Background: 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. Results: 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. Conclusion: 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.

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.

Acute respiratory infection (ARI) is a major killer of children in developing countries. Although the frequency of ARI is similar in both developed and developing countries, mortality due to ARI is 10–50 times higher in developing countries. Viruses are common causes of ARI among such children, yet the disease burden of these infections in rural communities is unknown. A prospective longitudinal study was carried out in children enrolled from two rural Indian villages at birth and followed weekly for the development of ARI, classified as upper respiratory infection, acute lower respiratory infection (ALRI), or severe ALRI. Respiratory syncytial virus (RSV), influenza, parainfluenza viruses and adenoviruses in nasopharyngeal aspirates were detected by direct fluorescent antibody testing (DFA) and, in addition, centrifugation enhanced culture for RSV was done. 281 infants enrolled in 39 months and followed until 42 months. During 440 child years of follow-up there were 1307 ARIs, including 236 ALRIs and 19 severe ALRIs. Virus specific incidence rates per 1000 child years for RSV were total ARI 234, ALRI 39, and severe ALRI 9; for influenza A total ARI 141, ALRI 39; for INF B total ARI 37; for PIV1 total ARI 23, for PIV2 total ARI 28, ALRI 5; for parainfluenza virus 3 total ARI 229, ALRI 48, and severe ALRI 5 and for adenovirus total ARI 18, ALRI 5. Repeat infections with RSV were seen in 18 children. RSV, influenza A and parainfluenza virus 3 were important causes of ARI among children in rural communities in India. These data will be useful for vaccine design, development and implementation purposes.

For efficient replication, viruses have developed mechanisms to evade innate immune responses, including the antiviral type-I interferon (IFN-I) system. Nipah virus (NiV), a highly pathogenic member of the Paramyxoviridae family (genus Henipavirus), is known to encode for four P gene-derived viral proteins (P/C/W/V) with IFN-I antagonist functions. Here we report that NiV matrix protein (NiV-M), which is important for virus assembly and budding, can also inhibit IFN-I responses. IFN-I production requires activation of multiple signaling components including the IκB kinase epsilon (IKKε). We previously showed that the E3-ubiquitin ligase TRIM6 catalyzes the synthesis of unanchored K48-linked polyubiquitin chains, which are not covalently attached to any protein, and activate IKKε for induction of IFN-I mediated antiviral responses. Using co-immunoprecipitation assays and confocal microscopy we show here that the NiV-M protein interacts with TRIM6 and promotes TRIM6 degradation. Consequently, NiV-M expression results in reduced levels of unanchored K48-linked polyubiquitin chains associated with IKKε leading to impaired IKKε oligomerization, IKKε autophosphorylation and reduced IFN-mediated responses. This IFN antagonist function of NiV-M requires a conserved lysine residue (K258) in the bipartite nuclear localization signal that is found in divergent henipaviruses. Consistent with this, the matrix proteins of Ghana, Hendra and Cedar viruses were also able to inhibit IFNβ induction. Live NiV infection, but not a recombinant NiV lacking the M protein, reduced the levels of endogenous TRIM6 protein expression. To our knowledge, matrix proteins of paramyxoviruses have never been reported to be involved in innate immune antagonism. We report here a novel mechanism of viral innate immune evasion by targeting TRIM6, IKKε and unanchored polyubiquitin chains. These findings expand the universe of viral IFN antagonism strategies and provide a new potential target for development of therapeutic interventions against NiV infections. Nipah virus (NiV) is a zoonotic paramyxovirus causing severe respiratory and encephalitic illness with case fatality rates of 40 to 90%. The host type-I interferon (IFN-I) system protects against viral infections; however, to establish productive infection NiV has developed mechanisms to evade these host antiviral responses. An important component of the IFN system is the IKKε kinase, which is directly involved in IFN-I production and IFN-I signaling. The activity of the IKKε kinase is regulated by unanchored K48-linked polyubiquitin chains, a novel form of ubiquitin that is not covalently attached to any protein and can induce activation of kinases by promoting protein oligomerization. These unanchored polyubiquitin chains that activate IKKε are generated by the E3-ubiquitin ligase TRIM6. Here we demonstrate that the matrix structural protein (M) of NiV, which is important for virus assembly and budding, also has IFN-I antagonist functions and interferes with the host antiviral response. We found that NiV-M interacts with TRIM6 and promotes its degradation. Consequently, association of unanchored polyubiquitin chains with IKKε is reduced leading to impaired IKKε activation and ineffective IFN responses. Since the matrix protein is present in the virions and is released immediately after virus entry into the cell, this provides an efficient mechanism to escape the host antiviral response. These data may help explain the highly pathogenic potential of these viruses.

Background: 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). Results: 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 0.1TCID₅₀ for RSV and influenza A and 1TCID₅₀ for 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). Conclusion: Multiplex PCR offers a rapid, sensitive and reasonably priced diagnostic method for common respiratory viruses.