Detection of H275Y Mutation of A(H1N1)pdmO9 Viruses Using a Real-time RT-PCR Assay in Mongolia

Objectives: Oseltamivir is recommended as a first-line drug for the prophylaxis and treatment of influenza A(H1N1)pdmO9 infection worldwide. However, oseltamivir-resistant influenza A(H1N1)pdmO9 viruses have been identified and are mostly associated with an H275Y substitution in the neuraminidase (NA) gene. Careful and rapid laboratory testing for antiviral resistance surveillance of influenza viruses are important to public health and clinical sectors. The aim of our study was to determine oseltamivir-resistant H275Y mutation in clinical specimens by the real-time reverse transcriptase polymerase chain reaction (rtRT-PCR) assay in Mongolia. Methods: A total of 215 clinical specimens tested positive for the A(H1N1)pdmO9 influenza virus by rtRT-PCR. The specimens were collected between January 2013 and August 2014 from patients who visited influenza surveillance sites. All collected specimens were tested for the presence of the oseltamivir-resistant H275Y mutation by rtRT-PCR and DNA sequencing on the NA gene was performed on four of the clinical samples. Results: For all specimens, the H275 (Ct value = 20.4-34.5) was sensitive to oseltamivir by rtRT-PCR assay for detection of the H275Y mutations in the NA gene. Four questionable clinical samples were sequenced for the full NA gene. The data of these samples are available in GISAID influenza virus database with accession numbers EPI533542, EPI462274, EPI462271 and EPI460844. Conclusion: The A(H1 N1)pdm09 viruses were susceptible to oseltamivir during the study period in Mongolia. The rtRT-PCR assay is useful for detection of the H275Y substitution but other possible mutations in the NA gene of the virus could limit the utility of this technique

Development of serological assay for detection of antibodies in the N protein of SARS-CoV-2 in human sera in Mongolia

The capability to detect Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection and identify immune responses among the population is crucial for managing the outbreak of the COVID-19 pandemic. Although PCR-based nucleic acid detection techniques are utilized to detect viral infection in people, alternative tests capable of distinguishing between exposure and infection are urgently needed beyond this restricted window of detectable viral replication. Antibodies are produced in human sera within a few days after viral infection, providing longer period for performing tests to acquire reliable database. Herewith, we provide the results of our in-house developed ELISA (Enzyme-Linked Immunosorbent Assay) that displays all of the properties necessary for high-throughput of human sera sample analysis. This test does not involve the handling of live viruses, although it detects a variety of antibody types in serum and plasma of human after exposure to the virus. For in-house development of the kit, the nucleocapsid (N) gene of SARS-CoV-2 virus was cloned in the prokaryotic expression vector pGEX-6P-1, and purified N protein was used to detect IgG antibodies in human sera samples. In total 76 human serum samples that were collected before novel coronavirus registry in Mongolia in March 2020, as well as 200 serum samples from patients who had been infected by SARS-CoV-2 virus, were used. Among 200 serum samples, 188 were positive and 12 were false negative, while in non-infected cases 69 were negative and 7 were false positive, suggesting 94 per cent sensitivity and 90.7 per cent specificity of the kit, with p-values of 0.02

Influenza B viruses circulated during last 5 years in Mongolia.

Influenza B virus-caused illness has recently been considered as an urgent public health problem due to substantial morbidity, mortality and life-threatening medical complications. In this study, we have reported the main characteristics of influenza B virus in Mongolia, including prevalence, lineages, suitability with vaccine strains and drug susceptibility against the virus. 15768 specimens were tested by qPCR for detecting influenza viruses. From positive specimens for influenza B virus, the clinical isolates were isolated using MDCK cells. Sequencing analysis, hemagglutination inhibition assay and Neuraminidase inhibitor (NAI) drug susceptibility testing were performed for the clinical isolates. Influenza B virus was around in 3.46% of the samples in Mongolia, and B/Victoria clade-1A and B/Yamagata clade-3 lineages were predominant. Importantly, it was confirmed that the lineages corresponded to the vaccine strains. Moreover, drug susceptibility tests revealed that some Mongolian clinical isolates showed reduced susceptibility to antiviral agents. Interestingly, G104R was identified as a novel mutation, which might have a significant role in drug resistance of the virus. These results describe the characteristics of influenza B viruses that have caused respiratory illness in the population of Mongolia between 2013 and 2017

Molecular epidemiology of SARS‐CoV‐2 in Mongolia, first experience with nanopore sequencing in lower‐ and middle‐income countries setting

Abstract Background Coronavirus disease (COVID‐19) has had a significant impact globally, and extensive genomic research has been conducted on severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) lineage patterns and its variants. Mongolia's effective response resulted in low prevalence until vaccinations became available. However, due to the lack of systematically collected data and absence of whole genome sequencing capabilities, we conducted a two‐stepped, nationally representative molecular epidemiologic study of SARS‐CoV‐2 in Mongolia for 2020 and 2021. Methods We used retrospective analysis of stored biological samples from November 2020 to October 2021 and a variant‐specific real‐time reverse transcription polymerase chain reaction (RT‐PCR) test to detect SARS‐CoV‐2 variants, followed by whole genome sequencing by Nanopore technology. Samples were retrieved from different sites and stored at −70°C deep freezer, and tests were performed on samples with cycle threshold <30. Results Out of 4879 nucleic acid tests, 799 whole genome sequencing had been carried out. Among the stored samples of earlier local transmission, we found the 20B (B.1.1.46) variant predominated in the earlier local transmission period. A slower introduction and circulation of alpha and delta variants were observed compared to global dynamics in 2020 and 2021. Beta or Gamma variants were not detected between November 2020 and September 2021 in Mongolia. Conclusions SARS‐CoV‐2 variants of concerns including alpha and delta were delayed in circulation potentially due to public health stringencies in Mongolia. We are sharing our initial experience with whole genome sequencing of SARS‐CoV‐2 from Mongolia, where sequencing data is sparse