P-body components LSM1, GW182, DDX3, DDX6 and XRN1 are recruited to WNV replication sites and positively regulate viral replication

AbstractIn mammalian cells, proteins involved in mRNA silencing and degradation localize to discrete cytoplasmic foci called processing or P-bodies. Here we show that microscopically visible P-bodies are greatly diminished following West Nile viral infection, but the component proteins are not depleted. On the other hand, many P-body components including LSM1, GW182, DDX3, DDX6 and XRN1, but not others like DCP1a and EDC4 are recruited to the viral replication sites, as evidenced by their colocalization at perinuclear region with viral NS3. Kinetic studies suggest that the component proteins are first released from P-bodies in response to WNV infection within 12h post-infection, followed by recruitment to the viral replication sites by 24–36 h post-infection. Silencing of the recruited proteins individually with siRNA interfered with viral replication to varying extents suggesting that the recruited proteins are required for efficient viral replication. Thus, the P-body proteins might provide novel drug targets for inhibiting viral infection

Concurrent infections by all four dengue virus serotypes during an outbreak of dengue in 2006 in Delhi, India

<p>Abstract</p> <p>Background</p> <p>Co-circulation of multiple dengue virus serotypes has been reported from many parts of the world including India, however concurrent infection with more than one serotype of dengue viruses in the same individual is rarely documented. An outbreak of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) occurred in and around Delhi in 2006. This is the first report from India with high percentage of concurrent infections with different dengue virus serotypes circulating during one outbreak.</p> <p>Results</p> <p>Acute phase sera from patients were tested for the presence of dengue virus RNA by RT-PCR assay. Of the 69 samples tested for dengue virus RNA, 48 (69.5%) were found to be positive. All the four dengue virus serotypes were found to be co-circulating in this outbreak with DENV-3 being the predominant serotype. In addition in 9 of 48 (19%) dengue virus positive samples, concurrent infection with more than one dengue virus serotype were identified.</p> <p>Conclusion</p> <p>This is the first report in which concurrent infections with different dengue virus serotypes is being reported during an outbreak from India. Delhi is now truly hyperendemic for dengue.</p

Ago-2-Mediated Slicer Activity Is Essential for Anti-Flaviviral Efficacy of RNAi

RNA interference can be mediated by fully complementary siRNA or partially complementary miRNA. siRNAs are widely used to suppress viral replication and the fully complementary siRNA bound Ago-2 in the RISC is known to degrade the target RNA. Although other argonaute proteins lacking slicer activity can also bind oligonucleotides with both si and miRNA structures, whether they can also contribute to antiviral effects is not entirely clear. We tested si and miRNA structured oligos for target repression in dual luciferase assays as well as for inhibition of Dengue and West Nile virus replication in ES cells expressing individual Ago proteins. In luciferase assays, both fully complementary and partially complementary oligos effectively repressed their targets in all individual Ago expressing cell lines, although the efficacy with fully complementary oligos was higher in Ago-2+ cells. However, partially complementary oligos had no effect on virus replication in any cell line, while fully complementary siRNAs were highly effective in Ago-2 expressing, but not in cells expressing other Ago proteins. This occurred irrespective of whether the target sequences were located in the coding region or 3′UTR of the virus. We conclude that Ago-2 slicer activity is essential for anti-viral efficacy of siRNAs and miRNA-mediated translational repression/transcript destabilization is too weak to suppress the abundantly expressed flaviviral proteins

Respiratory viral infections detected by multiplex PCR among pediatric patients with lower respiratory tract infections seen at an urban hospital in Delhi from 2005 to 2007

<p>Abstract</p> <p>Background</p> <p>Acute lower respiratory tract infections (ALRI) are the major cause of morbidity and mortality in young children worldwide. Information on viral etiology in ALRI from India is limited. The aim of the present study was to develop a simple, sensitive, specific and cost effective multiplex PCR (mPCR) assay without post PCR hybridization or nested PCR steps for the detection of respiratory syncytial virus (RSV), influenza viruses, parainfluenza viruses (PIV1–3) and human metapneumovirus (hMPV). Nasopharyngeal aspirates (NPAs) were collected from children with ALRI ≤ 5 years of age. The sensitivity and specificity of mPCR was compared to virus isolation by centrifugation enhanced culture (CEC) followed by indirect immunofluorescence (IIF).</p> <p>Results</p> <p>From April 2005–March 2007, 301 NPAs were collected from children attending the outpatient department or admitted to the ward of All India Institute of Medical Sciences hospital at New Delhi, India. Multiplex PCR detected respiratory viruses in 106 (35.2%) of 301 samples with 130 viruses of which RSV was detected in 61, PIV3 in 22, PIV2 in 17, hMPV in 11, PIV1 in 10 and influenza A in 9 children. CEC-IIF detected 79 viruses only. The sensitivity of mPCR was 0.1TCID₅₀for RSV and influenza A and 1TCID₅₀for hMPV, PIV1, PIV2, PIV3 and Influenza B. Mixed infections were detected in 18.8% of the children with viral infections, none detected by CEC-IIF. Bronchiolitis was significantly associated with both total viral infections and RSV infection (p < 0.05). History of ARI in family predisposed children to acquire viral infection (p > 0.05).</p> <p>Conclusion</p> <p>Multiplex PCR offers a rapid, sensitive and reasonably priced diagnostic method for common respiratory viruses.</p

Co-infections with Chikungunya Virus and Dengue Virus in Delhi, India

Aedes aegypti mosquitoes are common vectors for dengue virus and chikungunya virus. In areas where both viruses cocirculate, they can be transmitted together. During a dengue outbreak in Delhi in 2006, 17 of 69 serum samples were positive for chikungunya virus by reverse transcription–PCR; 6 samples were positive for both viruses

All 4 Ago proteins bind both fully complementary siRNA and partially complementary miRNA resembling oligos and repress target gene in reporter assays.

<p>Parental ES cells expressing all ago proteins and ES cells expressing individual Ago proteins were transfected with dual luciferase reporter vector containing dengue virus siRNA target sequences in the Renilla luciferase 3′UTR along with fully or partially complementary dengue virus siRNAs and the dual luciferase expression measured after 24 h of transfection. Rluc/Fluc expression normalized to control irrelevant GFP siRNA is shown. Error bars represent mean of quadruplicates +/− SD.</p

Flavivirally infected cells lose microscopic P bodies, but not GW182 and exhibit both si and miRNA-mediated target repression.

<p>A) Uninfected and dengue or West Nile virus infected HeLa cells were stained with DAPI, DCP1a and dengue/WNV antibody and examined for P bodies by microscopy. B) Control HeLa cells and HeLa cells expressing WNV replicon (HeLa WNR) were tested for GW182 isoform expression at mRNA level by QRT-PCR (left) and protein level by Western blot (right). C) West Nile replicon expressing HeLa cells were transfected with luciferase reporter with si/miRNA and analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027551#pone-0027551-g001" target="_blank">Fig 1a</a>. siDV1 represents control irrelevant siRNA. Error bars represent mean of quadruplicates +/− SD.</p

Sequence of siRNA and miRNA mimicking oligonucleotides.

<p>SiRNAs were designed to completely match the target sequence, while miRNA mimicking siRNAs were designed to contain central 3 mismatches (nts 9-11-nts depicted in bold letters).</p