One-step quantitative RT-PCR assay with armored RNA controls for detection of SARS-CoV-2.

Coronavirus disease 2019 (COVID-19) has become pandemic since March 11, 2020. Thus, development and integration in clinics of fast and sensitive diagnostic tools are essential. The aim of the study is a development and evaluation of a one-step quantitative reverse transcription-polymerase chain reaction (RT-qPCR) assay (COVID-19 Amp) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection with an armored positive control and internal controls constructed from synthetic MS2-phage-based RNA particles. The COVID-19 Amp assay limit of detection was 103 copies/ml, the analytical specificity was 100%. A total of 109 biological samples were examined using COVID-19 Amp and World Health Organization (WHO)-based assay. Discordance in nine samples was observed (negative by the WHO-based assay) and discordant samples were retested as positive according to the results obtained from the Vector-PCRrv-2019-nCoV-RG assay. The developed COVID-19 Amp assay has high sensitivity and specificity, includes virus particles-based controls, provides the direct definition of the SARS-CoV-2 RdRp gene partial sequence, and is suitable for any hospital and laboratory equipped for RT-qPCR.

Identification and Genetic Characterization of MERS-Related Coronavirus Isolated from Nathusius' Pipistrelle (Pipistrellus nathusii) near Zvenigorod (Moscow Region, Russia).

Being diverse and widely distributed globally, bats are a known reservoir of a series of emerging zoonotic viruses. We studied fecal viromes of twenty-six bats captured in 2015 in the Moscow Region and found 13 of 26 (50%) samples to be coronavirus positive. Of P. nathusii (the Nathusius' pipistrelle), 3 of 6 samples were carriers of a novel MERS-related betacoronavirus. We sequenced and assembled the complete genome of this betacoronavirus and named it MOW-BatCoV strain 15-22. Whole genome phylogenetic analysis suggests that MOW-BatCoV/15-22 falls into a distinct subclade closely related to human and camel MERS-CoV. Unexpectedly, the phylogenetic analysis of the novel MOW-BatCoV/15-22 spike gene showed the closest similarity to CoVs from Erinaceus europaeus (European hedgehog). We suppose MOW-BatCoV could have arisen as a result of recombination between ancestral viruses of bats and hedgehogs. Molecular docking analysis of MOW-BatCoV/15-22 spike glycoprotein binding to DPP4 receptors of different mammals predicted the highest binding ability with DPP4 of the Myotis brandtii bat (docking score -320.15) and the E. europaeus (docking score -294.51). Hedgehogs are widely kept as pets and are commonly found in areas of human habitation. As this novel bat-CoV is likely capable of infecting hedgehogs, we suggest hedgehogs can act as intermediate hosts between bats and humans for other bat-CoVs.

Current View on Genetic Relationships within the Bunyamwera Serological Group.

The Bunyamwera serological group includes a number of geographically widespread viruses that are related but not identical and have serological cross-reactivity. As the first group members were obtained in the pre-sequencing era, their classifications (group attribution, species differentiation) were originally based on serological reactions. At the same time, the accuracy of the typing in each case depended on the variety of viruses that the researcher had as a comparison panel. With the advent of sequencing techniques, it has become customary to use identity thresholds (nucleotide or amino acid composition) as demarcation criteria for the interspecific differentiation of viral species. Identity thresholds are determined by the International Committee on Taxonomy of Viruses (ICTV) and are regularly reviewed. Similar criteria were established for the Orthobunyavirus genus, which includes members of the Bunyamwera serological group. On the basis of these criteria, the species attributions of some members of the serological group need to be clarified. For this purpose, we analyzed sequences (available in NCBI GenBank) of viruses belonging to the Bunyamwera serological group in order to clarify their phylogenetic positions on the basis of the current demarcation criteria established by the ICTV.

Differential Laboratory Diagnosis of Acute Fever in Guinea: Preparedness for the Threat of Hemorrhagic Fevers.

Acute febrile illnesses occur frequently in Guinea. Acute fever itself is not a unique, hallmark indication (pathognomonic sign) of any one illness or disease. In the infectious disease context, fever's underlying cause can be a wide range of viral or bacterial pathogens, including the Ebola virus. In this study, molecular and serological methods were used to analyze samples from patients hospitalized with acute febrile illness in various regions of Guinea. This analysis was undertaken with the goal of accomplishing differential diagnosis (determination of causative pathogen) in such cases. As a result, a number of pathogens, both viral and bacterial, were identified in Guinea as causative agents behind acute febrile illness. In approximately 60% of the studied samples, however, a definitive determination could not be made.

Isolation and characterization of Wad Medani virus obtained in the tuva Republic of Russia.

Wad Medani virus (WMV) belongs to the genus Orbivirus and is a poorly studied arbovirus with unclear medical significance. Presently, a limited number of WMV strains are characterized and available in NCBI GenBank, some isolated many years ago. A new WMV strain was isolated in 2012 from Dermacentor nuttalli ticks collected from sheep in the Tuva Republic, Russia, and sequenced using high-throughput methods. Complete coding sequences were obtained revealing signs of multiple intersegment reassortments. These point to a high variability potential in WMV that may lead to the formation of strains with novel properties. These new data on WMV can promote better understanding of: ecological features of its circulation; relationships within the genus Orbivirus; and the medical significance of the virus.

Universal Library Preparation Protocol for Efficient High-Throughput Sequencing of Double-Stranded RNA Viruses.

This chapter reports a library preparation protocol for efficient high-throughput sequencing of double-stranded RNA viruses. The protocol consists of four main steps, viz., enzyme treatment, precipitation using lithium chloride, full-length amplification of cDNAs, and tailing adapters for high-throughput sequencing. This protocol will be useful for all double-stranded RNA viruses and for all of the high-throughput sequencing platforms.

Genetic diversity of Kemerovo virus and phylogenetic relationships within the Great Island virus genetic group.

Kemerovo virus (KEMV) is a member of the Great Island virus genetic group, belonging to the tick-borne arboviruses of the genus Orbivirus within the family Reoviridae. Nine strains of KEMV, which were isolated from various locations in Russia, were sequenced by high-throughput sequencing to study their intraspecific diversity and the interspecific relationships of viruses within the Great Island genetic group. For the first time, multiple reassortment within KEMV was reliably demonstrated. Different types of independently emerged alternative reading frames in segment 9 and heterogeneity of the viral population in one of the KEMV strains were found. The hypothesis of the role of an alternative open reading frame (ORF) in segment 9 in KEMV cellular tropism was not confirmed in this study.

Sequencing and genetic characterization of two strains Paramushir virus obtained from the Tyuleniy Island in the Okhotsk Sea (2015).

Paramushir virus belongs to Sakhalin virus genogroup within Orthonairovirus genus and is one of the poorly studied viruses with unknown pathogenicity. At the moment, only one nearly complete sequence of Paramushir virus genome, isolated in 1972, is available. Two new strains of PARV were isolated in 2015 from a sample collected at the Tyuleniy Island in the Okhotsk Sea and sequenced using a combination of high throughput sequencing and specific multiplex PCR. Both strains are closely related to the early sequenced PARV strain LEIV-1149 K. The signs of intersegment reassortment and probable recombination were revealed, which point to a high variability potential of Paramushir virus and may lead to the formation of strains with novel properties, different from those of the predecessors. The new data regarding Paramushir virus can promote a better understanding of the diversity and relations within Orthonairovirus genus and help define intragenic demarcation criteria, which have not yet been established.

Development and Evaluation of a One-Step Quantitative RT-PCR Assay for Detection of Lassa Virus.

Lassa fever is a severe viral hemorrhagic illness caused by Lassa virus. Based on estimates, the number of LASV infections ranges from 300,000 to 500,000 cases in endemic areas with a fatality rate of 1%. Development of fast and sensitive tools for the control and prevention of Lassa virus infection as well as for clinical diagnostics of Lassa fever are crucial. Here we reported development and evaluation of a one-step quantitative RT-qPCR assay for the Lassa virus detection - LASV-Fl. This assay is suitable for the detection of lineages I-IV of Lassa virus. The limit of detection of the assay ranged from 103 copies/ml to 105 copies/ml and has 96.4% diagnostic sensitivity, whereas analytical and diagnostic specificities both were 100%. Serum, whole blood and tissue are suitable for use with the assay. The assay contains all the necessary components to perform the analysis, including an armored positive control (ARC+) and an armored internal control (IC). The study was done during the mission of specialized anti-epidemic team of the Russian Federation (SAET) in the Republic of Guinea in 2015-2018. Based on sequencing data, LASV-specific assay was developed using synthetic MS2-phage-based armored RNA particles, RNA from Lassa virus strain Josiah, and further, evaluated in field conditions using samples from patients and Mastomys natalensis rodents.

The Study of Viral RNA Diversity in Bird Samples Using De Novo Designed Multiplex Genus-Specific Primer Panels.

Advances in the next generation sequencing (NGS) technologies have significantly increased our ability to detect new viral pathogens and systematically determine the spectrum of viruses prevalent in various biological samples. In addition, this approach has also helped in establishing the associations of viromes with many diseases. However, unlike the metagenomic studies using 16S rRNA for the detection of bacteria, it is impossible to create universal oligonucleotides to target all known and novel viruses, owing to their genomic diversity and variability. On the other hand, sequencing the entire genome is still expensive and has relatively low sensitivity for such applications. The existing approaches for the design of oligonucleotides for targeted enrichment are usually involved in the development of primers for the PCR-based detection of particular viral species or genera, but not for families or higher taxonomic orders. In this study, we have developed a computational pipeline for designing the oligonucleotides capable of covering a significant number of known viruses within various taxonomic orders, as well as their novel variants. We have subsequently designed a genus-specific oligonucleotide panel for targeted enrichment of viral nucleic acids in biological material and demonstrated the possibility of its application for virus detection in bird samples. We have tested our panel using a number of collected samples and have observed superior efficiency in the detection and identification of viral pathogens. Since a reliable, bioinformatics-based analytical method for the rapid identification of the sequences was crucial, an NGS-based data analysis module was developed in this study, and its functionality in the detection of novel viruses and analysis of virome diversity was demonstrated.

Sensitive Multiplex Real-time RT-qPCR Assay for the Detection of Filoviruses.

Filoviruses are important etiological agents of emergent diseases with high mortality rates. Traditionally, filovirus fever diseases have primarily been a burden of African countries; however, global interconnectedness has increased the probability of the worldwide spread of filoviruses. Therefore, national healthcare organizations need tools for managing filovirus risk, including diagnostic kits based on real-time reverse transcription PCR (RT-qPCR), as this is the most suitable method for diagnosing filovirus fever diseases. Here we describe a real-time RT-qPCR assay for filovirus detection. This assay is a further development of our previously reported EBOV (Zaire)-Fl kit. Two sets (FiloA-Fl and FiloB-Fl) of real-time RT-qPCR assays for the detection of filoviruses were developed and evaluated using armored RNA phage particles (ARs) as positive controls. The limit of detection of the assay was 5x102 copies/ml of the AR-positive control for the FiloA-Fl set and 5x103 copies/ml of the AR-positive control for the FiloB-Fl set. Our assay provides a rapid and sensitive tool for detecting filoviruses. The high specificity and sensitivity of the assay make it useful for clinical and epidemiologic investigations in the field of filovirus fever diseases and their etiological agents.

Complete Genome Sequence of Human Adenovirus 7 Associated with Fatal Adult Pneumonia.

Human adenovirus 7 (hAdv7) 19BOVLB/Volgograd/Rus/2014 was isolated from the autopsy material from an adult with fatal pneumonia in Volgograd, Russia, in March 2014. Whole-genome sequencing of the virus isolate was performed.