Acyclovir-resistant HSV-1 isolates among immunocompromised patients in southern Taiwan: Low prevalence and novel mutations.

Herpes simplex virus type 1 (HSV-1) can establish latency in humans and easily relapse in immunocompromised patients, with significant mortality. Treatment with acyclovir (ACV) can result in the emergence of HSV resistance. A total of 440 frozen HSV-1 isolates collected from 318 patients from January 2014 to July 2019 were obtained from National Cheng Kung University Hospital in southern Taiwan. These 440 isolates were subjected to phenotypic studies for ACV-resistance by initial screening with the plaque reduction assay (PRA) and further validation by the DNA reduction assay (DRA). The ACV-resistant strains were further investigated by Sanger sequencing for the full-length UL23 and UL30 genes, which encode thymidine kinase and DNA polymerase, respectively. Hematological malignancies or hematopoietic stem-cell transplantation patients accounted for 56.9% (124/218) among the immunocompromised patients (218/318) in this study. Repeated sampling for HSV testing was 50% (109/218) in immunocompromised patients. Only 1.38% (3/218) of immunocompromised patients and 0.9% (3/318) of all patients developed ACV-resistant HSV-1 as measured by phenotypic screening assays. It is noteworthy that a novel Y248D mutation in the UL23 gene from an immunocompromised patient was found by both PRA and DRA. In 3D protein predicting analysis, uncharged Y248 was located at an alpha-helix and substituted by negative-charged D248, which may alter the function of viral thymidine kinase. Besides, three unreported mutations related to natural polymorphism were found in virus isolates from two immunocompetent patients, including 683-688 deletion, R227H, and A351D in the UL30 gene. These data show that the prevalence of ACV-resistant HSV-1 among immunocompromised patients in southern Taiwan is low. These results will be helpful for the clinical management and treatment of HSV infections.

Novel Software for High-level Virological Testing: Self-Designed Immersive Virtual Reality Training Approach.

BACKGROUND: To ensure the timely diagnosis of emerging infectious diseases, high-tech molecular biotechnology is often used to detect pathogens and has gradually become the gold standard for virological testing. However, beginners and students are often unable to practice their skills due to the higher costs associated with high-level virological testing, the increasing complexity of the equipment, and the limited number of specimens from patients. Therefore, a new training program is necessary to increase training and reduce the risk of test failure. OBJECTIVE: The aim of the study is to (1) develop and implement a virtual reality (VR) software for simulated and interactive high-level virological testing that can be applied in clinical practice and skills building or training settings and (2) evaluate the VR simulation's effectiveness on reaction, learning, and behavior of the students (trainees). METHODS: Viral nucleic acid tests on a BD MAX instrument were selected for our VR project because it is a high-tech automatic detection system. There was cooperation between teachers of medical technology and biomedical engineering. Medical technology teachers were responsible for designing the lesson plan, and the biomedical engineering personnel developed the VR software. We designed a novel VR teaching software to simulate cognitive learning via various procedure scenarios and interactive models. The VR software contains 2D VR "cognitive test and learning" lessons and 3D VR "practical skills training" lessons. We evaluated students' learning effectiveness pre- and posttraining and then recorded their behavior patterns when answering questions, performing repeated exercises, and engaging in clinical practice. RESULTS: The results showed that the use of the VR software met participants' needs and enhanced their interest in learning. The average posttraining scores of participants exposed to 2D and 3D VR training were significantly higher than participants who were exposed solely to traditional demonstration teaching (P<.001). Behavioral assessments of students pre- and posttraining showed that students exposed to VR-based training to acquire relevant knowledge of advanced virological testing exhibited significantly improved knowledge of specific items posttraining (P<.01). A higher participant score led to fewer attempts when responding to each item in a matching task. Thus, VR can enhance students' understanding of difficult topics. CONCLUSIONS: The VR program designed for this study can reduce the costs associated with virological testing training, thus, increasing their accessibility for students and beginners. It can also reduce the risk of viral infections particularly during disease outbreaks (eg, the COVID-19 pandemic) and also enhance students' learning motivation to strengthen their practical skills.

RNA Interference Approach Is a Good Strategy against SARS-CoV-2.

COVID-19, caused by SARS-CoV-2, created a devastating outbreak worldwide and consequently became a global health concern. However, no verifiable, specifically targeted treatment has been devised for COVID-19. Several emerging vaccines have been used, but protection has not been satisfactory. The complex genetic composition and high mutation frequency of SARS-CoV-2 have caused an uncertain vaccine response. Small interfering RNA (siRNA)-based therapy is an efficient strategy to control various infectious diseases employing post-transcriptional gene silencing through the silencing of target complementary mRNA. Here, we designed two highly effective shRNAs targeting the conserved region of RNA-dependent RNA polymerase (RdRP) and spike proteins capable of significant SARS-CoV-2 replication suppression. The efficacy of this approach suggested that the rapid development of an shRNA-based therapeutic strategy might prove to be highly effective in treating COVID-19. However, it needs further clinical trials.

Increasing Cytomegalovirus Detection Rate from Respiratory Tract Specimens by a New Laboratory-Developed Automated Molecular Diagnostic Test.

Lots of automated molecular methods for detecting cytomegalovirus (CMV) DNA in the blood are available, but seldom for various clinical specimens. This study was designed to establish a highly sensitive automated assay to detect CMV DNA in non-blood specimens. We designed a new QMT assay using QIAGEN artus CMV RG polymerase chain reaction (Q-CMV PCR) kit applied on the BD MAX system and compared with the other assays, including an RGQ assay (LabTurbo auto-extraction combined Q-CMV PCR kit on Rotor-Gene-Q instrument), and in-house PCR assay. A total of 1067 various clinical samples, including 426 plasma, 293 respiratory tract specimens (RTS), 127 stool, 101 cerebral spinal fluid, 90 vitreous humours were analysed. Examining CMV DNA in simultaneous specimens of the same immunocompromised patient with respiratory symptoms, the detection rate of RTS (93.6%, 88/94) was significant higher than plasma (65.9%, 62/94). The positive rates for plasma samples with a low CMV viral load (<137 IU/mL) and diagnostic sensitivity of QMT, RGQ, and in-house assays were 65% and 99.1%, 45% and 100%, 5% and 65.5%, respectively. The QMT assay performs better, with shorter operational and turnaround time than the other assays, enabling the effective and early detection of CMV infection in various clinical specimens, particularly for RTS.

The Examination of Viral Characteristics of HIV-1 CRF07_BC and Its Potential Interaction with Extracellular Galectin-3.

HIV-1 CRF07_BC is a B' and C subtype recombinant emerging virus and many of its viral characteristics remain unclear. Galectin-3 (Gal3) is a ß-galactose binding lectin that has been reported as a pattern recognition receptor (PRR) and is known to mediate adhesion between cells and microbes. This study aims to examine the viral characteristics of HIV-1 CRF07_BC virus and the role of extracellular galectin-3 in HIV-1 CRF07_BC infection. A total of 28 HIV-1+ injecting drug users (IDUs) were recruited and 24 (85.7%) were identified as HIV-1 CRF07_BC. Results indicate that significant higher serum galectin-3 was measured in CRF07_BC infected patients and CRF07_BC infection triggered significant galectin-3 expression (p < 0.01). Viral characteristics demonstrate that CRF07_BC virions display a higher level of envelope gp120 spikes. The virus infectivity assay demonstrated that co-treatment with galectin-3 significantly promoted CRF07_BC attachment and internalization (p < 0.01). A co-immunoprecipitation assay showed that pulldown galectin-3 co-precipitated both CD4 and gp120 proteins. Results from an enzyme-linked immunosorbent assay (ELISA) indicate that the galectin-3 promoting effect occurs through enhancement of the interaction between gp120 and CD4. This study suggests that CRF07_BC was predominant in HIV-1+ IDUs and CRF07_BC utilized extracellular galectin-3 to enhance its infectivity via stabilization of the gp120-CD4 interaction.

Amino Acid Deletions in p6Gag Domain of HIV-1 CRF07_BC Ameliorate Galectin-3 Mediated Enhancement in Viral Budding.

HIV-1 CRF07_BC is a recombinant virus with amino acid (a.a.) deletions in p6Gag, which are overlapped with the Alix-binding domain. Galectin-3 (Gal3), a ß-galactose binding lectin, has been reported to interact with Alix and regulate HIV-1 subtype B budding. This study aims to evaluate the role of Gal3 in HIV-1 CRF07_BC infection and the potential effect of a.a. deletions on Gal3-mediated regulation. A total of 38 HIV-1+ injecting drug users (IDUs) were enrolled in the study. Viral characterization and correlation of Gal3 were validated. CRF07_BC containing 7 a.a. deletions and wild-type in the p6Gag (CRF07_BC-7d and -wt) were isolated and infectious clones were generated. Viral growth kinetic and budding assays using Jurkat-CCR5/Jurkat-CCR5-Gal3 cells infected with CRF07_BC were performed. Results indicate that 69.4% (25/38) of the recruited patients were identified as CRF07_BC, and CRF07_BC-7d was predominant. Slow disease progression and significantly higher plasma Gal3 were noted in CRF07_BC patients (p < 0.01). Results revealed that CRF07_BC infection resulted in Gal3 expression, which was induced by Tat. Growth dynamic and budding assays indicated that Gal3 expression in Jurkat-CCR5 cells significantly enhanced CRF07_BC-wt replication and budding (p < 0.05), while the promoting effect was ameliorated in CRF07_BC-7d. Co-immunoprecipitation found that deletions in the p6Gag reduced Gal-3-mediated enhancement of the Alix-Gag interaction.