Codon usage bias of goose circovirus and its adaptation to host.

Goose circovirus (GoCV), a potential immunosuppressive virus possessing a circular single-stranded DNA genome, is widely distributed in both domesticated and wild geese. This virus infection causes significant economic losses in the waterfowl industry. The codon usage patterns of viruses reflect the evolutionary history and genetic architecture, allowing them to adapt quickly to changes in the external environment, particularly to their hosts. In this study, we retrieved the coding sequences (Rep and Cap) and the genome of GoCV from GenBank, conducting comprehensive research to explore the codon usage patterns in 144 GoCV strains. The overall codon usage of the GoCV strains was relatively similar and exhibited a slight bias. The effective number of codons (ENC) indicated a low overall extent of codon usage bias (CUB) in GoCV. Combined with the base composition and relative synonymous codon usage (RSCU) analysis, the results revealed a bias toward A- and G-ending codons in the overall codon usage. Analysis of the ENC-GC3s plot and neutrality plot suggested that natural selection plays an important role in shaping the codon usage pattern of GoCV, with mutation pressure having a minor influence. Furthermore, the correlations between ENC and relative indices, as well as correspondence analysis (COA), showed that hydrophobicity and geographical distribution also contribute to codon usage variation in GoCV, suggesting the possible involvement of natural selection. In conclusion, GoCV exhibits comparatively slight CUB, with natural selection being the major factor shaping the codon usage pattern of GoCV. Our research contributes to a deeper understanding of GoCV evolution and its host adaptation, providing valuable insights for future basic studies and vaccine design related to GoCV.

Functional Involvement of circRNAs in the Innate Immune Responses to Viral Infection.

Effective viral clearance requires fine-tuned immune responses to minimize undesirable inflammatory responses. Circular RNAs (circRNAs) are a class of non-coding RNAs that are abundant and highly stable, formed by backsplicing pre-mRNAs, and expressed ubiquitously in eukaryotic cells, emerging as critical regulators of a plethora of signaling pathways. Recent progress in high-throughput sequencing has enabled a better understanding of the physiological and pathophysiological functions of circRNAs, overcoming the obstacle of the sequence overlap between circRNAs and their linear cognate mRNAs. Some viruses also encode circRNAs implicated in viral replication or disease progression. There is increasing evidence that viral infections dysregulate circRNA expression and that the altered expression of circRNAs is critical in regulating viral infection and replication. circRNAs were shown to regulate gene expression via microRNA and protein sponging or via encoding small polypeptides. Recent studies have also highlighted the potential role of circRNAs as promising diagnostic and prognostic biomarkers, RNA vaccines and antiviral therapy candidates due to their higher stability and lower immunogenicity. This review presents an up-to-date summary of the mechanistic involvement of circRNAs in innate immunity against viral infections, the current understanding of their regulatory roles, and the suggested applications.

MIR155HG Plays a Bivalent Role in Regulating Innate Antiviral Immunity by Encoding Long Noncoding RNA-155 and microRNA-155-5p.

MIR155HG encodes a precursor RNA of microRNA-155 (miRNA-155). We previously identified this RNA also as a long noncoding RNA (lncRNA) that we call lncRNA-155. To define the functions of miRNA-155 and lncRNA-155, we generated miRNA-155 knockout (KO) mice lacking only 19 bp of the miRNA-155 core sequence without affecting the expression of lncRNA-155. Surprisingly, compared with the miRNA-155KO mice, previously generated lncRNA-155KO mice were more susceptible to both influenza virus (RNA virus) and pseudorabies virus (DNA virus) infection, as characterized by lower survival rate, higher body weight loss, and higher viral load. We found that miRNA-155-5p enhanced antiviral responses by positively regulating activation of signal transducer and activator of transcription 1 (STAT1), but the STAT1 activity differed greatly in the animals (lncRNA-155KO < miRNA-155KO < wild type). In line with this, expression levels of several critical interferon-stimulated genes (ISGs) were also significantly different (lncRNA-155KO < miRNA-155KO < wild type). We found that lncRNA-155 augmented interferon beta (IFN-ß) production during the viral infection, but miRNA-155 had no significant effect on the virus-induced IFN-ß expression. Furthermore, we observed that lncRNA-155 loss in mice resulted in dramatic inhibition of virus-induced activation of interferon regulatory factor 3 compared to both miRNA-155KO and wild-type (WT) animals. Moreover, lncRNA-155 still significantly suppressed the viral infection even though the miRNA-155 derived from lncRNA-155 was deleted or blocked. These results reveal that lncRNA-155 and miRNA-155 regulate antiviral responses through distinct mechanisms, indicating a bivalent role for MIR155HG in innate immunity. IMPORTANCE Here, we found that lncRNA-155KO mice lacking most of the lncRNA-155 sequences along with pre-miRNA-155, were more susceptible to influenza virus or pseudorabies virus infection than miRNA-155KO mice lacking only 19 bp of the miRNA-155 core sequence without affecting the expression of lncRNA-155, as evidenced by faster body weight loss, poorer survival, and higher viral load, suggesting an additional role of lncRNA-155 in regulating viral pathogenesis besides via processing miRNA-155. Congruously, miRNA-155-deleted lncRNA-155 significantly attenuated the viral infection. Mechanistically, we demonstrated miRNA-155-5p potentiated antiviral responses by promoting STAT1 activation but could not directly regulate the IFN-ß expression. In contrast, lncRNA-155 enhanced virus-induced IFN-ß production by regulating the activation of interferon regulatory factor 3. This finding reveals a bivalent role of MIR155HG in regulating antiviral responses through encoding lncRNA-155 and miRNA-155-5p and provides new insights into complicated mechanisms underlying interaction between virus and host innate immunity.

Initial activation of STAT2 induced by IAV infection is critical for innate antiviral immunity.

STAT2 is an important transcription factor activated by interferons (IFNs) upon viral infection and plays a key role in antiviral responses. Interestingly, here we found that phosphorylation of STAT2 could be induced by several viruses at early infection stage, including influenza A virus (IAV), and such initial activation of STAT2 was independent of type I IFNs and JAK kinases. Furthermore, it was observed that the early activation of STAT2 during viral infection was mainly regulated by the RIG-I/MAVS-dependent pathway. Disruption of STAT2 phosphorylation at Tyr690 restrained antiviral response, as silencing STAT2 or blocking STAT2 Y690 phosphorylation suppressed the expression of several interferon-stimulated genes (ISGs), thereby facilitating viral replication. In vitro experiments using overexpression system or kinase inhibitors showed that several kinases including MAPK12 and Syk were involved in regulation of the early phosphorylation of STAT2 triggered by IAV infection. Moreover, when MAPK12 kinase was inhibited, expression of several ISGs was clearly decreased in cells infected with IAV at the early infection stage. Accordingly, inhibition of MAPK12 accelerated the replication of influenza virus in host. These results provide a better understanding of how initial activation of STAT2 and the early antiviral responses are induced by the viral infection.

Phenotypic detection of methicillin resistance, biofilm production, and inducible clindamycin resistance in Staphylococcus aureus clinical isolates in Kathmandu, Nepal.

INTRODUCTION: Methicillin resistance, inducible clindamycin resistance (ICR), biofilm production, and increased minimum inhibitory concentration (MIC) of vancomycin in Staphylococcus aureus are major causes of antibiotic treatment failure and increased morbidity and mortality. The surveillance of such isolates and the study of their antimicrobial pattern are essential in managing the infections caused by these isolates. This study aimed to determine methicillin resistance, biofilm production, and ICR in S. aureus isolates from a tertiary care hospital in Kathmandu, Nepal. MATERIALS AND METHODS: A total of 217 S. aureus isolated from different samples were processed following standard laboratory procedures. Antibiotic susceptibility testing was performed by the Kirby-Bauer disk diffusion technique. Methicillin-resistant S. aureus (MRSA) were identified by the cefoxitin disk diffusion test, and biofilm producers were examined using the microtiter plate technique. D-test and E-test were performed to determine inducible clindamycin resistance and minimum inhibitory concentration of vancomycin, respectively. RESULTS: Among the 217 S. aureus isolates, 78.3% were multidrug-resistant (MDR), 47.0% were MRSA, 62.2% were biofilm producers, and 50.7% showed ICR. All MRSA isolates exhibited MIC levels of vancomycin within the susceptible range. Biofilm producers and MRSA isolates showed elevated antimicrobial resistance. MRSA was significantly associated with MDR. Biofilm-producing and multidrug-resistant MRSA isolates showed significantly higher MIC levels of vancomycin (p = 0.0013 and < 0.0001, respectively), while ICR was significantly higher in MDR (p = 0.0001) isolates. CONCLUSION: High multidrug resistance, MRSA, and ICR in this study call for routine evaluation of antibiotic susceptibility patterns of S. aureus. Vancomycin can be used to treat serious staphylococcal infections. Clindamycin should be prescribed only after performing the D-test. Drugs like teicoplanin, chloramphenicol, doxycycline, amikacin, and levofloxacin can treat MRSA infections.

Epidemiology and Genetic Diversity of PCV2 Reveals That PCV2e Is an Emerging Genotype in Southern China: A Preliminary Study.

Porcine circovirus-associated disease (PCVAD), caused by porcine circovirus type 2 (PCV2), has ravaged the pig industry, causing huge economic loss. At present, PCV2b and PCV2d are highly prevalent genotypes worldwide, while in China, in addition to PCV2b and PCV2d, a newly emerged PCV2e genotype detected in the Fujian province has attracted attention, indicating that PCV2 genotypes in China are more abundant. A preliminary study was conducted to better understand the genetic diversity and prevalence of PCV2 genotypes in southern China. We collected 79 random lung samples from pigs with respiratory signs, from 2018 to 2021. We found a PCV2-positivity rate of 29.1%, and frequent co-infections of PCV2 with PCV3, Streptococcus suis (S. suis), and other porcine pathogens. All PCV2-positive samples were sequenced and subjected to whole-genome analysis. Phylogenetic analysis, based on the PCV2 ORF2 gene and complete genomes, found that PCV2 strains identified in this study belonged to genotypes PCV2a (1), PCV2b (6), PCV2d (10), and PCV2e (6). Importantly, PCV2e was identified for the first time in some provinces, including Guangdong and Jiangxi. Additionally, we found two positively selected sites in the ORF2 region, located on the previously reported antigenic epitopes. Moreover, codon 63, one of the positively selected sites, has different types of amino acids in different genotypes. In conclusion, this study shows that PCV2e is an emerging genotype circulating in southern China, which warrants urgent, specific surveillance to aid the development of prevention and control strategies in China.

Diagnostic performance of GeneXpert MTB/RIF in detecting MTB in smear-negative presumptive TB patients.

BACKGROUND: Tuberculosis (TB) is a leading cause of morbidity and mortality worldwide. Control of TB is lingering by the lack of diagnostic tests that are simple, rapid, yet accurate. Thus, smear-negative pulmonary TB often misses the diagnosis. The study evaluated the performance of GeneXpert MTB/RIF assay for the detection of Mycobacterium tuberculosis (MTB). METHODS: The study was carried out from June to December 2016 in Nepal Tuberculosis Center, Bhaktapur, Nepal. A total of 173 sputum samples were collected and processed by microscopy [Auramine-O staining and Ziehl-Neelsen (ZN) staining], followed by GeneXpert MTB/RIF assay and culture in Lowenstein-Jensen (LJ) medium. RESULTS: Of 173 sputum samples, 162 (93.6%) were smear-negative. Of 162 smear-negative sputum samples, 35 (21.6%) were confirmed to have MTB by culture, and 31 (19.1%) by GeneXpert MTB/RIF assay. Of 31 GeneXpert-positive samples, 25 (80.6%) were susceptible, 4 (12.9%) were resistant, and 2 (6.45%) were intermediate to rifampicin. The sensitivity, specificity, positive predictive value, and negative predictive value of GeneXpert MTB/RIF assay for smear-negative sputum samples were 74.3%, 96.6%, 86.7%, and 92%, respectively. The GeneXpert MTB/RIF has a substantial diagnostic agreement of 90.91% with culture (Cohen's Kappa coefficient = 0.73). CONCLUSION: The diagnostic performance of GeneXpert MTB/RIF assay was almost on par with culture, and thus can be relied upon for MTB detection in smear-negative sputum samples.

I226R Protein of African Swine Fever Virus Is a Suppressor of Innate Antiviral Responses.

African swine fever is one of the most devastating swine diseases caused by African swine fever virus (ASFV). Although ASFV encodes more than 160 viral proteins, the implication of a majority of ASFV proteins in regulating host immunity is yet to be explored, and the mechanisms of immune evasion by ASFV proteins are largely unknown. Here, we report that the I226R protein of ASFV significantly suppressed innate immune responses. The ectopic expression of ASFV I226R in 293T cells significantly inhibited the activation of interferon-stimulated response element promoters triggered by Sendai virus (SeV), poly(I:C), or cyclic GMP-AMP synthase (cGAS)/STING. The I226R protein caused a significant decrease in the expression of interferons and interferon-stimulating genes in cells infected with SeV. Similar results were obtained from experiments using I226R-overexpressed PK15 and 3D4/21 cells stimulated with vesicular stomatitis virus. We observed that I226R inhibited the activation of both nuclear factor-kappa B (NF-κB) and interferon regulatory factor 3 (IRF3). Furthermore, it was shown that overexpression of I226R suppressed IRF3 activation and caused the degradation of NF-κB essential modulator (NEMO) protein. The I226R-induced NEMO degradation could be prevented by treatment with MG132, a proteasome inhibitor. Together, these results reveal that the ASFV I226R protein impairs antiviral responses, likely through multiple mechanisms including the suppression of NF-κB and IRF3 activation, to counteract innate immune responses during the viral infection.

Syk Facilitates Influenza A Virus Replication by Restraining Innate Immunity at the Late Stage of Viral Infection.

Spleen tyrosine kinase (Syk) has recently come forth as a critical regulator of innate immune response. Previous studies identify Syk as a key kinase for STAT1 activation at the early stage of influenza A virus (IAV) infection that is involved in initial antiviral immunity. However, the involvement of Syk in host antiviral immunity during the late phase of IAV infection and its effect on pathogenesis of the virus remain unknown. Here, we found through time course studies that Syk restrained antiviral immune response at the late stage of IAV infection, thereby promoting viral replication. Depletion of Syk suppressed IAV replication in vitro, whereas ectopic expression of Syk facilitated viral replication. Moreover, Syk-deficient mice were employed, and we observed that knockout of Syk rendered mice more resistant to IAV infection, as evidenced by a lower degree of lung injury, slower body weight loss, and an increased survival rate of Syk knockout mice challenged with IAV. Furthermore, we revealed that Syk repressed the interferon response at the late stage of viral infection. Loss of Syk potentiated the expression of type I and III interferons in both Syk-depleted cells and mice. Mechanistically, Syk interacted with TBK1 and modulated its phosphorylation status, thereby impeding TBK1 activation and restraining innate immune signaling that governs interferon response. Together, these findings unveil a role of Syk in temporally regulating host antiviral immunity and advance our understanding of complicated mechanisms underlying regulation of innate immunity against viral invasion. IMPORTANCE Innate immunity must be tightly controlled to eliminate invading pathogens while avoiding autoimmune or inflammatory diseases. Syk is essential for STAT1 activation at the early stage of IAV infection, which is critical for initial antiviral responses. Surprisingly, here a time course study showed that Syk suppressed innate immunity during late phases of IAV infection and thereby promoted IAV replication. Syk deficiency enhanced the expression of type I and III interferons, inhibited IAV replication, and rendered mice more resistant to IAV infection. Syk impaired innate immune signaling through impeding TBK1 activation. These data reveal that Syk participates in the initiation of antiviral defense against IAV infection and simultaneously contributes to the restriction of innate immunity at the late stage of viral infection, suggesting that Syk serves a dual function in regulating antiviral responses. This finding provides new insights into complicated mechanisms underlying interaction between virus and host immune system.

sRNA23, a novel small RNA, regulates to the pathogenesis of Streptococcus suis serotype 2.

Streptococcus suis serotype 2 (S. suis 2) is an important ubiquitous zoonotic pathogen. To date, regulatory factors and their implication in S. suis pathogenesis are not fully understood. Small non-coding RNAs (sRNAs) have been proven to function as important regulatory factors in bacterial pathogenesis and stress adaptation. Here, we identified a differentially downregulated S. suis 05ZYH33 sRNA after iron starvation by RNA-seq, which we named sRNA23. The presence of sRNA23 was further confirmed by RACE and Northern blot. Expression of sRNA23 was significantly altered under different environmental stresses such as nutritional starvation, osmotic pressure, oxidative stress, and lysozymal exposure. A sRNA23-deleted mutant exhibited relatively shorter streptococcal chains and weakened biofilm-forming ability. The mutation also resulted in decreased adherence of the S. suis 05ZYH33 to human laryngeal epidermoid carcinoma (HEp-2) cells, increased sensitivity to phagocytosis by RAW264.7 macrophages, and significantly reduced hemolytic activity. Furthermore, we observed that a sRNA23-deleted mutant had a low survival rate in pig whole blood and attenuated virulence in a mouse model. Moreover, based on RNA pull-down and electrophoretic mobility shift assay, we found that sRNA23 can directly bind to two proteins involved in adhesion and biofilm formation, namely, moonlighting protein FBA (fructose diphosphate aldolase) and rplB (50S ribosomal protein L2), respectively. Collectively, sRNA23 enhances S. suis 2 pathogenicity and the binding between sRNA23 and FBA/rplB might play an essential role in the adherence and biofilm-forming ability of S. suis 2.

Deficiency of eIF4B Increases Mouse Mortality and Impairs Antiviral Immunity.

Eukaryotic translation initiation factor 4B (eIF4B) plays an important role in mRNA translation initiation, cell survival and proliferation in vitro. However, its function in vivo is poorly understood. Here, we identified that eIF4B knockout (KO) in mice led to embryonic lethality, and the embryos displayed severe liver damage. Conditional KO (CKO) of eIF4B in adulthood profoundly increased the mortality of mice, characterized by severe pathological changes in several organs and reduced number of peripheral blood lymphocytes. Strikingly, eIF4B CKO mice were highly susceptible to viral infection with severe pulmonary inflammation. Selective deletion of eIF4B in lung epithelium also markedly promoted replication of influenza A virus (IAV) in the lung of infected animals. Furthermore, we observed that eIF4B deficiency significantly enhanced the expression of several important inflammation-associated factors and chemokines, including serum amyloid A1 (Saa1), Marco, Cxcr1, Ccl6, Ccl8, Ccl20, Cxcl2, Cxcl17 that are implicated in recruitment and activation of neutrophiles and macrophages. Moreover, the eIF4B-deficient mice exhibited impaired natural killer (NK) cell-mediated cytotoxicity during the IAV infection. Collectively, the results reveal that eIF4B is essential for mouse survival and host antiviral responses, and establish previously uncharacterized roles for eIF4B in regulating normal animal development and antiviral immunity in vivo.

Prevalence of multidrug-resistance and bla VIM and bla IMP genes among gram-negative clinical isolates in tertiary care hospital, Kathmandu, Nepal.

BACKGROUND AND OBJECTIVES: Carbapenems have been the choice of antibiotics for the treatment of infections caused by multidrug-resistant bacteria. The main objective of this study was to determine the prevalence of carbapenemase (bla VIM and bla IMP ) producing isolates among Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii. MATERIALS AND METHODS: A total of 1,151 clinical samples were collected from the patients visiting Annapurna Neurological Institute and Allied Science and Annapurna Research Centre, Kathmandu, between June 2017 and January 2018. Antibiotic susceptibility testing (AST) was performed on the Enterobacteriaceae, P. aeruginosa and A. baumannii isolates using the Kirby-Bauer disk diffusion method. The modified Hodge test (MHT) was performed on the carbapenem-resistant isolates to confirm carbapenemase production. DNA was extracted and then screened for bla VIM and bla IMP genes by multiplex PCR. RESULTS: Of the total 1,151 clinical samples, 253 (22.0%) showed positive growth. Of them, 226 (89.3%) were identified as Enterobacteriaceae, P. aeruginosa, and A. baumannii. Among the 226 isolates, 106 (46.9%) were multidrug-resistant. Out of the 106, 97 (91.5%) isolates showed resistance to at least one of the carbapenem used. Among the 97 carbapenem-resistant isolates, 67 (69.1%) showed the modified Hodge test (MHT) positive results. bla VIM and bla IMP were detected in 40 and 38 isolates respectively using multiplex PCR assay. CONCLUSION: This study determined a high prevalence of MDR and carbapenem resistance among Enterobacteriaceae, P. aeruginosa, and A. baumannii as detected by the presence of bla VIM and bla IMP genes. This study recommends the use of rapid and advanced diagnostic tools along with conventional phenotypic detection methods in the clinical settings for early detection and management of drug-resistant pathogens to improve treatment strategies.

Prevalence of carbapenemase-producing Klebsiella pneumoniae at a tertiary care hospital in Kathmandu, Nepal.

AIM: Although carbapenem is the last-resort drug for treating drug-resistant Gram-negative bacterial infections, prevalence of carbapenem-resistant bacteria has substantially increased worldwide owing to irrational use of antibiotics particularly in developing countries like Nepal.  Therefore, this study was aimed to determine the prevalence of carbapenemase-producing K. pneumoniae and to detect the carbapenemase genes (blaNDM-2 and blaOXA-48) in at a tertiary care hospital in Nepal. MATERIALS AND METHODS: A hospital-based cross-sectional study was carried out from June 2018 to January 2019 at the Microbiology Laboratory of Annapurna Neurological Institute and Allied Sciences, Kathmandu, Nepal. Different clinical samples were collected and cultured in appropriate growth media. Biochemical tests were performed for the identification of K. pneumoniae. Antibiotic susceptibility testing (AST) was performed by the Kirby-Bauer disc diffusion method. The modified Hodge test (MHT) was performed to detect carbapenemase producers. The plasmid was extracted by the modified alkaline hydrolysis method. Carbapenemase-producing K. pneumoniae were further confirmed by detecting blaNDM-2 and blaOXA-48 genes by PCR using specific forward and reverse primers followed by gel electrophoresis. RESULTS: Out of the total 720 samples, 38.9% (280/720) were culture positive. K. pneumoniae was the most predominant isolate 31.4% (88/280). Of 88 K. pneumoniae isolates, 56.8% (50/88) were multi-drug resistant (MDR), and 51.1% (45/88) were MHT positive. Colistin showed the highest sensitivity (100%; 88/88), followed by tigecycline (86.4%; 76/88). blaNDM-2 and blaOXA-48 genes were detected in 24.4% (11/45) and 15.5% (7/45) of carbapenemase-producing K. pneumoniae isolates, respectively. CONCLUSION: The rate of MDR and carbapenemase production was high in the K. pneumoniae isolates. Colistin and tigecycline could be the drug of choice for the empirical treatments of MDR and carbapenemase-producing K. pneumoniae. Our study provides a better understanding of antibiotic resistance threat and enables physicians to select the most appropriate antibiotics.

Acute Infection of Viral Pathogens and Their Innate Immune Escape.

Viral infections can cause rampant disease in human beings, ranging from mild to acute, that can often be fatal unless resolved. An acute viral infection is characterized by sudden or rapid onset of disease, which can be resolved quickly by robust innate immune responses exerted by the host or, instead, may kill the host. Immediately after viral infection, elements of innate immunity, such as physical barriers, various phagocytic cells, group of cytokines, interferons (IFNs), and IFN-stimulated genes, provide the first line of defense for viral clearance. Innate immunity not only plays a critical role in rapid viral clearance but can also lead to disease progression through immune-mediated host tissue injury. Although elements of antiviral innate immunity are armed to counter the viral invasion, viruses have evolved various strategies to escape host immune surveillance to establish successful infections. Understanding complex mechanisms underlying the interaction between viruses and host's innate immune system would help develop rational treatment strategies for acute viral infectious diseases. In this review, we discuss the pathogenesis of acute infections caused by viral pathogens and highlight broad immune escape strategies exhibited by viruses.

Long Noncoding RNA IFITM4P Regulates Host Antiviral Responses by Acting as a Competing Endogenous RNA.

Long noncoding RNAs (lncRNAs) are involved in numerous cellular processes. Increasing evidence suggests that some lncRNAs function in immunity through various complex mechanisms. However, implication of a large fraction of lncRNAs in antiviral innate immunity remains uncharacterized. Here, we identified an lncRNA called lncRNA IFITM4P that was transcribed from interferon-induced transmembrane protein 4 pseudogene (IFITM4P), a pseudogene belonging to the interferon-induced transmembrane protein (IFITM) family. We found that expression of lncRNA IFITM4P was significantly induced by infection with several viruses, including influenza A virus (IAV). Importantly, lncRNA IFITM4P acted as a positive regulator of innate antiviral immunity. Ectopic expression of lncRNA IFITM4P significantly suppressed IAV replication in vitro, whereas IFITM4P deficiency promoted viral production. We further observed that expression of lncRNA IFITM4P was upregulated by interferon (IFN) signaling during viral infection, and altering the expression of this lncRNA had significant effects on the mRNA levels of several IFITM family members, including IFITM1, IFITM2, and IFITM3. Moreover, lncRNA IFITM4P was identified as a target of the microRNA miR-24-3p, which represses mRNA of IFITM1, IFITM2, and IFITM3. The experiments demonstrated that lncRNA IFITM4P was able to cross-regulate the expression of IFITM family members as a competing endogenous RNA (ceRNA), leading to increased stability of these IFITM mRNAs. Together, our results reveal that lncRNA IFITM4P, as a ceRNA, is involved in innate immunity against viral infection through the lncRNA IFITM4P-miR-24-3p-IFITM1/2/3 regulatory network. IMPORTANCE lncRNAs play important roles in various biological processes, but their involvement in host antiviral responses remains largely unknown. In this study, we revealed that the pseudogene IFITM4P belonging to the IFITM family can transcribe a functional long noncoding RNA termed lncRNA IFITM4P. Importantly, results showed that lncRNA IFITM4P was involved in innate antiviral immunity, which resembles some interferon-stimulated genes (ISGs). Furthermore, lncRNA IFITM4P was identified as a target of miR-24-3p and acts as a ceRNA to inhibit the replication of IAV through regulating the mRNA levels of IFITM1, IFITM2, and IFITM3. These data provide new insight into the role of a previously uncharacterized lncRNA encoded by a pseudogene in the host antiviral response and a better understanding of the IFITM antiviral network.

RDUR, a lncRNA, Promotes Innate Antiviral Responses and Provides Feedback Control of NF-κB Activation.

Influenza A virus (IAV), a highly infectious respiratory pathogen, remains a major threat to global public health. Numerous long non-coding RNAs (lncRNAs) have been shown to be implicated in various cellular processes. Here, we identified a new lncRNA termed RIG-I-dependent IAV-upregulated noncoding RNA (RDUR), which was induced by infections with IAV and several other viruses. Both in vitro and in vivo studies revealed that robust expression of host RDUR induced by IAV was dependent on the RIG-I/NF-κB pathway. Overexpression of RDUR suppressed IAV replication and downregulation of RDUR promoted the virus replication. Deficiency of mouse RDUR increased virus production in lungs, body weight loss, acute organ damage and consequently reduced survival rates of mice, in response to IAV infection. RDUR impaired the viral replication by upregulating the expression of several vital antiviral molecules including interferons (IFNs) and interferon-stimulated genes (ISGs). Further study showed that RDUR interacted with ILF2 and ILF3 that were required for the efficient expression of some ISGs such as IFITM3 and MX1. On the other hand, we found that while NF-κB positively regulated the expression of RDUR, increased expression of RDUR, in turn, inactivated NF-κB through a negative feedback mechanism to suppress excessive inflammatory response to viral infection. Together, the results demonstrate that RDUR is an important lncRNA acting as a critical regulator of innate immunity against the viral infection.

Prevalence of multidrug-resistant and extended-spectrum beta-lactamase producing Gram-negative isolates from clinical samples in a tertiary care hospital of Nepal.

BACKGROUND: The existence of multidrug-resistant organisms, including extended-spectrum beta-lactamases (ESBLs), is on rise across the globe and is becoming a severe problem. Knowledge of the prevalence and antibiogram profile of such isolates is essential to develop an appropriate treatment methodology. This study aimed to study the prevalence of Gram-negative isolates exhibiting ESBL at a tertiary care hospital and study their antibiogram profile. METHODS: A cross-sectional study was conducted at Shahid Gangalal National Heart Centre, Kathmandu, Nepal, from June 2018 to November 2018. A total of 770 clinical samples were collected and identified using the conventional biochemical tests following the Clinical and Laboratory Standard Institute (CLSI) guidelines. Antimicrobial susceptibility testing (AST) was performed using the standardized Kirby-Bauer disk diffusion method. The screening test for ESBL producers was performed as recommended by the CLSI and the confirmatory test was performed phenotypically using the E-test. RESULTS: Out of the 92 isolates, 84 (91.3%) were multidrug-resistant, and 47 (51.1%) were found to be potential ESBL producers. Of these, 16 isolates were confirmed ESBL producers by the E-test. Escherichia coli and Klebsiella pneumoniae were the predominant isolates and were also the major ESBL producers. Besides polymyxin B (100% sensitive), meropenem and imipenem showed high efficacy against the ESBL producers. CONCLUSION: Multidrug resistance was very high; however, ESBL production was low. Polymyxin B and carbapenems are the choice of drugs against ESBL producers but should be used only as the last line drugs.

Robust expression of p27Kip1 induced by viral infection is critical for antiviral innate immunity.

Influenza A virus (IAV) infection regulates the expression of numerous host genes. However, the precise mechanism underlying implication of these genes in IAV pathogenesis remains largely unknown. Here, we employed isobaric tags for relative and absolute quantification (iTRAQ) to identify host proteins regulated by IAV infection. iTRAQ analysis of mouse lungs infected or uninfected with IAV showed a total of 167 differentially upregulated proteins in response to the viral infection. Interestingly, we observed that p27Kip1, a potent cyclin-dependent kinase inhibitor, was markedly induced by IAV both at mRNA and protein levels through in vitro and in vivo studies. Furthermore, it was shown that innate immune signalling positively regulated p27Kip1 expression in response to IAV infection. Ectopic expression of p27Kip1 in A549 cells dramatically inhibited IAV replication, whereas, p27Kip1 knockdown significantly enhanced the virus replication. in vivo experiments demonstrated that p27Kip1 knockout (KO) mice were more susceptible to IAV than wild-type (WT) mice: exhibiting higher viral load in lung tissue, faster body-weight loss, reduced survival rate and more severe organ damage. Moreover, we found that p27Kip1 overexpression facilitated the degradation of viral NS1 protein, caused a dramatic STAT1 activation and promoted the expression of IFN-ß and several critical antiviral interferon-stimulated genes (ISGs). Increased p27Kip1 expression also restricted infections of several other viruses. Conversely, IAV-infected p27Kip1 KO mice exhibited a sharp increase in NS1 protein accumulation, reduced level of STAT1 activation and decreased expression of IFN-ß and the ISGs in the lung compared to WT animals. These findings reveal a key role of p27Kip1 in enhancing antiviral innate immunity.

Intestinal Parasitic Infections among Prison Inmates in Kathmandu Nepal.

BACKGROUND: Prison inmates are at high risk of intestinal parasitic infections. Thus, we studied intestinal parasitic infections among inmates of the Central Jail, Kathmandu, Nepal. METHODS: Morning stool samples from 400 inmates (M=282 and F=118), were collected in a clean, dry and wide-mouthed plastic container. The samples were transported to the research laboratory of Shi-Gan International College of Science and Technology, and were fixed using 10% formal saline. Then, samples were processed by formal ether sedimentation concentration technique and were observed microscopically by direct-smear technique. RESULTS: Six percent (24/400) samples were positive for intestinal parasites, with a gender ratio (M:F) 1.7:1. But, co-parasitism was not observed. Intestinal parasitic infections were higher among 21-40 years age-group, 3.5% (14/262). Similarly, intestinal parasitic infections were higher among 'Dalits' ethnic group, 21.1% (4/19). As compared to helminths, more protozoans, 62.5% (15/24), were observed. Giardia lamblia, 41.67% (10/24), was the most common protozoans while Trichuris trichiura, 25.0% (6/24), was the most common helminths. CONCLUSIONS: Intestinal parasitic infections were lower among the inmates of Central jail, but such conditions cannot be presumed in other peripheral settings. Pure and safe drinking water supply and the effective deworming campaign can further reduce this figure at this setting and peripheral jails across the country.

Identification of lncRNA-155 encoded by MIR155HG as a novel regulator of innate immunity against influenza A virus infection.

Long noncoding RNAs (lncRNAs) are single-stranded RNA molecules longer than 200 nt that regulate many cellular processes. MicroRNA 155 host gene (MIR155HG) encodes the microRNA (miR)-155 that regulates various signalling pathways of innate and adaptive immune responses against viral infections. MIR155HG also encodes a lncRNA that we call lncRNA-155. Here, we observed that expression of lncRNA-155 was markedly upregulated during influenza A virus (IAV) infection both in vitro (several cell lines) and in vivo (mouse model). Interestingly, robust expression of lncRNA-155 was also induced by infections with several other viruses. Disruption of lncRNA-155 expression in A549 cells diminished the antiviral innate immunity against IAV. Furthermore, knockout of lncRNA-155 in mice significantly increased IAV replication and virulence in the animals. In contrast, overexpression of lncRNA-155 in human cells suppressed IAV replication, suggesting that lncRNA-155 is involved in host antiviral innate immunity induced by IAV infection. Moreover, we found that lncRNA-155 had a profound effect on expression of protein tyrosine phosphatase 1B (PTP1B) during the infection with IAV. Inhibition of PTP1B by lncRNA-155 resulted in higher production of interferon-beta (IFN-ß) and several critical interferon-stimulated genes (ISGs). Together, these observations reveal that MIR155HG derived lncRNA-155 can be induced by IAV, which modulates host innate immunity during the virus infection via regulation of PTP1B-mediated interferon response.