RNA In Infected Cells Research Articles

Effective Antiviral Activity of the Tyrosine Kinase Inhibitor Sunitinib Malate against Zika Virus

IntroductionZika virus (ZIKV), a mosquito-borne flavivirus, causes the outbreaks of Latin America in 2015 - 2016, with the incidence of neurological complications. Sunitinib malate, an orally bioavailable malate salt of the tyrosine kinase inhibitor, is suggested as a broad-spectrum antiviral agent against emerging viruses like severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2.Materials and MethodsThis study investigated the antiviral efficacy and antiviral mechanisms of sunitinib malate against ZIKV infection using cytopathic effect reduction, virus yield, and time-of-addition assays.ResultsSunitinib malate concentration-dependently reduced ZIKV-induced cytopathic effect, the expression of viral proteins, and ZIKV yield in supernatant with 50% inhibitory concentration (IC50) value of 0.015 μM, and the selectivity index of greater than 100 against ZIKV infection, respectively. Sunitinib malate had multiple antiviral actions during entry and post-entry stages of ZIKV replication. Sunitinib malate treatment at entry stage significantly reduced the levels of ZIKV RNA replication with the reduction of (+) RNA to (-) RNA ratio and the production of new intracellular infectious particles in infected cells. The treatment at post-entry stage caused a concentration-dependent increase in the levels of ZIKV (+) RNA and (-) RNA in infected cells, along with enlarging the ratio of (+) RNA to (-) RNA, but caused a pointed increase in the titer of intracellular infectious particles by 0.01 and 0.1 μM, and a substantial decrease in the titer of intracellular infectious particles by 1 μM.ConclusionThe study discovered the antiviral actions of sunitinib malate against ZIKV infection, demonstrating a repurposed, host-targeted approach to identify potential antiviral drugs for treating emerging and global viral diseases.

The SARS-CoV-2 RNA\u2013protein interactome in infected human cells

Characterizing the interactions that SARS-CoV-2 viral RNAs make with host cell proteins during infection can improve our understanding of viral RNA functions and the host innate immune response. Using RNA antisense purification and mass spectrometry, we identified up to 104 human proteins that directly and specifically bind to SARS-CoV-2 RNAs in infected human cells. We integrated the SARS-CoV-2 RNA interactome with changes in proteome abundance induced by viral infection and linked interactome proteins to cellular pathways relevant to SARS-CoV-2 infections. We demonstrated by genetic perturbation that cellular nucleic acid-binding protein (CNBP) and La-related protein 1 (LARP1), two of the most strongly enriched viral RNA binders, restrict SARS-CoV-2 replication in infected cells and provide a global map of their direct RNA contact sites. Pharmacological inhibition of three other RNA interactome members, PPIA, ATP1A1, and the ARP2/3 complex, reduced viral replication in two human cell lines. The identification of host dependency factors and defence strategies as presented in this work will improve the design of targeted therapeutics against SARS-CoV-2.

Lipid droplets fuel SARS-CoV-2 replication and production of inflammatory mediators.

Viruses are obligate intracellular parasites that make use of the host metabolic machineries to meet their biosynthetic needs. Thus, identifying the host pathways essential for the virus replication may lead to potential targets for therapeutic intervention. The mechanisms and pathways explored by SARS-CoV-2 to support its replication within host cells are not fully known. Lipid droplets (LD) are organelles with major functions in lipid metabolism, energy homeostasis and intracellular transport, and have multiple roles in infections and inflammation. Here we described that monocytes from COVID-19 patients have an increased LD accumulation compared to SARS-CoV-2 negative donors. In vitro, SARS-CoV-2 infection were seen to modulate pathways of lipid synthesis and uptake as monitored by testing for CD36, SREBP-1, PPARγ, and DGAT-1 expression in monocytes and triggered LD formation in different human cell lines. LDs were found in close apposition with SARS-CoV-2 proteins and double-stranded (ds)-RNA in infected Vero cells. Electron microscopy (EM) analysis of SARS-CoV-2 infected Vero cells show viral particles colocalizing with LDs, suggestive that LDs might serve as an assembly platform. Pharmacological modulation of LD formation by inhibition of DGAT-1 with A922500 significantly inhibited SARS-CoV-2 replication as well as reduced production of mediators pro-inflammatory response. Taken together, we demonstrate the essential role of lipid metabolic reprograming and LD formation in SARS-CoV-2 replication and pathogenesis, opening new opportunities for therapeutic strategies to COVID-19.

Host-pathogen transcriptomics: Trypanosoma cruzi as a model for studying RNA contamination

Cellular infection assays constitute essential tools to understand host-pathogen interactions, particularly for intracellular microorganisms that are produced in cell lines are needed to propagate the microorganism. In this work, we demonstrate that RNA derived from Vero cells is an important contaminant to consider in order to avoid false positive results in transcriptomic experiments. We study the cross-contamination on a Trypanosoma cruzi cell infection model, the etiological agent of Chagas disease. We implemented the most frequently used trypanosome-purification protocols and, for all of them, we detected RNAs derived from Vero cells in trypomastigote extracts. For some of the protocols we also detected Vero RNAs in infected human cells. We also found this type of contamination in microarray experiments of human samples infected with T. cruzi. Concerning Illumina RNA-Seq data, we found that the contamination with Vero cells is probably introducing spurious results. Finally, we recommend a protocol to purify trypomastigotes, which showed a high percentage of trypomastigote recovery and the absence of Vero contamination in infected human samples. Avoiding this type of contamination should be an important factor to consider during experimental design, in order to minimize false positive results in transcriptomic studies as well as RNA contamination in vaccine production. SignificanceTranscriptomic studies are widely used to understand host-pathogen interactions. When the pathogen is an intracellular microorganism, an additional mammalian cell system can be needed to propagate it. In this work we demonstrate that pathogens purified from infected monolayers can carry RNAs from these mammalian cells, and that this ambient RNA contamination is probably producing false positive results in subsequent transcriptomic studies performed with qRT-PCR, microarrays or Next Generation Sequencing.

The Aedes aegypti Domino Ortholog p400 Regulates Antiviral Exogenous Small Interfering RNA Pathway Activity and ago-2 Expression.

Arboviruses are pathogens of humans and animals. A better understanding of the interactions between these pathogens and the arthropod vectors, such as mosquitoes, that transmit them is necessary to develop novel control measures. A major antiviral pathway in the mosquito vector is the exogenous small interfering RNA (exo-siRNA) pathway, which is induced by arbovirus-derived double-stranded RNA in infected cells. Although recent work has shown the key role played by Argonaute-2 (Ago-2) and Dicer-2 (Dcr-2) in this pathway, the regulatory mechanisms that govern these pathways have not been studied in mosquitoes. Here, we show that the Domino ortholog p400 has antiviral activity against the alphavirus Semliki Forest virus (Togaviridae) both in Aedes aegypti-derived cells and in vivo Antiviral activity of p400 was also demonstrated against chikungunya virus (Togaviridae) and Bunyamwera virus (Peribunyaviridae) but not Zika virus (Flaviviridae). p400 was found to be expressed across mosquito tissues and regulated ago-2 but not dcr-2 transcript levels in A. aegypti mosquitoes. These findings provide novel insights into the regulation of an important aedine exo-siRNA pathway effector protein, Ago-2, by the Domino ortholog p400. They add functional insights to previous observations of this protein's antiviral and RNA interference regulatory activities in Drosophila melanogasterIMPORTANCE Female Aedes aegypti mosquitoes are vectors of human-infecting arthropod-borne viruses (arboviruses). In recent decades, the incidence of arthropod-borne viral infections has grown dramatically. Vector competence is influenced by many factors, including the mosquito's antiviral defenses. The exogenous small interfering RNA (siRNA) pathway is a major antiviral response restricting arboviruses in mosquitoes. While the roles of the effectors of this pathway, Argonaute-2 and Dicer-2 are well characterized, nothing is known about its regulation in mosquitoes. In this study, we demonstrate that A. aegypti p400, whose ortholog Domino in Drosophila melanogaster is a chromatin-remodeling ATPase member of the Tip60 complex, regulates siRNA pathway activity and controls ago-2 expression levels. In addition, we found p400 to have antiviral activity against different arboviruses. Therefore, our study provides new insights into the regulation of the antiviral response in A. aegypti mosquitoes.

Characterization and functional analysis of chicken APOBEC4

The APOBEC proteins play significant roles in the innate and adaptive immune system, probably due to their deaminase activities. Because APOBEC1 (A1) and APOBEC3 (A3) are absent in the chicken genome, we were interested in determining whether chicken APOBEC4 (A4) possessed more complex functions than its mammalian homologs. In this study, chicken A4 (chA4) mRNA was identified and cloned for the first time. Based on bioinformatics analyses, the conserved zinc-coordinating motif (HXE … PC(X)2–6C) was identified on the surface of chA4 and contained highly conserved His97, Glu99, Pro130, Cys131 and Cys138 active sites. The highest expression levels of constitutive chA4 were detected in primary lymphocytes and bursa of Fabricius. Newcastle Disease (ND) is one of the most serious infectious diseases in birds, causing major economic losses to the poultry industry. In vitro, Newcastle Disease Virus (NDV) early infection induced significant increases in chA4 expression in the chicken B cell line, DT40, the macrophage cell line, HD11 and the CD4+ T cell line, MSB-1, but not the fibroblast cell line, DF-1. In vivo, the expression levels of chA4 were up-regulated in several tissues from NDV-infected chickens, especially the thymus, testicles, duodenum and kidney. The high level expression of exogenous chA4 displayed inhibitory effects on NDV and reduced viral RNA in infected cells. Taken together, these data demonstrate that chA4 is involved in the chicken immune system and may play important roles in host anti-viral responses.

The herpes simplex virus host shutoff (vhs) RNase limits accumulation of double stranded RNA in infected cells: Evidence for accelerated decay of duplex RNA.

The herpes simplex virus virion host shutoff (vhs) RNase destabilizes cellular and viral mRNAs and blunts host innate antiviral responses. Previous work demonstrated that cells infected with vhs mutants display enhanced activation of the host double-stranded RNA (dsRNA)-activated protein kinase R (PKR), implying that vhs limits dsRNA accumulation in infected cells. Confirming this hypothesis, we show that partially complementary transcripts of the UL23/UL24 and UL30/31 regions of the viral genome increase in abundance when vhs is inactivated, giving rise to greatly increased levels of intracellular dsRNA formed by annealing of the overlapping portions of these RNAs. Thus, vhs limits accumulation of dsRNA at least in part by reducing the levels of complementary viral transcripts. We then asked if vhs also destabilizes dsRNA after its initial formation. Here, we used a reporter system employing two mCherry expression plasmids bearing complementary 3’ UTRs to produce defined dsRNA species in uninfected cells. The dsRNAs are unstable, but are markedly stabilized by co-expressing the HSV dsRNA-binding protein US11. Strikingly, vhs delivered by super-infecting HSV virions accelerates the decay of these pre-formed dsRNAs in both the presence and absence of US11, a novel and unanticipated activity of vhs. Vhs binds the host RNA helicase eIF4A, and we find that vhs-induced dsRNA decay is attenuated by the eIF4A inhibitor hippuristanol, providing evidence that eIF4A participates in the process. Our results show that a herpesvirus host shutoff RNase destabilizes dsRNA in addition to targeting partially complementary viral mRNAs, raising the possibility that the mRNA destabilizing proteins of other viral pathogens dampen the host response to dsRNA through similar mechanisms.

Long non‐coding subgenomic flavivirus RNAs have extended 3D structures and are flexible in solution

Most mosquito‐borne flaviviruses, including Zika virus (ZIKV), Dengue virus (DENV), and West Nile virus (WNV), produce long non‐coding subgenomic RNAs (sfRNAs) in infected cells that link to pathogenicity and immune evasion. Until now, the structural characterization of these lncRNAs remains limited. Here, we studied the 3D structures of individual and combined subdomains of sfRNAs, and visualized the accessible 3D conformational spaces of complete sfRNAs from DENV2, ZIKV, and WNV by small angle X‐ray scattering (SAXS) and computational modeling. The individual xrRNA1s and xrRNA2s adopt similar structures in solution as the crystal structure of ZIKV xrRNA1, and all xrRNA1‐2s form compact structures with reduced flexibility. While the DB12 of DENV2 is extended, the DB12s of ZIKV and WNV are compact due to the formation of intertwined double pseudoknots. All 3′ stem‐loops (3′SLs) share similar rod‐like structures. Complete sfRNAs are extended and sample a large conformational space in solution. Our work not only provides structural insight into the function of flavivirus sfRNAs, but also highlights strategies of visualizing other lncRNAs in solution by SAXS and computational methods.

Subgenomic flavivirus RNA binds the mosquito DEAD/H-box helicase ME31B and determines Zika virus transmission by Aedes aegypti

Zika virus (ZIKV) is an arthropod-borne flavivirus predominantly transmitted by Aedes aegypti mosquitoes and poses a global human health threat. All flaviviruses, including those that exclusively replicate in mosquitoes, produce a highly abundant, noncoding subgenomic flavivirus RNA (sfRNA) in infected cells, which implies an important function of sfRNA during mosquito infection. Currently, the role of sfRNA in flavivirus transmission by mosquitoes is not well understood. Here, we demonstrate that an sfRNA-deficient ZIKV (ZIKVΔSF1) replicates similar to wild-type ZIKV in mosquito cell culture but is severely attenuated in transmission by Ae. aegypti after an infectious blood meal, with 5% saliva-positive mosquitoes for ZIKVΔSF1 vs. 31% for ZIKV. Furthermore, viral titers in the mosquito saliva were lower for ZIKVΔSF1 as compared to ZIKV. Comparison of mosquito infection via infectious blood meals and intrathoracic injections showed that sfRNA is important for ZIKV to overcome the mosquito midgut barrier and to promote virus accumulation in the saliva. Next-generation sequencing of infected mosquitoes showed that viral small-interfering RNAs were elevated upon ZIKVΔSF1 as compared to ZIKV infection. RNA-affinity purification followed by mass spectrometry analysis uncovered that sfRNA specifically interacts with a specific set of Ae. aegypti proteins that are normally associated with RNA turnover and protein translation. The DEAD/H-box helicase ME31B showed the highest affinity for sfRNA and displayed antiviral activity against ZIKV in Ae. aegypti cells. Based on these results, we present a mechanistic model in which sfRNA sequesters ME31B to promote flavivirus replication and virion production to facilitate transmission by mosquitoes.

Effect of RNA Interference on the Hepatitis C Virus Core Expression in HepG2/C3A Cells Using Genotype 4 Isolates from Saudi Patients.

To explore the effects of two different methodologies of RNA interference, namely small interfering RNA, and vector-based short hairpin RNA, on the expression levels of hepatitis C virus core RNA and protein of Saudi genotype 4 isolates. An experimental study. Laboratories of the College of Medicine Research Center, King Saud University, Saudi Arabia, from January to December 2018. Hepatitis C virus core small interfering RNA molecule and short hairpin RNA vector were designed against core region. Viral RNA expression was tested by RT-PCR; whereas, core protein was tested by flow cytometry and immunofluorescence. Results were statistically analysed by Chi-square analysis to calculate the p-value. Both molecules caused a reduction in core RNA and protein expression in infected cells. The effect of 100-pmol of small interfering RNA was more evident. For the vector-based short hairpin RNA, inhibition of core RNA expression was quite evident after 96 hours (p = 0.007). The results of flow cytometry and immunofluorescence showed a decline in core protein expression. The most dramatic effect was observed with 100-pmol small interfering RNA treatment of cells for 24 and 48 hours, which resulted in 63.5% and 91.1% core RNA expression reduction, respectively. RNA interference of hepatitis C virus core gene efficiently stopped viral replication and offer a promising therapeutic alternative against virus infection.

Analysis of Paramyxovirus Transcription and Replication by High-Throughput Sequencing.

We have developed a high-throughput sequencing (HTS) workflow for investigating paramyxovirus transcription and replication. We show that sequencing of oligo(dT)-selected polyadenylated mRNAs, without considering the orientation of the RNAs from which they had been generated, cannot accurately be used to analyze the abundance of viral mRNAs because genomic RNA copurifies with the viral mRNAs. The best method is directional sequencing of infected cell RNA that has physically been depleted of ribosomal and mitochondrial RNA followed by bioinformatic steps to differentiate data originating from genomes from viral mRNAs and antigenomes. This approach has the advantage that the abundance of viral mRNA (and antigenomes) and genomes can be analyzed and quantified from the same data. We investigated the kinetics of viral transcription and replication during infection of A549 cells with parainfluenza virus type 2 (PIV2), PIV3, PIV5, or mumps virus and determined the abundances of individual viral mRNAs and readthrough mRNAs. We found that the mRNA abundance gradients differed significantly between all four viruses but that for each virus the pattern remained relatively stable throughout infection. We suggest that rapid degradation of non-poly(A) mRNAs may be primarily responsible for the shape of the mRNA abundance gradient in parainfluenza virus 3, whereas a combination of this factor and disengagement of RNA polymerase at intergenic sequences, particularly those at the NP:P and P:M gene boundaries, may be responsible in the other viruses.IMPORTANCE High-throughput sequencing (HTS) of virus-infected cells can be used to study in great detail the patterns of virus transcription and replication. For paramyxoviruses, and by analogy for all other negative-strand RNA viruses, we show that directional sequencing must be used to distinguish between genomic RNA and mRNA/antigenomic RNA because significant amounts of genomic RNA copurify with poly(A)-selected mRNA. We found that the best method is directional sequencing of total cell RNA, after the physical removal of rRNA (and mitochondrial RNA), because quantitative information on the abundance of both genomic RNA and mRNA/antigenomes can be simultaneously derived. Using this approach, we revealed new details of the kinetics of virus transcription and replication for parainfluenza virus (PIV) type 2, PIV3, PIV5, and mumps virus, as well as on the relative abundance of the individual viral mRNAs.

The E3 ligase TRIM56 is a host restriction factor of Zika virus and depends on its RNA-binding activity but not miRNA regulation, for antiviral function.

Infection by Zika virus (ZIKV) is linked to microcephaly and other neurological disorders, posing a significant health threat. Innate immunity is the first line of defense against invading pathogens, but relatively little is understood regarding host intrinsic mechanisms that guard against ZIKV. Here, we show that host tripartite motif-containing protein 56 (TRIM56) poses a barrier to ZIKV infection in cells of neural, epithelial and fibroblast origins. Overexpression of TRIM56, but not an E3 ligase-dead mutant or one lacking a short C-terminal portion, inhibited ZIKV RNA replication. Conversely, depletion of TRIM56 increased viral RNA levels. Although the C-terminal region of TRIM56 bears sequence homology to NHL repeat of TRIM-NHL proteins that regulate miRNA activity, knockout of Dicer, which abolishes production of miRNAs, had no demonstrable effect on ZIKV restriction imposed by TRIM56. Rather, we found that TRIM56 is an RNA-binding protein that associates with ZIKV RNA in infected cells. Moreover, a recombinant TRIM56 fragment comprising the C-terminal 392 residues captured ZIKV RNA in cell-free reactions, indicative of direct interaction. Remarkably, deletion of a short C-terminal tail portion abrogated the TRIM56-ZIKV RNA interaction, concomitant with a loss in antiviral activity. Altogether, our study reveals TRIM56 is an RNA binding protein that acts as a ZIKV restriction factor and provides new insights into the antiviral mechanism by which this E3 ligase tackles flavivirus infections.

Hepatitis Delta Antigen Regulates mRNA and Antigenome RNA Levels during Hepatitis Delta Virus Replication.

Hepatitis delta virus (HDV) is a satellite of hepatitis B virus that increases the severity of acute and chronic liver disease. HDV produces three processed RNAs that accumulate in infected cells: the circular genome; the circular antigenome, which serves as a replication intermediate; and lesser amounts of the mRNA, which encodes the sole viral protein, hepatitis delta antigen (HDAg). The HDV genome and antigenome RNAs form ribonucleoprotein complexes with HDAg. Although HDAg is required for HDV replication, it is not known how the relative amounts of HDAg and HDV RNA affect replication, or whether HDAg synthesis is regulated by the virus. Using a novel transfection system in which HDV replication is initiated using in vitro-synthesized circular HDV RNAs, HDV replication was found to depend strongly on the relative amounts of HDV RNA and HDAg. HDV controls these relative amounts via differential effects of HDAg on the production of HDV mRNA and antigenome RNA, both of which are synthesized from the genome RNA template. mRNA synthesis is favored at low HDAg levels but becomes saturated at high HDAg concentrations. Antigenome RNA accumulation increases linearly with HDAg and dominates at high HDAg levels. These results provide a conceptual model for how HDV antigenome RNA production and mRNA transcription are controlled from the earliest stage of infection onward and also demonstrate that, in this control, HDV behaves similarly to other negative-strand RNA viruses, even though there is no genetic similarity between them.IMPORTANCE Hepatitis delta virus (HDV) is a satellite of hepatitis B virus that increases the severity of liver disease; approximately 15 million people are chronically infected worldwide. There are no licensed therapies available. HDV is not related to any known virus, and few details regarding its replication cycle are known. One key question is whether and how HDV regulates the relative amounts of viral RNA and protein in infected cells. Such regulation might be important because the HDV RNA and protein form complexes that are essential for HDV replication, and the proper stoichiometry of these complexes could be critical for their function. Our results show that the relative amounts of HDV RNA and protein in cells are indeed important for HDV replication and that the virus does control them. These observations indicate that further study of these regulatory mechanisms is required to better understand replication of this serious human pathogen.

B-104 Presence of HIV-1 RNA in extracellular vesicles from HIV-1 cART-treated cells

HIV-1 is a complex retrovirus that produces chronic infection affecting approximately 40 million people worldwide and 1.1 million new infections each year. We have previously shown that during infection, EVs contain a non-coding HIV-1 RNA, which has been shown to elicit responses in recipient cells (1–3). First, we investigated the effect of exosomes derived from uninfected cells on latent HIV-1–infected cells and found that there is an abundant level of short noncoding RNA in infected cells, arguing that a true “transcriptional latency” may not exist in vivo since cells are constantly in contact with exosomes from uninfected cells. A possible mechanism for this finding revealed that the exosomes cause an increase in RNA Polymerase II loading onto the DNA within the infected cells; this was possibly driven by the presence of specific kinases in the exosomes, some of which were unique to T-cell-derived and myeloid-derived exosomes. Subsequently transcribed viral transcripts, which include TAR and a novel RNA termed “TAR-gag”, can then be packaged into exosomes and potentially be exported to neighboring uninfected cells. TAR-gag is a long, noncoding RNA that is 615 bases long, contains the TAR element, and terminates within the p17 region of HIV-1 gag (1). In the presence of transcription inhibitors F07#13 (a Tat peptide mimetic) and CR8#13 (an ATP-binding analog of Cdk9), TAR-gag is capable of binding to SWI/SNF components in the nucleus, including the mSin3A/HDAC-1 complex, and serves as a scaffolding RNA. Additionally, TAR-gag can recruit suppressive factors and RNA-binding proteins to the HIV-1 promoter, resulting in transcriptional gene silencing (TGS) (2). In our most recent data, we found altered levels of genomic RNA within EVs while other RNAs, such as TAR RNA, remained unaffected by the addition of cART treatment in both cell lines, primary macrophages, and patient biofluids. Furthermore, we determined possible mechanisms involved in the selective packaging of HIV-1 RNA products into EVs specifically, an increase in EV-associated RNA-binding protein hnRNPA2/B1, and a “reader” for methylated RNA.

Quantitative PCR-Based Assessment of Vaccinia Virus RNA and DNA in Infected Cells.

Quantitative PCR-based methods have proven to be easy-to-use, cost-effective procedures for the quantification of viral gene expression and viral genome numbers. Quantitative PCR (qPCR) and quantitative reverse transcriptase-PCR (qRT-PCR) are rapid and sensitive approaches that can be used to pinpoint defects in viral DNA replication and transcriptional activity, respectively. Due to the significant nucleotide overlap between Poxviridae these methods can be employed across a wide range of viruses from this family. Here we provide methods for the quantification of vaccinia DNA replication by qPCR and quantification of the three classes of vaccinia gene transcription by qRT-PCR.

Contemporary Zika Virus Isolates Induce More dsRNA and Produce More Negative-Strand Intermediate in Human Astrocytoma Cells.

The recent emergence and rapid geographic expansion of Zika virus (ZIKV) poses a significant challenge for public health. Although historically causing only mild febrile illness, recent ZIKV outbreaks have been associated with more severe neurological complications, such as Guillain-Barré syndrome and fetal microcephaly. Here we demonstrate that two contemporary (2015) ZIKV isolates from Puerto Rico and Brazil may have increased replicative fitness in human astrocytoma cells. Over a single infectious cycle, the Brazilian isolate replicates to higher titers and induces more severe cytopathic effects in human astrocytoma cells than the historical African reference strain or an early Asian lineage isolate. In addition, both contemporary isolates induce significantly more double-stranded RNA in infected astrocytoma cells, despite similar numbers of infected cells across isolates. Moreover, when we quantified positive- and negative-strand viral RNA, we found that the Asian lineage isolates displayed substantially more negative-strand replicative intermediates than the African lineage isolate in human astrocytoma cells. However, over multiple rounds of infection, the contemporary ZIKV isolates appear to be impaired in cell spread, infecting a lower proportion of cells at a low MOI despite replicating to similar or higher titers. Taken together, our data suggests that contemporary ZIKV isolates may have evolved mechanisms that allow them to replicate with increased efficiency in certain cell types, thereby highlighting the importance of cell-intrinsic factors in studies of viral replicative fitness.

Irradiation by ultraviolet light-emitting diodes inactivates influenza a viruses by inhibiting replication and transcription of viral RNA in host cells

Influenza A viruses (IAVs) pose a serious global threat to humans and their livestock, especially poultry and pigs. This study aimed to investigate how to inactivate IAVs by using different ultraviolet-light-emitting diodes (UV-LEDs). We developed sterilization equipment with light-emitting diodes (LEDs) those peak wavelengths were 365 nm (UVA-LED), 310 nm (UVB-LED), and 280 nm (UVC-LED). These UV-LED irradiations decreased dose fluence-dependent plaque-forming units of IAV H1N1 subtype (A/Puerto Rico/8/1934) infected Madin-Darby canine kidney (MDCK) cells, but the inactivation efficiency of UVA-LED was significantly lower than UVB- and UVC-LED. UV-LED irradiations did not alter hemagglutination titer, but decreased accumulation of intracellular total viral RNA in infected MDCK cells was observed. Additionally, UV-LED irradiations suppressed the accumulation of intracellular mRNA (messenger RNA), vRNA (viral RNA), and cRNA (complementary RNA), as measured by strand-specific RT-PCR. These results suggest that UV-LEDs inhibit host cell replication and transcription of viral RNA. Both UVB- and UVC-LED irradiation decreased focus-forming unit (FFU) of H5N1 subtype (A/Crow/Kyoto/53/2004), a highly pathogenic avian IAV (HPAI), in infected MDCK cells, and the amount of FFU were lower than the H1N1 subtype. From these results, it appears that IAVs may have different sensitivity among the subtypes, and UVB- and UVC-LED may be suitable for HPAI virus inactivation.

Molecular and genetic characterization of peste des petits ruminants virus Kurdistan 2011 strain based on the haemagglutinin and fusion protein genes sequences

Severe outbreaks of peste des petits ruminants virus (PPRV) occurred during 2011 in wild goats in the Kurdistan region in Iraq. Based on a partial sequence of the nucleoprotein (N) gene the causative virus, named PPRV Kurdistan 2011, was classified as PPRV lineage IV genotype. For further characterization, the open reading frames (ORFs) coding for the haemagglutinin (H) and fusion (F) protein of PPRV Kurdistan 2011 were amplified from infected-cell RNA by RT-PCR. Sequencing of the ORFs revealed sizes of 1830 nucleotides (nt) for the H-ORF and 1641 nt for the F-ORF. This study represents the first report describing the full length ORFs of H and F for a PPRV strain from Kurdistan, Iraq.Comparative sequence analysis performed for the entire H- and F- ORFs of Kurdistan 2011 with respective sequences of PPRV isolates showed the highest homology with PPRV lineage IV isolates which is also reflected by comparative amino acid analysis. Phylogenetic analysis performed based on the partial and complete nucleotide sequences of both H- and F-ORFs clustered Kurdistan 2011 within PPRV lineage IV genotype. H-gene based phylogenetic trees aligned Kurdistan 2011 strain along with Turkey/TR/AFYON/2011-35H and Turkey/2000 strains whereas F-gene based phylogenetic trees aligned Kurdistan 2011 strain along with Qatar/37/10-02, Kurdistan/2012 and Egypt/2009 strains.

The conserved stem-loop II structure at the 3' untranslated region of Japanese encephalitis virus genome is required for the formation of subgenomic flaviviral RNA.

Flaviviruses accumulate abundant subgenomic RNA (sfRNA) in infected cells. It has been reported that sfRNA results from stalling of host 5’-to-3’ exoribonuclease XRN1 at the highly structured RNA of the 3’ untranslated region (UTR). Although XRN1 digestion of a 3’-terminal 800-nt RNA could stall at a position to generate the sfRNA in vitro, we found that knocking out XRN1 had no effect on the accumulation of sfRNA in Japanese encephalitis virus (JEV) infected cells. Mutagenesis studies revealed that the stemloop II (SLII) at the 3’ UTR is required for the accumulation of sfRNA. According to the results of an in vitro RNA-dependent RNA polymerase (RdRp) assay, the (-)10431-10566 RNA fragment, containing the putative promoter on the antigenome for the sfRNA transcription, binds to RdRp protein and exhibits a strong promoter activity. Taken together, our results indicate that the JEV sfRNA could be transcribed initially and then be trimmed by XRN1 or other unidentified exoribonucleases.

Anti-influenza virus activity of a salcomine derivative mediated by inhibition of viral RNA synthesis.

Influenza virus infection is a major threat to global health. Although vaccines and anti-influenza virus drugs are available, annual influenza virus epidemics result in severe illness, and an influenza pandemic occurs every 20-30years. To identify candidate anti-influenza virus compounds, we screened approximately 5,000 compounds in an in-house library. We identified MZ7465, a salcomine derivative, as a potent inhibitor of influenza virus propagation. We analyzed the antiviral propagation mechanism of the hit compound by determining the amounts of viral proteins and RNA in infected cells treated with or without the hit compound. Treatment of infected cells with MZ7465 decreased both viral protein and RNA synthesis. In addition, an in vitro assay showed that viral RNA synthesis was directly inhibited by MZ7465. These results suggest that salcomine and its derivatives are potential candidates for the treatment of influenza virus infections.