The previous outbreaks of SARS-CoV and MERS-CoV have led researchers to study the role of diagnostics in impediment of further spread and transmission. With the recent emergence of the novel SARS-CoV-2, the availability of rapid, sensitive, and reliable diagnostic methods is essential for disease control. Hence, we have developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the specific detection of SARS-CoV-2. The primer sets for RT-LAMP assay were designed to target the nucleocapsid gene of the viral RNA, and displayed a detection limit of 10² RNA copies close to that of qRT-PCR. Notably, the assay has exhibited a rapid detection span of 30 min combined with the colorimetric visualization. This test can detect specifically viral RNAs of the SARS-CoV-2 with no cross-reactivity to related coronaviruses, such as HCoV-229E, HCoV-NL63, HCoV-OC43, and MERS-CoV as well as human infectious influenza viruses (type B, H1N1pdm, H3N2, H5N1, H5N6, H5N8, and H7N9), and other respiratory disease-causing viruses (RSVA, RSVB, ADV, PIV, MPV, and HRV). Furthermore, the developed RT-LAMP assay has been evaluated using specimens collected from COVID-19 patients that exhibited high agreement to the qRT-PCR. Our RT-LAMP assay is simple to perform, less expensive, time-efficient, and can be used in clinical laboratories for preliminary detection of SARS-CoV-2 in suspected patients. In addition to the high sensitivity and specificity, this isothermal amplification conjugated with a single-tube colorimetric detection method may contribute to the public health responses and disease control, especially in the areas with limited laboratory capacities.

Reassortment events among influenza viruses occur naturally and may lead to the development of new and different subtypes which often ignite the possibility of an influenza outbreak. Between 2008 and 2010, highly pathogenic avian influenza (HPAI) H5 of the N1 subtype from the A/goose/Guangdong/1/96-like (Gs/GD) lineage generated novel reassortants by introducing other neuraminidase (NA) subtypes reported to cause most outbreaks in poultry. With the extensive divergence of the H5 hemagglutinin (HA) sequences of documented viruses, the WHO/FAO/OIE H5 Evolutionary Working Group clustered these viruses into a systematic and unified nomenclature of clade 2.3.4.4 currently known as “H5Nx” viruses. The rapid emergence and circulation of these viruses, namely, H5N2, H5N3, H5N5, H5N6, H5N8, and the regenerated H5N1, are of great concern based on their pandemic potential. Knowing the evolution and emergence of these novel reassortants helps to better understand their complex nature. The eruption of reports of each H5Nx reassortant through time demonstrates that it could persist beyond its usual seasonal activity, intensifying the possibility of these emerging viruses’ pandemic potential. This review paper provides an overview of the emergence of each novel HPAI H5Nx virus as well as its current epidemiological distribution.

MicroRNAs (miRNAs) are known to regulate various biological processes, including expression of cellular gene and virus-induced inflammation. Recently, studies have indicated that some miRNAs could regulate influenza virus replication. Due to differential sensitivities of influenza A virus strains to different species (avian and mammalian), variations in host responses may be observed. Therefore, we investigated and compared the differences in global host miRNA expression in mouse lungs infected with wild type low pathogenicity A/Aquatic bird/Korea/w81/2005 (H5N2) (w81) or mouse-adapted virulent A/Aquatic bird /Korea/ma81/2007 (H5N2) (ma81) virus. Although the mice infected with ma81 exhibited much greater mortality than w81-infected mice, the parental w81 virus induced a higher number of differentially expressed miRNAs compared to the ma81 virus. Between these 2 viruses, a total of 27 and 20 miRNAs were commonly expressed at 1 dpi and 3 dpi, respectively. It is noteworthy that only 9 miRNAs (miR-100-5p, miR-130a-5p, miR-146b-3p, miR-147-3p, miR-151-5p, miR-155-3p, miR-223-3p, miR-301a-3p, and miR-495-3p) were significantly upregulated in both lungs infected with either wild type w81 or the mouse-adapted ma81 strain at both time points. Notably, expression levels of miR-147-3p, miR-151-5p, miR-155-3p, and miR-223-3p were higher in the lungs of mice infected with the ma81 virus than those infected with the w81 virus. To identify potential roles of these miRNAs in regulating influenza virus replication, each group of mice was intranasally treated with each inhibitor of specifically targeting 4 miRNAs, and then challenged with 5 mouse lethal dose 50% (MLD50) of the virulent ma81 virus on the following day. Although the specific miRNA inhibitors could not completely attenuate mortality or reduce viral replication, the miR-151-5p- and miR-223-3p-inhibitors reduced mortality of inoculated mice to 70% and substantially delayed death. Our results suggest that the mammalian adaptation of avian influenza A virus results in a different miRNA expression pattern in lungs of virus-infected mice compared with its parental strain, and use of specific miRNA inhibitors to target genes associated with the immune response or cell death may affect virulence and virus replication.

Several subtypes of avian influenza viruses (AIVs) are emerging as novel human pathogens, and the frequency of related infections has increased in recent years. Although neuraminidase (NA) inhibitors (NAIs) are the only class of antiviral drugs available for therapeutic intervention for AIV-infected patients, studies on NAI resistance among AIVs have been limited, and markers of resistance are poorly understood. Previously, we identified unique NAI resistance substitutions in AIVs of the N3, N7, and N9 NA subtypes. Here, we report profiles of NA substitutions that confer NAI resistance in AIVs of the N4, N5, N6, and N8 NA subtypes using gene-fragmented random mutagenesis. We generated libraries of mutant influenza viruses using reverse genetics (RG) and selected resistant variants in the presence of the NAIs oseltamivir carboxylate and zanamivir in MDCK cells. In addition, two substitutions, H274Y and R292K (N2 numbering), were introduced into each NA gene for comparison. We identified 37 amino acid substitutions within the NA gene, 16 of which (4 in N4, 4 in N5, 4 in N6, and 4 in N8) conferred resistance to NAIs (oseltamivir carboxylate, zanamivir, or peramivir) as determined using a fluorescence-based NA inhibition assay. Substitutions conferring NAI resistance were mainly categorized as either novel NA subtype specific (G/N147V/I, A246V, and I427L) or previously reported in other subtypes (E119A/D/V, Q136K, E276D, R292K, and R371K). Our results demonstrate that each NA subtype possesses unique NAI resistance markers, and knowledge of these substitutions in AIVs is important in facilitating antiviral susceptibility monitoring of NAI resistance in AIVs.IMPORTANCEThe frequency of human infections with avian influenza viruses (AIVs) has increased in recent years. Despite the availability of vaccines, neuraminidase inhibitors (NAIs), as the only available class of drugs for AIVs in humans, have been constantly used for treatment, leading to the inevitable emergence of drug-resistant variants. To screen for substitutions conferring NAI resistance in AIVs of N4, N5, N6, and N8 NA subtypes, random mutations within the target gene were generated, and resistant viruses were selected from mutant libraries in the presence of individual drugs. We identified 16 NA substitutions conferring NAI resistance in the tested AIV subtypes; some are novel and subtype specific, and others have been previously reported in other subtypes. Our findings will contribute to an increased and more comprehensive understanding of the mechanisms of NAI-induced inhibition of influenza virus and help lead to the development of drugs that bind to alternative interaction motifs.