Hesperetin blocks poxvirus replication with a low tendency to select for drug-resistant viral variants.

In this study, we demonstrated the antiviral efficacy of hesperetin against multiple poxviruses, including buffalopox virus (BPXV), vaccinia virus (VACV), and lumpy skin disease virus (LSDV). The time-of-addition and virus step-specific assays indicated that hesperetin reduces the levels of viral DNA, mRNA, and proteins in the target cells. Further, by immunoprecipitation (IP) of the viral RNA from BPXV-infected Vero cells and a cell-free RNA-IP assay, we demonstrated that hesperetin-induced reduction in BPXV protein synthesis is also consistent with diminished interaction between eukaryotic translation initiation factor eIF4E and the 5' cap of viral mRNA. Molecular docking and MD simulation studies were also consistent with the binding of hesperetin to the cap-binding pocket of eIF4E, adopting a conformation similar to m7GTP binding. Furthermore, in a BPXV egg infection model, hesperetin was shown to suppress the development of pock lesions on the chorioallantoic membrane and associated mortality in the chicken embryos. Most importantly, long-term culture of BPXV in the presence of hesperetin did not induce the generation of drug-resistant viral mutants. In conclusion, we, for the first time, demonstrated the antiviral activity of hesperetin against multiple poxviruses, besides providing some insights into its potential mechanisms of action.

Resistance Evolution against Host-directed Antiviral Agents: Buffalopox Virus Switches to Use p38-ϒ under Long-term Selective Pressure of an Inhibitor Targeting p38-α.

Host-dependency factors have increasingly been targeted to minimize antiviral drug resistance. In this study, we have demonstrated that inhibition of p38 mitogen-activated protein kinase (a cellular protein) suppresses buffalopox virus (BPXV) protein synthesis by targeting p38-MNK1-eIF4E signaling pathway. In order to provide insights into the evolution of drug resistance, we selected resistant mutants by long-term sequential passages (P; n = 60) in the presence of p38 inhibitor (SB239063). The P60-SB239063 virus exhibited significant resistance to SB239063 as compared to the P60-Control virus. To provide mechanistic insights on the acquisition of resistance by BPXV-P60-SB239063, we generated p38-α and p38-ϒ (isoforms of p38) knockout Vero cells by CRISPR/Cas9-mediated genome editing. It was demonstrated that unlike the wild type (WT) virus which is dependent on p38-α isoform, the resistant virus (BPXV-P60-SB239063) switches over to use p38-ϒ so as to efficiently replicate in the target cells. This is a rare evidence wherein a virus was shown to bypass the dependency on a critical cellular factor under selective pressure of a drug.

miRNA profiling of primary lamb testicle cells infected with lumpy skin disease virus.

In this study, miRNA profiling of cells infected with lumpy skin disease virus (LSDV) was conducted for the first time. When compared to mock-infected cells, LSDV-infected primary lamb testicle (LT) cells showed dysregulation of 64, 85, and 85 miRNAs at 12 hours postinfection (hpi), 48 hpi, and 72 hpi, respectively. While some of these miRNAs were found to be dysregulated at a particular time point following LSDV infection, others were dysregulated at all three time points. Analysis of the differentially expressed miRNA-mRNA interaction networks, Gene Ontology analysis of the predicted targets, and KEGG analysis of highly enriched pathways revealed several cellular factors/pathways involved in protein/ion/enzyme binding, cell differentiation, movement of subcellular components, calcium reabsorption, aldosterone synthesis and secretion, and melanogenesis. Some selected upregulated (oar-mir-379-5p, oar-let-7d, Chr10-18769, Chr2_5162 and oar-miR-493-5p) and downregulated (ChrX-33741, Chr3_8257 and Chr26_32680) miRNAs were further confirmed by quantitative real-time PCR. These findings contribute to our understanding of virus replication, virus-host interactions, and disease pathogenesis, and the differentially expressed miRNAs and their cellular targets may serve as biomarkers as well as novel targets for therapeutic intervention against LSDV.

Role of p38 mitogen-activated protein kinase signalling in virus replication and potential for developing broad spectrum antiviral drugs.

Mitogen-activated protein kinases (MAPKs) play a key role in complex cellular processes such as proliferation, development, differentiation, transformation and apoptosis. Mammals express at least four distinctly regulated groups of MAPKs which include extracellular signal-related kinases (ERK)-1/2, p38 proteins, Jun amino-terminal kinases (JNK1/2/3) and ERK5. p38 MAPK is activated by a wide range of cellular stresses and modulates activity of several downstream kinases and transcription factors which are involved in regulating cytoskeleton remodeling, cell cycle modulation, inflammation, antiviral response and apoptosis. In viral infections, activation of cell signalling pathways is part of the cellular defense mechanism with the basic aim of inducing an antiviral state. However, viruses can exploit enhanced cell signalling activities to support various stages of their replication cycles. Kinase activity can be inhibited by small molecule chemical inhibitors, so one strategy to develop antiviral drugs is to target these cellular signalling pathways. In this review, we provide an overview on the current understanding of various cellular and viral events regulated by the p38 signalling pathway, with a special emphasis on targeting these events for antiviral drug development which might identify candidates with broad spectrum activity.

Prevalence of Antineutrophil Cytoplasmic Antibodies and Antinuclear Antibodies in Patients with Pulmonary Tuberculosis: A Tertiary Care Center Experience from North India.

BACKGROUND: Tuberculosis (TB) can have manifestations closely mimicking autoimmune diseases. The prevalence of autoantibodies in TB varies among different populations. OBJECTIVES: To study the prevalence of anti-neutrophilic cytoplasmic antibodies (ANCA) and antinuclear antibodies (ANA) in pulmonary tuberculosis (PTB). METHODS: This was a cross-sectional, observational study. Subjects with microbiologically confirmed PTB, either via smear or culture positivity on sputum or bronchoalveolar lavage (BAL) fluid, or positive rapid diagnostic tests were included. ANCA against proteinase-3 (PR3), myeloperoxidase (MPO), lactoferrin, and elastase were tested using an enzyme-linked immunosorbent assay (ELISA). ANA was detected using indirect immunofluorescence (IIF). RESULTS: Eighty-nine subjects with a median [interquartile range (IQR)] age of 28 (20-46) years, 67.4% males, were recruited. Eighty-one subjects had microbiological confirmation on sputum examination, and eight required examination of BAL fluid. Sera were drawn from 62 treatment-naïve subjects, the rest (27) were on antitubercular therapy (ATT). Eighty-six (96.6%) subjects tested positive for anti-elastase antibody, seven of which were also positive for anti-PR3. None were positive for anti-MPO and anti-lactoferrin. Six (6.7%) subjects tested positive for ANA. None of the subjects had features of underlying connective tissue disease or vasculitis. CONCLUSION: PTB patients showed a high prevalence of anti-elastase and a low prevalence of ANA and anti-PR3 antibodies. ANCA positivity should be interpreted with caution in TB endemic areas. The role of anti-elastase antibodies in differentiating TB from ANCA-associated vasculitis (AAV) needs further research.

Scrub typhus cholecystitis.

Acute acalculous cholecystitis is usually seen in association with systemic medical illness, or after surgery, trauma or burn and is considered as a more severe disease than acute calculous cholecystitis. We recently had the opportunity of observing a patient who was admitted in a surgical emergency with clinical features of acute cholecystitis and was found to have Orientia tsutsugamushi infection after a strong clinical suspicion of acute tropical fever illness.

Molecular Diagnostic Field Test for Point-of-Care Detection of Ebola Virus Directly From Blood.

A molecular diagnostic method for robust detection of Ebola virus (EBOV) at the point of care (POC) directly from blood samples is described. This assay is based on reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) of the glycoprotein gene of EBOV. Complete reaction formulations were lyophilized in 0.2-mL polymerase chain reaction tubes. RT-LAMP reactions were performed on a battery-operated isothermal instrument. Limit of detection of this RT-LAMP assay was 2.8 × 102 plaque-forming units (PFU)/test and 1 × 103 PFU/test within 40 minutes for EBOV-Kikwit and EBOV-Makona, respectively. This assay was found to be specific for the detection of EBOV, as no nonspecific amplification was detected in blood samples spiked with closely related viruses and other pathogens. These results showed that this diagnostic test can be used at the point of care for rapid and specific detection of EBOV directly from blood with high sensitivity within 40 minutes.

Molecular Epidemiology of Novel Pathogen "Brachyspira hampsonii" Reveals Relationships between Diverse Genetic Groups, Regions, Host Species, and Other Pathogenic and Commensal Brachyspira Species.

Outbreaks of bloody diarrhea in swine herds in the late 2000s signaled the reemergence of an economically significant disease, swine dysentery, in the United States. Investigations confirmed the emergence of a novel spirochete in swine, provisionally designated "Brachyspira hampsonii," with two genetically distinct clades. Although it has since been detected in swine and migratory birds in Europe and North America, little is known about its genetic diversity or its relationships with other Brachyspira species. This study characterizes B. hampsonii using a newly developed multilocus sequence typing (MLST) approach and elucidates the diversity, distribution, population structure, and genetic relationships of this pathogen from diverse epidemiological sources globally. Genetic characterization of 81 B. hampsonii isolates, originating from six countries, with our newly established MLST scheme identified a total of 20 sequence types (STs) belonging to three clonal complexes (CCs). B. hampsonii showed a heterogeneous population structure with evidence of microevolution locally in swine production systems, while its clustering patterns showed associations with its epidemiological origins (country, swine production system, and host species). The close genetic relatedness of B. hampsonii isolates from different countries and host species highlights the importance of strict biosecurity control measures. A comparative analysis of 430 isolates representing seven Brachyspira species (pathogens and commensals) from 19 countries and 10 host species depicted clustering by microbial species. It revealed the close genetic relatedness of B. hampsonii with commensal Brachyspira species and also provided support for the two clades of B. hampsonii to be considered a single species.

Does the management of patients with myocardial infarction with nonobstructive coronary arteries (MINOCA) changes with advanced diagnostic workup beyond coronary angiography? Results from the "Evaluation of the clinical Profile, Investigations and Cardiac Imaging of the Patients with MINOCA (EPIC-MINOCA Study)".

BACKGROUND: Evaluation of the patients with MINOCA and identifying the underlying aetiology remains challenging. However, investigation in most patients remains limited to coronary angiography (CAG). The study aimed to assess the clinical profile, investigations and cardiac imaging of the patients with MINOCA and its outcomes. RESULTS: Out of 55 patients with MINOCA, CAG was normal in 16 (29.1%), while 39 (69.9%) had nonobstructive coronary artery disease. Of 55 patients, 34 had limited workup (Group 1) and only 21 had advanced workup (Group 2). In comparison to Group 1, Group 2 had a significantly higher association with the identification of possible underlying aetiology (16 vs. 4, p < 0.001) and a change in the management (10 vs. 3, p = 0.002). CONCLUSION: Diagnostic workup in patients with MINOCA was limited to CAG in 61.8% of patients in this study. However, patients with advanced workup had a significantly higher association with the change in the treatment and identifying possible underlying aetiology in such patients.

p38-MAPK is prerequisite for the synthesis of SARS-CoV-2 protein.

The inhibition of p38 mitogen-activated protein kinase (p38-MAPK) by small molecule chemical inhibitors was previously shown to impair severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication, however, mechanisms underlying antiviral activity remains unexplored. In this study, reduced growth of SARS-CoV-2 in p38-α knockout Vero cells, together with enhanced viral yield in cells transfected with construct expressing p38α, suggested that p38-MAPK is essential for the propagation of SARS-CoV-2. The SARS-CoV-2 was also shown to induce phosphorylation (activation) of p38, at time when transcription/translational activities are considered to be at the peak levels. Further, we demonstrated that p38 supports viral RNA/protein synthesis without affecting viral attachment, entry, and budding in the target cells. In conclusion, we provide mechanistic insights on the regulation of SARS-CoV-2 replication by p38 MAPK.

First Isolation and Genetic Characterization of Avian Nephritis Virus 4 from Commercial Poultry in India.

Avian nephritis virus (ANV), which belongs to the family Astroviridae, is associated with different clinical manifestations (enteric and kidney disorders) in poultry. Despite being a significant pathogen of the avian industry worldwide, information regarding genetic features of these viruses in India is scarce. In this study, 386 intestinal samples collected from 37 slaughterhouses in two north Indian states (Rajasthan and Haryana) were screened for ANV with reverse transcription PCR (RT-PCR) targeting the conserved ORF1b gene, followed by nucleotide sequencing of the amplified product. RT-PCR and sequencing confirmed the presence of ANV in 32 clinical samples (8.29%), with concurrent infections of infectious bronchitis virus, chicken astrovirus, and fowl adenoviruses observed in some clinical samples (n = 4). Virus isolations were successful from four out of 12 ANV-positive clinical samples passaged via the yolk-sac route in specific-pathogen-free embryonated chicken eggs. Additionally, the near-complete genomes of two viruses were determined through sequencing. Phylogenetic analysis based on full-length capsid protein sequences classified the viruses into ANV genotype 4 (ANV4), and to the best of our knowledge this is the first report of ANV4 from India. This study revealed the presence and circulation of new strains of ANV in Indian poultry. Genetic profiling and isolation of the viruses in this study will not only aid in the development of diagnostic tools and vaccines for ANV but also offer valuable insights into its epidemiology.

Primer aislamiento y caracterización genética del virus de la nefritis aviar 4 en la avicultura comercial de la India El virus de la nefritis aviar (ANV), que pertenece a la familia Astroviridae, se asocia con diferentes manifestaciones clínicas (trastornos entéricos y renales) en la avicultura. A pesar de ser un patógeno importante de la industria avícola en todo el mundo, la información sobre las características genéticas de estos virus en la India es escasa. En este estudio, se analizaron 386 muestras intestinales recolectadas en 37 plantas de procesamiento en dos estados del norte de la India (Rajasthan y Haryana) para detectar al virus de la nefritis aviar mediante un método de RT-PCR dirigido al gene conservado ORF1b, seguido de la secuenciación de nucleótidos del producto amplificado. El método de RT-PCR y la secuenciación confirmaron la presencia del virus de la nefritis aviar en 32 muestras clínicas (8.29%), observándose infecciones concurrentes por el virus de la bronquitis infecciosa, astrovirus del pollo y adenovirus de las aves en algunas muestras clínicas (n = 4). Los aislamientos del virus tuvieron éxito en cuatro de las 12 muestras clínicas positivas para el virus de la nefritis aviar inoculadas a través de la ruta del saco vitelino en huevos de gallina embrionados libres de patógenos específicos. Además, se determinaron los genomas casi completos de dos virus mediante secuenciación. El análisis filogenético basado en secuencias completas de proteínas de la cápside clasificó los virus en el genotipo 4 del virus de la nefritis aviar (ANV4) y hasta donde se sabe, este es el primer informe del virus de la nefritis aviar 4 en la India. Este estudio reveló la presencia y circulación de nuevas cepas del virus de la nefritis aviar en la avicultura comercial de la India. El perfil genético y el aislamiento de los virus en este estudio no solo ayudarán en el desarrollo de herramientas de diagnóstico y vacunas para el virus de la nefritis aviar, sino que también ofrecerán información valiosa sobre su epidemiología.

Identification of miR-29a as a novel biomarker for lumpy skin disease virus exposure in cattle.

Micro RNAs (miRNAs) have been implicated in the regulation of maturation, proliferation, differentiation, and activation of immune cells. In this study, we demonstrated that miR-29a antagonizes IFN-γ production at early times post-LSDV infection in cattle. miR-29a was predicted to target upstream IFN-γ regulators, and its inhibition resulted in enhanced IFN-γ production in sensitized peripheral blood mononuclear cells (PBMCs). Further, stimulation of PBMCs with LSDV antigen exhibited lower levels of miR-29a, concomitant with a potent cell-mediated immune response (CMI), characterized by an increase in LSDV-specific CD8+ T cell counts and enhanced levels of IFN-γ, which eventually facilitated virus clearance. In addition, a few immunocompromised cattle (developed secondary LSDV infection at ~ 6 months) that failed to mount a potent cell-mediated immune response, were shown to maintain higher miR-29a levels. Furthermore, as compared to the sensitized crossbred cattle, PBMCs from sensitized Rathi (a native Indian breed) animals exhibited lower levels of miR-29a along with an increase in CD8+ T cell counts and enhanced levels of IFN-γ. Finally, we analysed that a ≥ 60% decrease in miR-29a expression levels in the PBMCs of sensitized cattle correlated with a potent CMI response. In conclusion, miR-29a expression is involved in antagonizing the IFN-γ response in LSDV-infected cattle and may serve as a novel biomarker for the acute phase of LSDV infection, as well as predicting the functionality of T cells in sensitized cattle. In addition, Rathi cattle mount a more potent CMI response against LSDV than crossbred cattle.

Isolation and molecular characterisation of a pestivirus from goats in Egypt.

Nine fetuses and neonates from sheep and goats in Egypt were screened for pestiviruses using immunohistochemistry (IHC), virus isolation, and reverse transcription-polymerase chain reaction (RT-PCR). Two goat kids with typical border disease (BD) were positive for pestivirus infection by immunohistochemistry (IHC) using polyclonal anti-BDV serum but not when four different monoclonal antibodies (MAbs) were used. On inoculation in MDBK cells, a cytopathic bovine viral diarrhoea virus (BVDV) was isolated from one of the two kids. PCR amplification followed by sequencing of the 5'-UTR region confirmed it as BVDV subtype 1b. Although the circulating virus in Egypt is considered to be BVDV 1a, this report confirms the existence of BVDV 1b in addition to BVDV 1a. To our knowledge, this is the first report of isolation of a pestivirus from goats in Egypt and is probably the second report worldwide of a goat kid showing central nervous signs associated with border disease.

A novel HRM-based gap-qRT-PCR for identification and quantitation of the vaccine and field strain(s) of lumpy skin disease virus.

Lumpy skin disease (LSD) has become the most important animal health problem in India due to high morbidity, mortality and production losses caused by it. A homologous live-attenuated LSD vaccine (Lumpi-ProVacInd) was recently developed by using a local LSD virus (LSDV) strain (LSDV/2019/India/Ranchi) in India which is likely to replace the existing practice of vaccinating cattle with goatpox vaccine. It is essential to differentiate the vaccine and field strains, if a live-attenuated vaccine has been used for control and eradication of the disease. As compared to the prevailing vaccine and field/virulent strains, the Indian vaccine strain (Lumpi-ProVacInd) has a unique deletion of 801 nucleotides in its inverted terminal repeat (ITR) region. We exploited this unique feature and developed a novel high resolution melting-based gap quantitative real-time PCR (HRM-gap-qRT-PCR) for rapid identification and quantitation of the vaccine and field strain(s) of LSDV.

Evidence of lumpy skin disease virus infection in camels.

Countries in the Indian subcontinent are currently facing a deadly epidemic of lumpy skin disease (LSD).  LSD is primarily a disease of cattle. Buffaloes may sometimes develop mild illness, however, other domestic animals are considered resistant to LSD. We confirmed the LSDV infection in camels as evidenced by skin nodules on the body surface of the affected camels, isolation of LSD virus (LSDV) and amplification of LSDV-specific gene segments from the skin nodules (PCR), nucleotide sequencing of the viral genome and, demonstration of anti-LSDV antibodies in serum. Phylogenetic analysis based on nucleotide sequencing of ORF011, ORF012 and ORF036 revealed that the virus (LSDV/Camel/India/2022/Bikaner) is related to the historical NI-2490/Kenya/KSGP-like field strains which are predominantly circulating in the Indian subcontinent. This is the first report wherein LSDV has been to infect camels.

Evaluation of the safety, immunogenicity and efficacy of a new live-attenuated lumpy skin disease vaccine in India.

Lumpy skin disease (LSD) was reported for the first time in India in 2019 and since then, it has become endemic. Since a homologous (LSD-virus based) vaccine was not available in the country, goatpox virus (GPV)-based heterologous vaccine was authorized for mass immunization to induce protection against LSD in cattle. This study describes the evaluation of safety, immunogenicity and efficacy of a new live-attenuated LSD vaccine developed by using an Indian field strain, isolated in 2019 from cattle. The virus was attenuated by continuous passage (P = 50) in Vero cells. The vaccine (50th LSDV passage in Vero cells, named as Lumpi-ProVacInd) did not induce any local or systemic reaction upon its experimental inoculation in calves (n = 10). At day 30 post-vaccination (pv), the vaccinated animals were shown to develop antibody- and cell-mediated immune responses and exhibited complete protection upon virulent LSDV challenge. A minimum Neethling response (0.018% animals; 5 out of 26,940 animals) of the vaccine was observed in the field trials conducted in 26,940 animals. There was no significant reduction in the milk yield in lactating animals (n = 10108), besides there was no abortion or any other reproductive disorder in the pregnant animals (n = 2889). Sero-conversion was observed in 85.18% animals in the field by day 30 pv.

A multiplex RT-PCR for the detection of astrovirus, rotavirus, and reovirus in turkeys.

This study was undertaken to develop and validate a multiplex reverse transcription-polymerase chain reaction (mRT-PCR) for simultaneous detection of avian rotavirus, turkey astrovirus-2 (TAstV-2), and avian reovirus. Primers targeting the conserved regions of NSP4 gene of avian rotavirus, polymerase gene of TAstV-2, and S4 gene of avian reovirus were used. The position of bands at 630, 802, and 1120 base pairs on agarose gel confirmed the presence of rotavirus, TAstV-2, and reovirus, respectively. This mRT-PCR was found to be specific as no amplification was observed with avian influenza virus, Newcastle disease virus, turkey coronavirus, avian metapneumovirus, and intestinal contents of uninfected turkey poults. Intestinal contents of poults from flocks suspected of exhibiting "poult enteritis syndrome" were pooled and tested. Of the 120 pooled samples tested, 70% were positive for TAstV-2, 45% for avian rotavirus, and 18% for avian reovirus. These three viruses were detected alone or in different combinations. Of the samples tested, 20% were negative for these three viruses, 38% were positive for a single virus (TAstV or rotavirus or reovirus), and 42% were positive for two or three viruses. This single-tube mRT-PCR assay has the potential to serve as a rapid diagnostic method for the simultaneous detection of the three enteric viruses in turkeys.

ROCK1/MLC2 inhibition induces decay of viral mRNA in BPXV infected cells.

Rho-associated coiled-coil containing protein kinase 1 (ROCK1) intracellular cell signaling pathway regulates cell morphology, polarity, and cytoskeletal remodeling. We observed the activation of ROCK1/myosin light chain (MLC2) signaling pathway in buffalopox virus (BPXV) infected Vero cells. ROCK1 depletion by siRNA and specific small molecule chemical inhibitors (Thiazovivin and Y27632) resulted in a reduced BPXV replication, as evidenced by reductions in viral mRNA/protein synthesis, genome copy numbers and progeny virus particles. Further, we demonstrated that ROCK1 inhibition promotes deadenylation of viral mRNA (mRNA decay), mediated via inhibiting interaction with PABP [(poly(A)-binding protein] and enhancing the expression of CCR4-NOT (a multi-protein complex that plays an important role in deadenylation of mRNA). In addition, ROCK1/MLC2 mediated cell contraction, and perinuclear accumulation of p-MLC2 was shown to positively correlate with viral mRNA/protein synthesis. Finally, it was demonstrated that the long-term sequential passage (P = 50) of BPXV in the presence of Thiazovivin does not select for any drug-resistant virus variants. In conclusion, ROCK1/MLC2 cell signaling pathway facilitates BPXV replication by preventing viral mRNA decay and that the inhibitors targeting this pathway may have novel therapeutic effects against buffalopox.

S-adenosylmethionine-dependent methyltransferase inhibitor DZNep blocks transcription and translation of SARS-CoV-2 genome with a low tendency to select for drug-resistant viral variants.

We report the in vitro antiviral activity of DZNep (3-Deazaneplanocin A; an inhibitor of S-adenosylmethionine-dependent methyltransferase) against SARS-CoV-2, besides demonstrating its protective efficacy against lethal infection of infectious bronchitis virus (IBV, a member of the Coronaviridae family). DZNep treatment resulted in reduced synthesis of SARS-CoV-2 RNA and proteins without affecting other steps of viral life cycle. We demonstrated that deposition of N6-methyl adenosine (m6A) in SARS-CoV-2 RNA in the infected cells recruits heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), an RNA binding protein which serves as a m6A reader. DZNep inhibited the recruitment of hnRNPA1 at m6A-modified SARS-CoV-2 RNA which eventually suppressed the synthesis of the viral genome. In addition, m6A-marked RNA and hnRNPA1 interaction was also shown to regulate early translation to replication switch of SARS-CoV-2 genome. Furthermore, abrogation of methylation by DZNep also resulted in defective synthesis of the 5' cap of viral RNA, thereby resulting in its failure to interact with eIF4E (a cap-binding protein), eventually leading to a decreased synthesis of viral proteins. Most importantly, DZNep-resistant mutants could not be observed upon long-term sequential passage of SARS-CoV-2 in cell culture. In summary, we report the novel role of methylation in the life cycle of SARS-CoV-2 and propose that targeting the methylome using DZNep could be of significant therapeutic value against SARS-CoV-2 infection.

Sequence analysis of the neuraminidase genes of avian influenza A viruses isolated from live bird markets in the United States.

Neuraminidase (NA) gene of avian influenza viruses isolated from Live Bird Markets (LBMs) on the east coast of the United States was sequenced and analyzed for mutations associated with antiviral resistance. In total, 189 isolates collected from 1994 to 2005 were used in this study. Full length sequences of the NA gene were obtained from 183 of the 189 isolates. Four different lengths of NA gene were observed; 40 isolates had full length (about 1400 nt), 132 isolates had a deletion of 48 nt, 10 isolates had a deletion of 66 nt, and one isolate had a deletion of 72 nt. Amino acid analysis of the sequence data showed point mutations distributed throughout the gene length. None of these deletions was in the catalytic region and most of the mutations were observed in the flanking regions. None of the isolates had mutations which are known to confer antiviral resistance. These results indicate that though NA gene is prone to mutational changes, much of those changes occur outside the catalytic domain.