Detection Of Viral RNA Research Articles

A novel and efficient method for identifying cotton leafroll dwarf virus infection in upland cotton (Gossypium hirsutum)

AbstractWe report the development of a novel and more efficient, rapid, cost‐effective and simple technique than current PCR‐based identification methods for screening cotton (Gossypium hirsutum) plants for the presence of cotton leafroll dwarf virus (CLRDV). This protocol takes advantage of the PACE (PCR Allele Competitive Extension) system and uses PCR amplification of cDNA, coupled with sequence‐specific fluorescent probes to differentiate between infected and uninfected cotton plants. This procedure has the potential for application in detection of other RNA viruses in a variety of other crops, by using primers specific for the RNA‐dependent RNA polymerase (RdRP) gene and a widely conserved housekeeping gene in the host organism; in this case, the G. hirsutum polyubiquitin gene (GhUB).

Kinetics and magnitude of viral RNA shedding as indicators for Influenza A virus transmissibility in ferrets

The ferret transmission model is routinely used to evaluate the pandemic potential of newly emerging influenza A viruses. However, concurrent measurement of viral load in the air is typically not a component of such studies. To address this knowledge gap, we measured the levels of virus in ferret nasal washes as well as viral RNA emitted into the air for 14 diverse influenza viruses, encompassing human-, swine-, and avian-origin strains. Here we show that transmissible viruses display robust replication and fast release into the air. In contrast, poorly- and non-transmissible viruses show significantly reduced or delayed replication along with lower detection of airborne viral RNA at early time points post inoculation. These findings indicate that efficient ferret-to-ferret transmission via the air is directly associated with fast emission of virus-laden particles; as such, quantification of viral RNA in the air represents a useful addition to established assessments of new influenza virus strains.

Prevalence of HIV and Viral Hepatitis Markers among Healthcare Workers in the Republic of Guinea.

Healthcare workers are much more likely to be infected with HIV and hepatitis viruses compared to the general population. Although healthcare workers are more aware of HIV and hepatitis viruses, several countries in Africa lack a comprehensive grasp of disease routes and transmission risks. The aim of this study was to assess the prevalence of the serological and molecular biological markers of HIV and viral hepatitis among healthcare workers in the Republic of Guinea. The study material was 74 blood serum samples collected from healthcare workers who received additional training at the Institute of Applied Biological Research of Guinea (IRBAG, Kindia, Republic of Guinea). The markers examined included HBsAg, HBeAg, anti-HBs IgG, anti-HBcore IgG, anti-HCV qualitative determination, anti-HEV IgM and IgG, anti-HAV IgM and IgG, and anti-HIV. For viral DNA and RNA detection, nucleic acids were extracted from blood serum, and viral presence was inferred using real-time PCR with hybridization fluorescence detection. A high prevalence of viral hepatitis B markers was shown, and significantly fewer cases of viral hepatitis C and HIV were detected. Almost all examined medical workers had anti-HAV IgG antibodies, but no antibodies to hepatitis E virus. Apparently, the identified markers depend on the general prevalence of certain pathogens in the region and are associated with the traditions and characteristics of the country's residents.

Qualitative hepatitis C virus RNA assay identifies active infection with sufficient viral load for treatment among Phetchabun residents in Thailand.

The World Health Organization envisions the elimination of viral hepatitis by 2030 through reducing prevalence and transmission, increasing diagnostic screening, and expanding treatment coverage. Efforts to micro-eliminate hepatitis in Phetchabun province in Thailand, a region where the prevalence of hepatitis C virus (HCV) infection and liver cancer is higher than elsewhere in the country, began with evaluating the province-wide burden of HCV. Here, we describe a feasibility study to assess active HCV infection by screening Phetchabun residents ages 35 to 69 years for anti-HCV antibodies by using a rapid diagnostic test (RDT) at the point of care. Positive anti-HCV results were further evaluated for active infection using qualitative HCV RNA assay, followed by quantitative HCV viral load determination in a subset of samples. Currently, we have identified 6.2% (10,621/170,163) anti-HCV positive individuals, of whom 74.9% (3,930/5,246) demonstrated detectable viral RNA. Quantitative test found that 97.5% (1,001/1,027) had HCV viral load ≥5,000 IU/mL. Thus, primary screening with anti-HCV RDT followed by qualitative HCV RNA evaluation could identify active and chronic HCV infection in almost all individuals with a viral load ≥5,000 IU/mL, which is the current threshold for treatment dictated by Thailand's National Health Security Office. Our data suggest that qualitative HCV RNA evaluation may obviate the need for the more expensive quantitative HCV viral load test and reduce a significant barrier toward HCV elimination in a middle-income country.

Prognostic factors for SARS-CoV 2 nasopharyngeal swab negativity: a multicentric study.

Background : The diagnosis of Covid 19 is made by the detection of viral RNA by PCR on nasopharyngeal swabs. In some patients the test is falsely negative, while other biological samples are positive. The aim of the study is to identify characteristics and prognostic factors for swab negativity in COVID-19 patients with BA-confirmed disease.
 Materials and methods : Multicentric retrospective case-control study of patients admitted for COVID-19 between March and November 2020 in two internal medicine units of the AOU Careggi and in the Internal Medicine of the Hospital of Varese. Were enrolled patients aged ≥18 years hospitalized for COVID-19 with viral RNA isolation on biological specimen, considering as cases the patients negative to swab but positive to BA. For each case, four controls with positive swab at admission were enrolled
 Results : the study included 95 patients, 19 cases and 76 controls. The mean time between symptoms onset and swab was 2.65±1.9 days in cases, with a statistically significant (p= 0.003) difference compared to controls (5.53±3.0 days). Patients with negative swab had a longer mean length of stay and more frequent adverse outcome than controls.
 Conclusions : Swabbing within a short time of symptoms onset is a predictor for false negative. Patients with repeated negative swabs have a worse clinical picture with longer hospital stay, greater need for non-invasive ventilation and higher frequency of adverse outcome.

Detection of West Nile Virus Lineage 2 in Eastern Romania and First Identification of Sindbis Virus RNA in Mosquitoes Analyzed using High-Throughput Microfluidic Real-Time PCR.

The impact of mosquito-borne diseases on human and veterinary health is being exacerbated by rapid environmental changes caused mainly by changing climatic patterns and globalization. To gain insight into mosquito-borne virus circulation from two counties in eastern and southeastern Romania, we have used a combination of sampling methods in natural, urban and peri-urban sites. The presence of 37 mosquito-borne viruses in 16,827 pooled mosquitoes was analyzed using a high-throughput microfluidic real-time PCR assay. West Nile virus (WNV) was detected in 10/365 pools of Culex pipiens (n = 8), Culex modestus (n = 1) and Aedes vexans (n = 1) from both studied counties. We also report the first molecular detection of Sindbis virus (SINV) RNA in the country in one pool of Culex modestus. WNV infection was confirmed by real-time RT-PCR (10/10) and virus isolation on Vero or C6/36 cells (four samples). For the SINV-positive pool, no cytopathic effectwas observed after infection of Vero or C6/36 cells, but no amplification was obtained in conventional SINV RT-PCR. Phylogenetic analysis of WNV partial NS5 sequences revealed that WNV lineage 2 of theCentral-Southeast European clade, has a wider circulation in Romania than previously known.

Post-translational modifications and regulations of RLR signaling molecules in cytokines-mediated response in fish

Teleosts rely on innate immunity to recognize and defense against pathogenic microorganisms. RIG-I-like receptor (RLR) family is the major pattern recognition receptor (PRR) to detect RNA viruses. After recognition of viral RNA components, these cytosolic sensors activate downstream signaling cascades to induce the expression of type I interferons (IFNs) and other cytokines firing antiviral responses. Meanwhile, numerous molecules take part in the complex regulation of RLR signals by various methods, such as post-translational modification (PTM), to produce an immune response that is appropriately balanced. In this review, we summarize our recent understanding of PTMs and other regulatory proteins in modulating RLR signaling pathway, which is helpful for systematically studying the regulatory mechanism of antiviral innate immunity of teleost fish.

Detection of RNA viruses in potato seed-tubers from northern Antioquia (Colombia)

Detection of RNA viruses in potato seed-tubers from northern Antioquia (Colombia)

Method Validation Study for SARS-CoV-2 Viral RNA Detection in Cervical, Rectal, Amniotic Fluid, Placental, Umbilical Cord Blood, and Breastmilk Specimens in a Cohort of Unvaccinated Women in Manila, Philippines.

To validate a method in detecting SARS-CoV-2 via RT-qPCR in pregnant and non-pregnant samples other than nasopharyngeal swabs and/or oropharyngeal swabs such as cervical, rectal, amniotic fluid, placental, umbilical cord blood, and breastmilk. We performed a validation experiment using MGI easy extraction kits and BGI PCR kits on non-conventional specimens, including cervical, rectal, amniotic fluid, placental, umbilical cord blood, and breastmilk to detect and confirm the presence of SARS-CoV-2. In addition, we tested the validated method on 572 purposively sampled field-collected non-conventional specimens from a cohort of 109 unvaccinated pregnant and 47 unvaccinated non-pregnant women to assess which candidate non-conventional maternal- and fetal-associated specimens may contribute to maternal-fetal viral vertical transmission. Positive detection of SARS-CoV-2 viral RNA in non-conventional specimens was demonstrated and verified. Of the 572 non-conventional samples tested, 1.8% (10/572) were positively validated by RT-qPCR for SARS-CoV-2 in the maternal-associated specimens particularly the rectal (5), placental (1), and cervical (4) swabs among six pregnant and four non-pregnant individuals. In contrast, no SARS-CoV-2 viral RNA was detected in fetal-associated specimens. The results of the validation study may serve as an additional diagnostic screening layer to support maternal-child care. Furthermore, viral detection in these non-conventional maternal specimens may also be utilized to provide guidance in the clinical management of neonates, and pregnant women during delivery.

Serum biomarker panel for disease severity and prognosis in patients with COVID-19.

Coronavirus disease-2019 (COVID-19) has become a worldwide emergency and has had a severe impact on human health. Inflammatory factors have the potential to either enhance the efficiency of host immune responses or damage the host organs with immune overreaction in COVID-19. Therefore, there is an urgent need to investigate the functions of inflammatory factors and serum markers that participate in disease progression. In total, 54 COVID-19 patients were enrolled in this study. Disease severity was evaluated by clinical evaluation, laboratory tests, and computed tomography (CT) scans. Data were collected at: admission, 3-5 days after admission, when severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA detection became negative, and composite endpoint. We found that the positive rate in sputum was three times higher than that in throat swabs. Higher levels of C-reactive protein (CRP), lactate dehydrogenase (LDH), D-dimer (D-D), interleukin-6 (IL-6) and neutrophil-to-lymphocyte ratio (NLR) or lower lymphocyte counts suggested more severe disease, and the levels of cytokines and serum markers were intrinsically correlated with disease progression. When SARS-CoV-2 RNA detection became negative, the receiver operating characteristic (ROC) curve demonstrated that LDH had the highest sensitivity independently, and four indicators (NLR, CRP, LDH, and D-D) when combined had the highest sensitivity in distinguishing critically ill patients from mild ones. Monitoring dynamic changes in NLR, CRP, LDH, IL-6, and D-D levels, combined with CT imaging and viral RNA detection in sputum, could aid in severity evaluation and prognosis prediction and facilitate COVID-19 treatment.

Multiple gene detection using a selective fluorophore probe-RNA hybridization/graphene oxide quenching system.

Herein, we introduce a novel assay for multiple-gene recognition by ligation-double transcription mediated fluorometric profiling. We demonstrated the capability of the system to identify potential multi-gene classifiers for diagnostic use by employing a combination of a ligation-double transcription approach with a selective fluorophore probe-RNA hybridization/graphene oxide quenching system. The system is efficient and requires only 45 minutes for total experimentation and offers high sensitivity (369.6, 408, and 407.8 copies per mL for the O, E, and N genes of SARS-CoV-2, respectively) and specificity (selective to until two mismatches). Our system is expected to expedite the precise diagnosis of RNA-virus-related diseases with multiple gene classifiers. By focusing on distinct viral genes, our method allowed for the detection of various RNA viruses in a variety of sample pools.

Compact wide-field femtoliter-chamber imaging system for high-speed and accurate digital bioanalysis.

The femtoliter-chamber array is a bioanalytical platform that enables highly sensitive and quantitative analysis of biological reactions at the single-molecule level. This feature has been considered a key technology for "digital bioanalysis" in the biomedical field; however, its versatility is limited by the need for a large and expensive setup such as a fluorescence microscope, which requires a long time to acquire the entire image of a femtoliter-chamber array. To address these issues, we developed a compact and inexpensive wide-field imaging system (COWFISH) that can acquire fluorescence images with a large field of view (11.8 mm × 7.9 mm) and a high spatial resolution of ∼ 3 μm, enabling high-speed analysis of sub-million femtoliter chambers in 20 s. Using COWFISH, we demonstrated a CRISPR-Cas13a-based digital detection of viral RNA of SARS-CoV-2 with an equivalent detection sensitivity (limit of detection: 480 aM) and a 10-fold reduction in total imaging time, as compared to confocal fluorescence microscopy. In addition, we demonstrated the feasibility of COWFISH to discriminate between SARS-CoV-2-positive and -negative clinical specimens with 95% accuracy, showing its application in COVID-19 diagnosis. Therefore, COWFISH can serve as a compact and inexpensive imaging system for high-speed and accurate digital bioanalysis, paving a way for various biomedical applications, such as diagnosis of viral infections.

A point-of-care microfluidic biosensing system for rapid and ultrasensitive nucleic acid detection from clinical samples.

Rapid and ultrasensitive point-of-care RNA detection plays a critical role in the diagnosis and management of various infectious diseases. The gold-standard detection method of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is ultrasensitive and accurate yet limited by the lengthy turnaround time (1-2 days). On the other hand, an antigen test offers rapid at-home detection (typically ~15 min) but suffers from low sensitivity and high false-negative rates. An ideal point-of-care diagnostic device would combine the merits of PCR-level sensitivity and rapid sample-to-result workflow comparable to antigen testing. However, the existing detection platforms typically possess superior sensitivity or rapid sample-to-result time, but not both. This paper reports a point-of-care microfluidic device that offers ultrasensitive yet rapid detection of viral RNA from clinical samples. The device consists of a microfluidic chip for precisely manipulating small volumes of samples, a miniaturized heater for viral lysis and ribonuclease inactivation, a Cas13a-electrochemical sensor for target preamplification-free and ultrasensitive RNA detection, and a smartphone-compatible potentiostat for data acquisition. As demonstrations, the devices achieve the detection of heat-inactivated SARS-CoV-2 samples with a limit of detection down to 10 aM within 25 minutes, which is comparable to the sensitivity of RT-PCR and rapidness of an antigen test. The platform also successfully distinguishes all nine positive unprocessed clinical SARS-CoV-2 nasopharyngeal swab samples from four negative samples within 25 minutes of sample-to-result time. Together, this device provides a point-of-care solution that can be deployed in diverse settings beyond laboratory environments for rapid and accurate detection of RNA from clinical samples. The device can potentially be expandable to detect other viral targets, such as human immunodeficiency virus self-testing and Zika virus, where rapid and ultrasensitive point-of-care detection is required.

VPrimer: A Method of Designing and Updating Primer and Probe With High Variant Coverage for RNA Virus Detection.

Fatal infectious diseases caused by RNA viruses, such as COVID-19, have emerged around the world. RT-PCR is widely employed for virus detection, and its accuracy depends on the primers and probes since RT-PCR can detect a virus only when the primers and probes bind to the target gene of the virus. Most of primer design methods are for a single host and so require a great deal of effort to design for RNA virus detection, including homology tests among the host and all the viruses for the host using BLAST-like tools. Furthermore, they do not consider variant sequences, which are very common in viruses. In this study, we describe VPrimer, a method of designing high-quality primer-probe sets for RNA viruses. VPrimer can find primer-probe sets that cover more than 95% of the variants of a target virus but do not cover any sequences of other viruses or the host. With VPrimer, we found 381,698,582 primer-probe sets for 3,104 RNA viruses. Multiplex PCR assays using the top 2 primer-probe sets suggested by VPrimer usually cover 100% of variants. To address the rapid changes in viral genomes, VPrimer finds the best and up-to-date primer-probe sets incrementally against the most recently reported variants.

PddCas: A Polydisperse Droplet Digital CRISPR/Cas-Based Assay for the Rapid and Ultrasensitive Amplification-Free Detection of Viral DNA/RNA.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based assays have been an emerging diagnostic technology for pathogen diagnosis. In this work, we developed a polydisperse droplet digital CRISPR-Cas-based assay (PddCas) for the rapid and ultrasensitive amplification-free detection of viral DNA/RNA with minimum instruments. LbaCas12a and LbuCas13a were used for the direct detection of viral DNA and RNA, respectively. The reaction mixtures were partitioned with a common vortex mixer to generate picoliter-scale polydisperse droplets in several seconds. The limit of detection (LoD) for the target DNA and RNA is approximately 100 aM and 10 aM, respectively, which is about 3 × 104-105 fold more sensitive than corresponding bulk CRISPR assays. We applied the PddCas to successfully detect severe acute respiratory syndrome coronavirus (SARS-CoV-2) and human papillomavirus type 18 (HPV 18) in clinical samples. For the 23 HPV 18-suspected cervical epithelial cell samples and 32 nasopharyngeal swabs for SARS-CoV-2, 100% sensitivity and 100% specificity were demonstrated. The dual-gene virus detection with PddCas was also established and verified. Therefore, PddCas has potential for point-of-care application and is envisioned to be readily deployed for frequent testing as part of an integrated public health surveillance program.

Detection of SARS-CoV-2 in Human Breast Milk.

Certain viral pathogens can be shed into the human breast milk and cause infections in the infant upon breastfeeding. Thus, it is important to clarify whether viral RNA as well as infectious virus can be found in breast milk. The complexity of this body fluid poses several challenges for viral RNA isolation and detection of infectious virus. We here provide a protocol that allowed the identification of SARS-CoV-2 RNA in breast milk and the isolation of infectious virus after the virus has been artificially spiked into milk samples.

Infectivity of SARS-CoV-2 on Inanimate Surfaces: Don’t Trust Ct Value

SARS-CoV-2 RNA is frequently identified in patient rooms and it was speculated that the viral load quantified by PCR might correlate with infectivity of surfaces. To evaluate Ct values for the prediction of infectivity, we investigated contaminated surfaces and Ct-value changes after disinfection. Viral RNA was detected on 37 of 143 investigated surfaces of an ICU. However, virus isolation failed for surfaces with a high viral RNA load. Also, SARS-CoV-2 could not be cultivated from surfaces artificially contaminated with patient specimens. In order to evaluate the significance of Ct values more precisely, we used surrogate enveloped bacteriophage Φ6. A strong reduction in Φ6 was achieved by three different disinfection methods. Despite a strong reduction in viability almost no change in the Ct values was observed for UV-C and alcoholic surface disinfectant. Disinfection using ozone resulted in a lack of Φ6 recovery as well as a detectable shift in Ct values indicating strong degradation of the viral RNA. The observed lack of significant effects on the detectable viral RNA after effective disinfection suggest that quantitative PCR is not suitable for predicting the infectivity of SARS-CoV-2 on inanimate surfaces. Ct values should therefore not be considered as markers for infectivity in this context.

Liver alterations and detection of SARS-CoV-2 RNA and proteins in COVID-19 autopsies

The most severe alterations in Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) infection are seen in the lung. However, other organs also are affected. Here, we report histopathologic findings in the liver and detection of viral proteins and RNA in COVID-19 autopsies performed at the Semmelweis University (Budapest, Hungary). Between March 2020 through March 2022, 150 autopsies on patients who died of COVID-19 were analyzed. Cause-of-death categories were formed based on the association with SARS-CoV-2 as strong, contributive, or weak. Samples for histopathologic study were obtained from all organs, fixed in formalin, and embedded in paraffin (FFPE). Immunohistochemical study (IHC) to detect SARS-CoV-2 spike protein and nucleocapsid protein (NP), CD31, claudin-5, factor VIII, macrosialin (CD68), and cytokeratin 7, with reverse transcriptase polymerase chain reaction (RT-PCR), and in situ hybridization (ISH, RNAscope®) for SARS-CoV-2 RNA were conducted using FFPE samples of livers taken from 20 autopsies performed ≤ 2 days postmortem. All glass slides were scanned; the digital images were evaluated by semiquantitative scoring and scores were analyzed statistically. Steatosis, single-cell and focal/zonal hepatocyte necrosis, portal fibrosis, and chronic inflammation were found in varying percentages. Sinusoidal ectasia, endothelial cell disruption, and fibrin-filled sinusoids were seen in all cases; these were assessed semiquantitatively for severity (SEF scored). SEF scores did not correlate with cause-of-death categories (p = 0.92) or with severity of lung alterations (p = 0.96). SARS-CoV-2 RNA was detected in 13/20 cases by PCR and in 9/20 by ISH, with IHC demonstration of spike protein in 4/20 cases and NP in 15/20. Viral RNA and proteins were located in endothelial and Kupffer cells, and in portal macrophages, but not in hepatocytes and cholangiocytes. In conclusion, endothelial damage (SEF scores) was the most common alteration in the liver and was a characteristic, but not specific alteration in COVID-19, suggesting an important role in the pathogenesis of COVID-19-associated liver disease. Detection of SARS-CoV-2 RNA and viral proteins in liver non-parenchymal cells suggests that while the most extended primary viral cytotoxic effect occurs in the lung, viral components are present in other organs too, as in the liver. The necrosis/apoptosis and endothelial damage associated with viral infection in COVID-19 suggest that those patients who survive more severe COVID-19 may face prolonged liver repair and accordingly should be followed regularly in the post-COVID period.

Breastfeeding by chikungunya virus-infected dams confers resistance to challenge in the offspring

Vertical transmission of Chikungunya virus (CHIKV) has been reported in humans, but the transmission routes have not been completely understood, and experimental animal models are needed to enable detailed investigation of the transmission and pathogenesis of congenital infections. The intertwining of immune response and virus components at the gestation/breastfeeding interfaces between mother and fetus/newborn may have effects during the offspring development. An experimental model of CHIKV was established by infecting pregnant BALB/c female mice that enabled confirmation that dams inoculated up to the 10th gestational day transmit CHIKV transplacentally to approximately 8.4% of the fetuses, resulting in severe teratogenic effects. CHIKV neutralizing antibodies were detected in sera from adult mice born to healthy females and breastfed by CHIKV-infected dams, while no neutralization was detected in sera from animals born to CHIKV-infected dams. Moreover, adult mice born to healthy dams and cross-fostered for breastfeeding by CHIKV-infected dams were resistant to challenge with CHIKV on the 90th day after birth. The animals also had reduced viral loads in brain and spleen as compared to controls. There was expression of fluorescent CHIKV non-structural protein, and detection of viral RNA by RT-PCR in breast tissue from infected dams. CHIKV RNA and proteins were also detected in breast milk retrieved from the stomachs of recently fed newborns. The experimental results were also complemented by the finding of CHIKV RNA in 6% of colostrum samples from healthy lactating women in a CHIKV-endemic area. Breastfeeding induces immune protection to challenge with CHIKV in mice.

Matrix-Encoding Gene Diversity of 624 Influenza A/H3N2 Genomes Does Not Show Association with Impaired Viral Detection by Commercialized qPCR Assays

As for the case of SARS-CoV-2, genome sequencing of influenza viruses is of potential interest to raise and address virological issues. Recently, false-negativity of real-time reverse transcription-PCR (qPCR) assays that detect influenza A/H3N2 virus RNA were reported and associated with two mutations (A37T and C161T) in the Matrix-encoding (M1) gene located on viral segment 7. This triggered a national alert in France. The present study sought to assess the association between the presence of these mutations and potential false negative results of influenza A/H3N2 virus RNA detection by commercialized qPCR assays at the clinical virology laboratory of our university hospitals in southern France. This study focused on the genetic diversity in the M1 gene and segment 7 of 624 influenza A/H3N2 virus genomes obtained from respiratory samples having tested qPCR-positive with M1 gene-targeting assays in our clinical virology laboratory. A total of 585 among the 624 influenza A/H3N2 virus genomes (93.7%) were of clade 3C.2a1b.2a.2, and 39 (6.3%) were of clade 3C.2a1b.1a. M1 gene substitutions A37T and C161T were both present in 582 (93.3%) genomes, only of clade 3C.2a1b.2a.2. Substitution A37T was present in 621 (99.5%) genomes. Substitution C161T was present in 585 genomes (93.8%), all of clade 3C.2a1b.2a.2. Moreover, 21 other nucleotide positions were mutated in ≥90% of the genomes. The present study shows that A37T/C and C161T mutations, and other mutations in the M1 gene and segment 7, were widely present in influenza A/H3N2 virus genomes recovered from respiratory samples diagnosed qPCR-positive with commercialized assays.