RNase P ribozyme can be engineered to be a sequence-specific gene-targeting agent with promising application in both basic research and clinical settings. By using an in vitro selection system, we have previously generated RNase P ribozyme variants that have better catalytic activity in cleaving an mRNA sequence than the wild type ribozyme. In this study, one of the variants was used to target the mRNA encoding human cytomegalovirus (HCMV) essential transcription factor immediate-early protein 2 (IE2). The variant was able to cleave IE2 mRNA in vitro 50-fold better than the wild type ribozyme. A reduction of about 98% in IE2 expression and a reduction of 3500-fold in viral production was observed in HCMV-infected cells expressing the variant compared to a 75% reduction in IE2 expression and a 100-fold reduction in viral production in cells expressing the ribozyme derived from the wild type sequence. These results suggest that ribozyme variants that are selected to be highly active in vitro are also more effective in inhibiting the expression of their targets in cultured cells. Our study demonstrates that RNase P ribozyme variants are efficient in reducing HCMV gene expression and growth and are potentially useful for anti-viral therapeutic application.

Coronavirus Disease 2019 (COVID-19) caused by SARS-CoV-2 poses a significant threat to global public health. Early detection with reliable, fast, and simple assays is crucial to contain the spread of SARS-CoV-2. The real-time reverse transcription-polymerase chain reaction (RT-PCR) assay is currently the gold standard for SARS-CoV-2 detection; however, the reverse transcription loop-mediated isothermal amplification method (RT-LAMP) assay may allow for faster, simpler and cheaper screening of SARS-CoV-2. In this study, the triple-target RT-LAMP assay was first established to simultaneously detect three different target regions (ORF1ab, N and E genes) of SARS-CoV-2. The results revealed that the developed triplex RT-LAMP assay was able to detect down to 11 copies of SARS-CoV-2 RNA per 25 µL reaction, with greater sensitivity than singleplex or duplex RT-LAMP assays. Moreover, two different indicators, hydroxy naphthol blue (HNB) and cresol red, were studied in the colorimetric RT-LAMP assay; our results suggest that both indicators are suitable for RT-LAMP reactions with an obvious color change. In conclusion, our developed triplex colorimetric RT-LAMP assay may be useful for the screening of COVID-19 cases in limited-resource areas.

Background: COVID-19, caused by SARS-CoV-2, has spread worldwide and become a global pandemic. Objectives: Studies of the earliest events of the COVID-19 pandemic are critical in understanding how the pandemic started and providing insight into the spread of SARS-CoV-2 and its infection in humans. Methods: In this report, we studied the epidemiological characteristics of all 34 confirmed COVID-19 cases in Wuhu, China, from January 3 to March 19, 2020. Results: Our study indicated that cases in male patients (61.76%, 21/34) outnumbered those in female patients (38.24%, 13/34). Studies of the age distribution revealed that most COVID-19 patients were 15 to 59 years of age (26/34, 76%), while more than 14% (5/34) were ≥ 60 years old, and less than 9% (3/34) were ≤ 14 years old. Notably, 32 of the 34 confirmed cases were (a) people who had recently resided in or traveled to Wuhan or had close contact with Wuhan residents or visitors (22 cases); and (b) people who had close contact with these 22 confirmed COVID-19 patients (10 cases). Conclusions: This study revealed the epidemiological characteristics of COVID-19 outbreaks in Wuhu between January and March 2020 and provided insight into the earliest events of the COVID-19 pandemic out of Wuhan in China. Our analyses suggested that the COVID-19 cases confirmed in Wuhu from January to March 2020 were directly related to or originated from the outbreaks in Wuhan.

Background/Aims: This research explores the effectiveness of a new cytotoxic T-cell epitope peptide specific for HLA-A2402 in enhancing cellular immune responses to SARS-CoV-2 infections. HLA molecules play a key role in presenting antigenic epitopes to T cells, with genetic polymorphisms resulting in varying immune responses among individuals. The study aimed to investigate whether loading this epitope peptide into dendritic cells (DCs) from HLA-A2402(+) individuals could improve immune responses. Methods: DCs were sensitized with varying doses of the peptide (2–12 µg/mL), with optimal results observed at 8 µg/mL. T-cell responses, proliferation, differentiation, Th cytokine secretion, CTL function, and apoptotic response were compared among the HLA-A2402(+), HLA-A2402(-), PBS control, DC only, and epitope-only groups. Results: A significant enhancement in DC maturation, antigen presentation, T-cell activation, and proliferation was observed in the HLA-A2402(+) group compared to the HLA-A2402(-) control. Conclusion: These findings suggest that HLA-A2402-restricted epitope peptides can enhance cellular immunity, offering potential for improving allele-specific SARS-CoV-2 vaccines and other molecular therapies, advancing precision medicine for infectious diseases.

Background: More and more variants of concern (VOCs) of SARS-CoV-2 continued to emerge in different regions worldwide. Objectives: Studies on COVID-19 outbreaks caused by different VOCs are critical for understanding their infections in humans in different regions. Methods: In this descriptive study, the individual information of 64 confirmed COVID-19 cases reported between March 3 and April 26, 2022, was collected, and the epidemiological features were analyzed. Results: We found COVID-19 epidemic caused by Omicron subvariant BA.2 in 2022 had unique characteristics compared to the earliest COVID-19 outbreaks in 2020 in Wuhu city. First, the geographic distribution of COVID-19 cases was highly concentrated, and most infections (58/64, 90.62%) were identified in one district in this study. Second, the mean age of COVID-19 cases in this study was 39.57 ± 16.39 years old, which was younger than the COVID-19 cases identified in Wuhan and Wuhu cities in 2020. Third, the spanned period of the COVID-19 outbreak in this study was shorter than the COVID-19 outbreak in 2020 in Wuhu city (13 days vs. 34 days). Conclusions: This study illustrates the epidemic of COVID-19 in Wuhu city from March to April 2022, and our result provides valuable information about COVID-19 outbreak caused by omicron subvariant BA.2. It also provides insights into developing effective strategies, such as high coverage of COVID-19 vaccination, lockdown policies, Ankang code, and population-bases COVID-19 screening, for successfully controlling the potential outbreaks caused by Omicron or future novel variants of SARS-CoV-2 in the medium-sized city like Wuhu.

Background: Seasonal influenza A virus (IAV) continues to pose a substantial global health threat because of its high mutation rate, widespread prevalence, and the ongoing challenge of timely detection, particularly in resource-limited settings. Objectives: To address this gap, we developed and analytically evaluated a novel colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid, sensitive, and visually interpretable detection of seasonal IAV. Methods: Ten reported LAMP primer sets previously reported were initially screened for amplification efficiency using certified reference RNA materials from H1N1 and H3N2 as templates. Analytical sensitivity was assessed using serial dilutions of quantified RNA standards, and specificity was rigorously evaluated through both in silico analysis and experimental testing with a panel of common respiratory pathogens. Results: Sensitivity analyses based on triplicate serial dilution testing revealed preliminary limits of detection as low as 67 copies per reaction for H1N1 and 42 copies per reaction for H3N2, with performance matching or exceeding that reported in previous LAMP-based studies. No cross-reactivity with other respiratory pathogens was observed, confirming the high specificity of the assay. The assay targets the conserved matrix protein gene and detects IAV broadly; however, it does not differentiate between the H1N1 and H3N2 subtypes in a single reaction. Notably, incorporation of hydroxy naphthol blue (HNB) enabled direct visual detection via a distinct color change from violet to sky blue within 45 minutes under isothermal conditions, thereby eliminating the need for advanced instrumentation. Conclusions: The assay demonstrated high sensitivity and specificity, supporting future clinical validation studies to establish its utility for point-of-care testing (POCT) deployment.