RESUMEN
Baculoviruses are arthropod-specific pathogens, and find extensive applications in pest control strategies and recombinant protein expression. Spodoptera litura nucleopolyhedrovirus (SpltNPV) infects the tropical armyworm Spodoptera litura, which is an important polyphagous crop pest widely distributed in regions of Asia and Oceania. Using next-generation sequencing, we report stage-specific profiling of SpltNPV-encoded microRNAs (miRNAs) at different time intervals post-infection (p.i.) of Sf21 cells. Sequence length distribution analysis of the small RNA libraries revealed a significant increase in 20 nt reads and a reduction of other size fractions during late phases of infection. In silico miRNA prediction tools identified 48 novel SpltNPV-encoded miRNAs, of which 10 were validated experimentally in Sf21 cells using Northern blot analysis and TaqMan quantitative real-time (qRT)-PCR. The viral miRNAs were also found to be expressed in fat-body and mid-gut tissues of infected fifth-instar S. litura larva. qRT-PCR analysis confirmed that expression of most viral miRNAs was triggered 12 h p.i. and continued thereafter. Gene Ontology and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway annotation of computationally predicted targets of the reported miRNAs suggested a major impact of these miRNAs on cell signalling, protein translation and metabolic processes.
Asunto(s)
Regulación Viral de la Expresión Génica/fisiología , MicroARNs/metabolismo , Nucleopoliedrovirus/metabolismo , ARN Viral/metabolismo , Transcriptoma , Animales , Línea Celular , MicroARNs/genética , Mariposas Nocturnas , Nucleopoliedrovirus/genética , ARN Viral/genética , Factores de TiempoRESUMEN
The host-mediated RNAi pathways restrict replication of viruses in plant, invertebrate and vertebrate systems. However, comparatively little is known about the interplay between RNAi and various viral infections in mammalian hosts. We show in the present study that the siRNA-mediated silencing of Drosha, Dicer and Ago2 [argonaute RISC (RNA-induced silencing complex) catalytic component 2] transcripts in Huh7 cells resulted in elevated levels of HBV (hepatitis B virus)-specific RNAs and, conversely, we observed a decrease in mRNA and protein levels of same RNAi components in HepG2 cells infected with HBV. Similar reductions were also detectable in CHB (chronic hepatitis B) patients. Analysis of CHB liver biopsy samples, with high serum HBV DNA load (>log108 IU/ml), revealed a reduced mRNA and protein levels of Drosha, Dicer and Ago2. The low expression levels of key RNAi pathway components in CHB patient samples as well as hepatic cells established a link between HBV replication and RNAi components. The HBV proteins were also examined for RSS (RNA-silencing suppressor) properties. Using GFP-based reversion of silencing assays, in the present study we found that HBx is an RSS protein. Through a series of deletions and substitution mutants, we found that the full-length HBx protein is required for optimum RSS activity. The in vitro dicing assays revealed that the HBx protein inhibited the human Dicer-mediated processing of dsRNAs into siRNAs. Together, our results suggest that the HBx protein might function as RSS to manipulate host RNAi defence, in particular by abrogating the function of Dicer. The present study may have implications in the development of newer strategies to combat HBV infection.
Asunto(s)
Virus de la Hepatitis B/fisiología , Interferencia de ARN , Transactivadores/fisiología , Adulto , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Línea Celular Tumoral , Femenino , Células HEK293 , Hepatitis B Crónica/metabolismo , Humanos , Hígado/metabolismo , Masculino , Mutación , Sistemas de Lectura Abierta , ARN Bicatenario/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Transactivadores/genética , Proteínas Reguladoras y Accesorias Virales , Replicación Viral , Adulto JovenRESUMEN
Aedes aegypti and Aedes albopictus are principal vectors for the transmission of chikungunya virus (CHIKV). India is a hub for both dengue and chikungunya infections and there are several reports of co-infection of dengue and chikungunya virus in the clinical scenario. The present pilot entomological survey was conducted to evaluate vertical transmission of CHIKV in Aedes field populations. Aedes immature (larvae and pupae) collection was done in 2012, over a period of six months from selected sites in Delhi and Haryana, India. The immatures collected were reared for adult emergence and species identification was done. A. aegypti male and female mosquitoes were separated and pooled collection spot-wise, RNA extracted and RT PCR performed to test for the presence of CHIKV in the pools. Container index (CI) and minimum infection rate (MIR) were estimated. From study areas that tested positive for CHIKV, adult collections were made and females upon feeding on uninfected blood in laboratory were allowed to lay eggs. The progeny that emerged from these field-collected mothers were tested for CHIKV presence. Our pilot survey showed the existence of A. aegypti population even during peak summer season in a few foci which eventually helped the mosquitoes to tide over adverse environmental conditions and with the start of rainfall, the population exploded within a short period of time. Immatures collected from field and progeny of adults collected from the field were CHIKV positive demonstrating the presence of vertical transmission of chikungunya virus in field population of A. aegypti. The present study further demonstrates the importance of identifying permanent breeding sites for proper Aedes species control.