Small Drugs, Huge Impact: The Extraordinary Impact of Antisense Oligonucleotides in Research and Drug Development

Antisense oligonucleotides (ASOs) are an increasingly represented class of drugs. These small sequences of nucleotides are designed to precisely target other oligonucleotides, usually RNA species, and are modified to protect them from degradation by nucleases. Their specificity is due to their sequence, so it is possible to target any RNA sequence that is already known. These molecules are very versatile and adaptable given that their sequence and chemistry can be custom manufactured. Based on the chemistry being used, their activity may significantly change and their effects on cell function and phenotypes can differ dramatically. While some will cause the target RNA to decay, others will only bind to the target and act as a steric blocker. Their incredible versatility is the key to manipulating several aspects of nucleic acid function as well as their process, and alter the transcriptome profile of a specific cell type or tissue. For example, they can be used to modify splicing or mask specific sites on a target. The entire design rather than just the sequence is essential to ensuring the specificity of the ASO to its target. Thus, it is vitally important to ensure that the complete process of drug design and testing is taken into account. ASOs’ adaptability is a considerable advantage, and over the past decades has allowed multiple new drugs to be approved. This, in turn, has had a significant and positive impact on patient lives. Given current challenges presented by the COVID-19 pandemic, it is necessary to find new therapeutic strategies that would complement the vaccination efforts being used across the globe. ASOs may be a very powerful tool that can be used to target the virus RNA and provide a therapeutic paradigm. The proof of the efficacy of ASOs as an anti-viral agent is long-standing, yet no molecule currently has FDA approval. The emergence and widespread use of RNA vaccines during this health crisis might provide an ideal opportunity to develop the first anti-viral ASOs on the market. In this review, we describe the story of ASOs, the different characteristics of their chemistry, and how their characteristics translate into research and as a clinical tool.

Detection of Suspended SARS-CoV-2 in Indoor Air Using an Electrostatic Sampler

A prototype virus sampler using electrostatic precipitation has been developed to investigate aerosol infection by SARS-CoV-2. The sampler consists of a discharge electrode placed inside a vial, and a thin layer of viral lysis buffer at the bottom, working as a collection electrode. The sampler was operated with the sampling air flow rate of 40 L/min. Collection efficiency of the sampler is about 80% for 25nm to 5.0µm diameter particles. We sampled the air of a food court of a commercial facility, a connecting corridor of a clouded train station, and two office rooms (A and B) in September 2021, just after the 5th peak of COVID-19 in Japan. The analysis using a RT-qPCR detected the virus RNA in the air of the office A, B and the food court. Estimated concentration of the virus in the air determined by calibration curve was 2.0 x 102, 7.8 x 102, and 0.6 - 2.4 x 102 copies/m3, in the office A, B, and the food court, respectively. These results indicate that the sampler using electrostatic precipitation can detect SARS-CoV-2 in indoor air. It could be developed as a risk assessment method for aerosol infection.

Atypical skin manifestation in severe acute chikungunya infection in a pregnant woman: a case report

Introduction Patients with chikungunya virus infection commonly present with fever, skin rash, and severe joint pain. The vesiculobullous rash is rare in adults but common in infants. In addition, septic shock and acute respiratory distress syndrome are rare complications of atypical and severe acute chikungunya infection. Case presentation We report the presence of an 18-year-old Thai female, at 31 weeks gestation, with fever, maculopapular rash, and polyarthritis. The rash later progressed to a vesiculobullous pattern, and she developed septic shock and acute respiratory distress syndrome. Skin biopsy and blood were positive for chikungunya virus RNA. The patient was intubated with a mechanical ventilator and subsequently fully recovered. Conclusion Atypical skin manifestation and severe acute disease is likely due to immune response attenuation in pregnancy. The possibility of progression to severe or atypical disease in pregnant women suffering chikungunya should always be considered.

Regressing SARS-CoV-2 Sewage Measurements Onto COVID-19 Burden in the Population: A Proof-of-Concept for Quantitative Environmental Surveillance

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an RNA virus, a member of the coronavirus family of respiratory viruses that includes severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and the Middle East respiratory syndrome (MERS). It has had an acute and dramatic impact on health care systems, economies, and societies of affected countries during the past 8 months. Widespread testing and tracing efforts are being employed in many countries in attempts to contain and mitigate this pandemic. Recent data has indicated that fecal shedding of SARS-CoV-2 is common and that the virus RNA can be detected in wastewater. This indicates that wastewater monitoring may provide a potentially efficient tool for the epidemiological surveillance of SARS-CoV-2 infection in large populations at relevant scales. In particular, this provides important means of (i) estimating the extent of outbreaks and their spatial distributions, based primarily on in-sewer measurements, (ii) managing the early-warning system quantitatively and efficiently, and (iii) verifying disease elimination. Here we report different virus concentration methods using polyethylene glycol (PEG), alum, or filtration techniques as well as different RNA extraction methodologies, providing important insights regarding the detection of SARS-CoV-2 RNA in sewage. Virus RNA particles were detected in wastewater in several geographic locations in Israel. In addition, a correlation of virus RNA concentration to morbidity was detected in Bnei-Barak city during April 2020. This study presents a proof of concept for the use of direct raw sewage-associated virus data, during the pandemic in the country as a potential epidemiological tool.

Computational Study of SARS-CoV-2 RNA Dependent RNA Polymerase Allosteric Site Inhibition

The COVID-19 pandemic has caused millions of fatalities since 2019. Despite the availability of vaccines for this disease, new strains are causing rapid ailment and are a continuous threat to vaccine efficacy. Here, molecular docking and simulations identify strong inhibitors of the allosteric site of the SARS-CoV-2 virus RNA dependent RNA polymerase (RdRp). More than one hundred different flavonoids were docked with the SARS-CoV-2 RdRp allosteric site through computational screening. The three top hits were Naringoside, Myricetin and Aureusidin 4,6-diglucoside. Simulation analyses confirmed that they are in constant contact during the simulation time course and have strong association with the enzyme’s allosteric site. Absorption, distribution, metabolism, excretion and toxicity (ADMET) data provided medicinal information of these top three hits. They had good human intestinal absorption (HIA) concentrations and were non-toxic. Due to high mutation rates in the active sites of the viral enzyme, these new allosteric site inhibitors offer opportunities to drug SARS-CoV-2 RdRp. These results provide new information for the design of novel allosteric inhibitors against SARS-CoV-2 RdRp.

HEPATITIS E VIRUS MONITORING RESULTS AND ITS LABORATORY SCREENING ALGORITHM

Background. Individual cases of viral hepatitis E are recorded in the Republic of Belarus annually indicating the need for the pathogen monitoring at both the population and reservoir levels. Objective. To consolidate the monitoring data on hepatitis E virus over the period of 2018 - 2021, as well as to work out an effective algorithm for its laboratory screening. Material and methods. As part of the study, 345 samples were analyzed, including 227 human biological samples, 37 samples of biological materials of domestic pigs, 22 samples of food and 59 samples of waste water. Results. According to the results of serum diagnostics, in the group of kidney recipients (n = 29), the detection rate of IgM and IgG to hepatitis E virus was 6.9% [0.85%; 23.03%] and 17.2% [7.13%; 35, 02%] respectively; in the group of patients with pregnancy pathology (n = 44) - 6.8% [1.68%; 18.89%] and 11.4% [4.5%; 24.43%] respectively. In patients with acute hepatitis of unknown etiology (n = 26), antiviral IgM was not detected, while the frequency of antiviral IgG detection reached 7.7% [1.02%; 25.26]. In control group (blood donors, n = 53) IgM and IgG were detected in 1.9% [0.6%; 10.88%] and 5.7% [1.35%; 15.97] of those examined respectively. Hepatitis E virus RNA was detected in 8 human biological samples (3.8%) from kidney recipients. The identified hepatitis E viruses were represented by genotype GIII and belonged to a previously unidentified subgenotype (GIIIa - GIIIi). In the studied samples of biological material from pigs, as well as in samples of food and waste water, hepatitis E virus RNA was not detected. Conclusions. An algorithm for hepatitis E virus laboratory screening has been developed and tested. Its section concerning the diagnosis of viral hepatitis E is set out in the Instructions for use "Algorithm for laboratory diagnosis of viral hepatitis E" (No. 148-1220 from January 28, 2021).

Results of isolation and genotyping of measles viruses that circulated in 2012–2017 in the Odesa region

Background. The circulation of different strains of the measles virus is closely related to the region and the incidence rate since circulating strains can change during epidemic outbreaks and in interepidemic periods. According to the WHO, the B3 strain is most common during outbreaks worldwide. Therefore, typing of circulating strains of measles virus, especially during an epidemic outbreak, is an important process, inclu­ding for predicting the development of an epidemic. The study was aimed to identify and determine the genotype of measles virus types that circulate in Ukraine during 2012–2019. Materials and methods. Materials of the reporting documentation of the State Institution “Odessa Regional Laboratory Center of the Ministry of Health of Ukraine” in the Odessa region during 2012–2019 were used and analyzed. Materials from patients with suspected measles were used for molecular biological, genetic, analytical, and statistical approaches investigation. Following the standard WHO protocol for sequencing and phylogenetic analysis, circulating measles virus strains were isolated from the patient in a special culture of Vero/SLAM cells. Measles virus RNA was isolated from the resulting virus-containing material after cultivation and RT-PCR was performed. The resulting cDNA was sent for genotyping, which was carried out at the WHO reference labo­ratory for the diagnosis of measles and rubella in Luxembourg (WHO RRL). Results. Twenty strains of measles virus from 45 samples (urine and nasopharyngeal swabs) from patients diagnosed with measles were isolated during 2012–2014. Virus isolation was not carried out in 2015–2016 due to isolated cases of the disease. Twenty-four virus strains from 164 samples were isolated in 2017. Conclusions. The results obtained at the State Institution “Odessa Regional Laboratory Center” demonstrated that during the interepidemic period of 2012–2014, the D4 geno­type circulated in the region. But since 2017, when there was an increas of cases associated with a new epidemic outbreak, B3, genetic line MVs/Kabul.AFG/20.2014/3 B3 mainly circulates in the region of southern Ukraine. As you can see, these data completely coin­cide with the data about circulating genotypes that were found at a certain time in the European Region, according to the data from the literature.

Intrauterine Fetal Demise After Uncomplicated COVID-19: What Can We Learn from the Case?

Background: SARS-CoV-2 infection in pregnant women can lead to placental damage and transplacental infection transfer, and intrauterine fetal demise is an unpredictable event. Case study: A 32-year-old patient in her 38th week of pregnancy reported loss of fetal movements. She overcame mild COVID-19 with positive PCR test 22 days before. A histology of the placenta showed deposition of intervillous fibrinoid, lympho-histiocytic infiltration, scant neutrophils, clumping of villi, and extant infarctions. Immunohistochemistry identified focal SARS-CoV-2 nucleocapsid and spike protein in the syncytiotrophoblast and isolated in situ hybridization of the virus’ RNA. Low ACE2 and TMPRSS2 contrasted with strong basigin/CD147 and PDL-1 positivity in the trophoblast. An autopsy of the fetus showed no morphological abnormalities except for lung interstitial infiltrate, with prevalent CD8-positive T-lymphocytes and B-lymphocytes. Immunohistochemistry and in situ hybridization proved the presence of countless dispersed SARS-CoV-2-infected epithelial and endothelial cells in the lung tissue. The potential virus-receptor protein ACE2, TMPRSS2, and CD147 expression was too low to be detected. Conclusion: Over three weeks’ persistence of trophoblast viral infection lead to extensive intervillous fibrinoid depositions and placental infarctions. High CD147 expression might serve as the dominant receptor for the virus, and PDL-1 could limit maternal immunity in placental tissue virus clearance. The presented case indicates that the SARS-CoV-2 infection-induced changes in the placenta lead to ischemia and consecutive demise of the fetus. The infection of the fetus was without significant impact on its death. This rare complication of pregnancy can appear independently to the severity of COVID-19’s clinical course in the pregnant mother.