonella Enteritidis resistance in laying hens.

This experiment was conducted to evaluate diets containing a high level of corn silage and alfalfa meal in inducing molt and reducing susceptibility to Salmonella Enteritidis (SE) colonization in laying hens. Thirty-two healthy hens were examined by cloacal swab samples to be free of Salmonella. Then they were weighed individually and distributed to 4 experimental groups containing 8 hens each, including Full-fed (control, FF); total feed withdrawal (positive control for molt induction, FW); 80% corn silage (CS) + 20% layer diet (CS80), and 80% alfalfa meal (AM) + 20% layer diet (AM80). The molting program was initiated at 71 wk of age. On d 4 of the experiment, all hens were inoculated with SE by oral gavage. All hens were first weighed at the ending molting period on d 10 and then euthanized by CO2 gas. The internal organs including the ovary, oviduct, liver, and spleen, were excised aseptically and weighed. Cloacal swab and feed samples at the beginning and organ samples (liver, ovary, spleen, and cecum) were collected from each hen at the end of the experiment and examined for SE colonies. Molted birds lost roughly 14 to 27%t of their body weight and had significantly lower organ weight and egg production compared to FF group (P < 0.05). No significant difference was observed in the number of days to zero egg production between molted treatments. The SE positive organs did not significantly differ between CS80 and AM80 with FF treatment. Treatment CS80 had the lowest crop pH and differed substantially from treatment FW. In conclusion, results indicate that using corn silage and alfalfa meal, can improve resistance to salmonella Enteritidis during molt inducing compared to traditional feed withdrawal.

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.

Advanced sequence optimization for the high efficient yield of human group A rotavirus VP6 recombinant protein in Escherichia coli and its use as immunogen.

Rotavirus is the important etiological agents of infectious diarrhea among children under 5 years old. Rotaviruses are divided into 10 serogroups (A-J) and each group is based on genetic properties of major structural protein VP6. We designed a novel VP6 sequence optimization to increase the expression level of this protein. Numerous factors such as codon adaptation index, codon pair bias, and guanine-cytosine content were adapted based on Escherichiacoli codon usage. In addition, the ribosome binding site (RBS) of pET-15b was redesigned by the RBS calculator and the secondary structure of VP6 messenger RNA was optimized in the whole length of the coding sequence. Various factors including isopropyl beta- d-thiogalactoside (IPTG) concentration, temperature, and induction time were analyzed for the optimization of the best expression in E. coli by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting. The recombinant VP6 (rVP6) protein was purified by the Ni-sepharose and then the hyperimmune sera were generated against rVP6 in rabbits. Among three different temperatures, IPTG concentrations, and postinductions, the level of rVP6 was higher at 37°C, 1 mM of IPTG, and 8 h, respectively. Also, the high expression level of rVP6 was obtained in the insoluble aggregate form (43.8 g/L). After purification, the yield of rVP6 was 10.83 g/L. The rVP6 specific antiserum was confirmed by both immunofluorescent and western blotting. The versatile sequence optimization was the reason to produce a high level of rVP6 compared to other reports and can potentially apply to produce cheaper commercial kits to diagnose serological tests and new rotavirus vaccines.

Effect of sodium alginate coating incorporated with nisin, Cinnamomum zeylanicum, and rosemary essential oils on microbial quality of chicken meat and fate of Listeria monocytogenes during refrigeration.

The present study was conducted to preserve the microbial quality of chicken meat fillets during storage time by using sodium alginate active coating solutions incorporated with different natural antimicrobials including nisin, Cinnamomum zeylanicum (cinnamon), and rosemary essential oils (EOs) which were added individually and in combination. The samples were stored in refrigeration condition for 15days and were analyzed for total viable count, Enterobacteriaceae count, lactic acid bacteria count, Pseudomonas spp. count, psychrotrophic count, and yeast and mold count, as well as fate of inoculated Listeria monocytogenes at 3-day intervals. Results indicated that values of tested microbial indicators in all samples increased during storage. Antimicrobial agents, when used in combination, had stronger effect in preserving the microbial quality of chicken meat samples rather than their individual use and the strongest effect was observed in samples coated with alginate solution containing both cinnamon and rosemary EOs (CEO+REO). However, all treatments significantly inhibited microbial growth when compared to the control (P<0.05). Therefore, based on the results of this study, application of alginate coating solutions containing nisin, cinnamon, and rosemary EOs as natural preservatives is recommended in meat products especially in chicken meats.

Cloning and Functional Assessment of the Recombinant Human Hepcidin-25 in the Baculovirus Expression System

Hepcidin is the primary regulatory hormone responsible for lowering the iron content in the blood circulation. Due to its biodegradability and low cytotoxicity, hepcidin is considered as an alternative for iron chelators. The baculovirus expression system may be suitable for human hepcidin production because the expressed proteins generally exhibit proper folding, post-translational modifications, and oligomerization. Using data from two vector maps, pFastBac1 and pFastBac HTB, a unique vector was designed encoding human hepcidin-25 as fusion recombinant peptide. Expression analysis showed that it was expressed as a peptide with a molecular weight near to 5 kDa. After purification and TEV treatment, findings revealed that recombinant human hepcidin-25 was functional and its effect was dose dependent (P=0.001). It was concluded that baculovirus expression was a suitable expression system for production of functional recombinant human hepcidin-25.

Genistein induces a protective immunomodulatory effect in a mouse model of cervical cancer.

BACKGROUND: Genistein (GEN), a naturally occurring flavonoid present in soy bean, has attracted scientific interest for its possible benefits in cancer. OBJECTIVE: The potential immunomodulatory effects of genistein on the immune system and against TC-1 tumor cell line were evaluated in adult female C57BL/6 mice. METHODS: Mice were treated with GEN 10 days before to 10 days after the tumor induction. Thirty days after the last GEN treatment, lymphocyte proliferation, Lactase Dehydrogenase (LDH) cytolytic activity and cytokine secretion were analyzed in GEN and control groups. RESULTS: The results showed that ingestion of genistein significantly increased lymphocyte proliferation and LDH release. Furthermore, the treatment with genistein also caused a significant increment in interferon gamma (IFN-γ). In addition, the treatment achieved significant therapeutic effect in tumor models compared to the control group. These results indicated that the effect of GEN on tumor growth may be attributed to its effect on lymphocyte proliferation, cytolytic activity and IFN-γ production. CONCLUSION: These results demonstrate that GEN exerts an immunomodulatory effect in a mouse model of Human Papillomavirus (HPV) associated-cervical cancer.