Analysis of Novel Drug-Resistant Human Cytomegalovirus DNA Polymerase Mutations Reveals the Role of a DNA-Binding Loop in Phosphonoformic Acid Resistance.

The appearance of drug-resistant mutations in UL54 DNA polymerase and UL97 kinase genes is problematic for the treatment of human cytomegalovirus (HCMV) diseases. During treatment of HCMV infection in a pediatric hematopoietic cell transplant recipient, H600L and T700A mutations and E576G mutation were independently found in the UL54 gene. Foscarnet (FOS; phosphonoformic acid) resistance by T700A mutation is reported. Here, we investigated the role of novel mutations in drug resistance by producing recombinant viruses and a model polymerase structure. The H600L mutant virus showed an increase in resistance to ganciclovir (GCV) by 11-fold and to FOS and cidofovir (CDV) by 5-fold, compared to the wild type, while the E756G mutant virus showed an increase in resistance to FOS by 9-fold and modestly to CDV by 2-fold. With the FOS-resistant T700A mutation, only H600L produced increased FOS resistance up to 37-fold, indicating an additive effect of these mutations on FOS resistance. To gain insight into drug resistance mechanisms, a model structure for UL54 polymerase was constructed using the yeast DNA polymerase as a template. In this model, HCMV DNA polymerase contains a long palm loop domain of which H600 and T700 are located on each end and T700 interacts with the FOS binding pocket. Our results demonstrate that H600L and E756G mutations in UL54 polymerase are novel drug-resistant mutations and that the acquisition of both H600L and T700A mutations in the DNA-binding loop confers increased resistance to FOS treatment, providing novel insights for the mechanism acquiring foscarnet resistance.

Genetic Analysis of the Measles Virus From the Outbreaks in South Korea, 2019.

Three genotypes (B3, D8, and H1) of the measles virus (MeV) have recently caused global outbreaks. In Korea, four measles outbreaks were reported during 2018-2019 and most patients were infants and health care workers in their 20s and 30s. To investigate the genetic characteristics and molecular epidemiology of the outbreaks, we analyzed the sequence of MeVs by targeting the N-450, MF-NCR, and/or H gene regions. Considering their phylogenetic relationships, besides the N-450 and MF-NCR sequences that are commonly used for genotyping MeVs, the MF-NCR-H sequence was related to the dynamics for identifying the transmission of MeVs. Phylogenetic clustering patterns reconstructed from the MF-NCR-H sequence set revealed that genotype D8 caused three of the four outbreaks, while B3 seemed to have induced the fourth outbreak. These results suggest that the MF-NCR-H sequence is useful for rapid confirmation of measles outbreaks and to identify the epidemiological routes of MeVs.

Detection and Isolation of SARS-CoV-2 in Serum, Urine, and Stool Specimens of COVID-19 Patients from the Republic of Korea.

OBJECTIVES: Coronavirus Disease-19 (COVID-19) is a respiratory infection characterized by the main symptoms of pneumonia and fever. It is caused by the novel coronavirus severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2), which is known to spread via respiratory droplets. We aimed to determine the rate and likelihood of SARS-CoV-2 transmission from COVID-19 patients through non-respiratory routes. METHODS: Serum, urine, and stool samples were collected from 74 hospitalized patients diagnosed with COVID-19 based on the detection of SARS-CoV-2 in respiratory samples. The SARS-CoV-2 RNA genome was extracted from each specimen and real-time reverse transcription polymerase chain reaction performed. CaCo-2 cells were inoculated with the specimens containing the SARS-COV-2 genome, and subcultured for virus isolation. After culturing, viral replication in the cell supernatant was assessed. RESULTS: Of the samples collected from 74 COVID-19 patients, SARS-CoV-2 was detected in 15 serum, urine, or stool samples. The virus detection rate in the serum, urine, and stool samples were 2.8% (9/323), 0.8% (2/247), and 10.1% (13/129), and the mean viral load was 1,210 ± 1,861, 79 ± 30, and 3,176 ± 7,208 copy/µL, respectively. However, the SARS-CoV-2 was not isolated by the culture method from the samples that tested positive for the SARS-CoV-2 gene. CONCLUSION: While the virus remained detectable in the respiratory samples of COVID-19 patients for several days after hospitalization, its detection in the serum, urine, and stool samples was intermittent. Since the virus could not be isolated from the SARS-COV-2-positive samples, the risk of viral transmission via stool and urine is expected to be low.

Genetic Characterization of Middle East Respiratory Syndrome Coronavirus, South Korea, 2018.

We evaluated genetic variation in Middle East respiratory syndrome coronavirus (MERS-CoV) imported to South Korea in 2018 using specimens from a patient and isolates from infected Caco-2 cells. The MERS-CoV strain in this study was genetically similar to a strain isolated in Riyadh, Saudi Arabia, in 2017.

A large healthcare-associated outbreak of hepatitis C virus genotype 1a in a clinic in Korea.

BACKGROUND: In November 2015, reuse of needles and syringes in conjunction with an increase in cases of HCV at a clinic in Korea was reported and investigated by public health authorities. Patients who received injections at the clinic from the first time this infection control breach may have occurred in 2008 through 2015 when the practice was stopped were offered screening for HCV and other blood-borne pathogens such as HIV, HTLV, HBV, syphilis, and malaria. OBJECTIVES: The aim of this study was to assess whether an outbreak of hepatitis C had occurred among the potentially exposed clinic patients due to this infection control breach. STUDY DESIGN: We performed hepatitis C viral RNA load tests and genotyping using plasma from hepatitis C antibody-positive individuals who had visited the clinic between May 2008 and November 2015. We analyzed the core-E2 and NS5B regions of the virus from RNA-positive samples by constructing a phylogenetic tree based on maximum likelihood analysis. To identify transmission risk factors and epidemiological relationships among the patients, we reviewed their medical records, assessed staff infection control practices and performed environmental inspection of the clinic. Environmental samples from medication room surfaces and medication vial contents were tested for HCV RNA. RESULTS AND CONCLUSIONS: Among the 1721 patients tested, 96 were IgG-positive and 70 were viral RNA-positive. Among the 61 patients whose viral loads were greater than the detection limit, 41 (67.2%) were classified as genotype 1a, 1 (1.6%) as genotype 1b, 18 (29.5%) as genotype 1, and one (1.6%) as genotype 2. After sequencing, 12 genotype 1 cases were further classified as genotype 1a (11) or 1b (1). The sequences of the core-E2 and NS5B regions of 45 patients formed a monophyletic cluster distinct from genotype 1a. The hepatitis C virus sequences from patients and environmental specimens were well-matched in the partial E1 gene region. We detected genotype 1a RNA in environmental specimens, indicating a healthcare-associated outbreak caused by reuse of syringes and contaminated multi-dose vials. Our molecular epidemiological investigation of hepatitis C genotype 1a, rare in Korea, will aid investigations of infection sources during future pathogen outbreaks.