Molecular determinants and mechanisms of arthropod-borne flavivirus transmission to the vertebrate host are poorly understood. In this study, we show for the first time that a cell line from medically important arthropods, such as ticks, secretes extracellular vesicles (EVs) including exosomes that mediate transmission of flavivirus RNA and proteins to the human cells. Our study shows that tick-borne Langat virus (LGTV), a model pathogen closely related to tick-borne encephalitis virus (TBEV), profusely uses arthropod exosomes for transmission of viral RNA and proteins to the human- skin keratinocytes and blood endothelial cells. Cryo-electron microscopy showed the presence of purified arthropod/neuronal exosomes with the size range of 30 to 200 nm in diameter. Both positive and negative strands of LGTV RNA and viral envelope-protein were detected inside exosomes derived from arthropod, murine and human cells. Detection of Nonstructural 1 (NS1) protein in arthropod and neuronal exosomes further suggested that exosomes contain viral proteins. Viral RNA and proteins in exosomes derived from tick and mammalian cells were secured, highly infectious and replicative in all tested evaluations. Treatment with GW4869, a selective inhibitor that blocks exosome release affected LGTV loads in both arthropod and mammalian cell-derived exosomes. Transwell-migration assays showed that exosomes derived from infected-brain-microvascular endothelial cells (that constitute the blood-brain barrier) facilitated LGTV RNA and protein transmission, crossing of the barriers and infection of neuronal cells. Neuronal infection showed abundant loads of both tick-borne LGTV and mosquito-borne West Nile virus RNA in exosomes. Our data also suggest that exosome-mediated LGTV viral transmission is clathrin-dependent. Collectively, our results suggest that flaviviruses uses arthropod-derived exosomes as a novel means for viral RNA and protein transmission from the vector, and the vertebrate exosomes for dissemination within the host that may subsequently allow neuroinvasion and neuropathogenesis.

Introduction: Enteric fever is endemic in Nepal and poses a significant public health burden. The first-line drugs ampicillin, chloramphenicol, and cotrimoxazole have not been part of empirical therapy for two decades due to the development of multidrug-resistant Salmonella strains. The objective of this study was to determine the antibiogram pattern of Salmonella serovars isolated from the blood of clinically suspected enteric fever patients. Methodology: A cross sectional study was carried out in a tertiary care hospital in Lalitpur, Nepal, between July 2011 and February 2012. Standard microbiological procedures were followed during collection and processing of blood samples, isolation and identification of Salmonella serotypes. The antimicrobial sensitivity of ampicillin, chloramphenicol, cotrimoxazole, nalidixic acid, and ciprofloxacin was determined using a modified Kirby-Bauer disk diffusion method as per the guidelines of the Clinical and Laboratory Standards Institute. Results: Out of 86 Salmonella isolates, 56 (65.1%) were Salmonella Typhi and 30 (34.9%) were Salmonella Paratyphi A. Salmonella Typhi were 100% sensitive to chloramphenicol, cotrimoxazole, and ciprofloxacin and 98.2% sensitive to ampicillin. Similarly, Salmonella Paratyphi A isolates were 100% sensitive to ampicillin and cotrimoxazole and 96.7% sensitive to chloramphenicol and ciprofloxacin. More than 90.0% of isolates were nalidixic acid resistant and none of the Salmonella isolates were multi-drug resistant. Conclusions: This study revealed the increasing frequency of nalidixic acid-resistant Salmonella isolates, indicating the possibility of fluoroquinolone resistance in near future. Furthermore, re-emergence of susceptibility to conventional first-line drugs ampicillin, chloramphenicol, and cotrimoxazole supports the possibility of using these drugs in empirical therapy.

A small percentage of babies born to Zika virus (ZIKV)-infected mothers manifest severe defects at birth, including microcephaly. Among those who appeared healthy at birth, there are increasing reports of postnatal growth or developmental defects. However, the impact of congenital ZIKV infection in postnatal development is poorly understood. Here, we report that a mild congenital ZIKV-infection in pups born to immunocompetent pregnant mice did not display apparent defects at birth, but manifested postnatal growth impediments and neurobehavioral deficits, which include reduced locomotor and cognitive deficits that persisted into adulthood. We found that the brains of these pups were smaller, had a thinner cortical layer 1, displayed increased astrogliosis, decreased expression of microcephaly- and neuron development- related genes, and increased pathology as compared to mock-infected controls. In summary, our results showed that even a mild congenital ZIKV infection in immunocompetent mice could lead to postnatal deficits, providing definitive experimental evidence for a necessity to closely monitor postnatal growth and development of presumably healthy human infants, whose mothers were exposed to ZIKV infection during pregnancy.

Interferons (IFNs) are the key components of innate immunity and are crucial for host defense against viral infections. Here, we report a novel role of interleukin-17A (IL-17A) in inhibiting IFN-α2 expression thus promoting chikungunya virus (CHIKV) infection. CHIKV infected IL-17A deficient ( Il17a −/− ) mice expressed a higher level of IFN-α2 and developed diminished viremia and milder footpad swelling in comparison to wild-type (WT) control mice, which was also recapitulated in IL-17A receptor-deficient ( Il17ra −/− ) mice. Interestingly, IL-17A selectively blocked IFN-α2 production during CHIKV, but not West Nile virus (WNV) or Zika virus (ZIKV), infections. Recombinant IL-17A treatment inhibited CHIKV-induced IFN-α2 expression and enhanced CHIKV replication in both human and mouse cells. We further found that IL-17A inhibited IFN-α2 production by modulating the expression of Interferon Regulatory Factor-5 (IRF-5), IRF-7, IFN-stimulated gene 49 (ISG-49), and Mx1 expression during CHIKV infection. Neutralization of IL-17A in vitro leads to the increase of the expression of these antiviral molecules and decrease of CHIKV replication. Collectively, these results suggest a novel function of IL-17A in inhibiting IFN-α2–mediated antiviral responses during CHIKV infection, which may have broad implications in viral infections and other inflammatory diseases.

Abstract. Chikungunya virus (CHIKV) is an emerging alphaviral disease and a public health problem in South Asia including Nepal in recent years. In this study, sera were collected from patients presenting with fever, headache, muscular pain, fatigue, and joint pain of both upper and lower extremities. A total of 169 serum samples were tested for CHIKV and dengue virus (DENV) by using Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibody using enzymelinked immunosorbent assay (ELISA) method during August to November 2013. Results showed that 3.6% and 27.8% samples were positive for CHIKV and DENV IgM positive, respectively. Similarly, results of IgG showed 3.0% samples were positive for CHIKV IgG and 29.0% were for DENV IgG positive. Further, a 50% focal reduction neutralization test (FRNT 50 ) was performed to confirm the presence of CHIKV, which demonstrated that 8.9% of CHIKV IgM and/or IgG ELISA positive possessed neutralizing anti-CHIK antibodies. To our knowledge, this is the first report in which the presence of CHIKV is confirmed in Nepalese patients by FRNT 50 . Basic scientists and clinicians need to consider CHIKV as a differential diagnosis in febrile Nepalese patients, and policy makers should consider appropriate surveillance and actions for control strategies.Chikungunya virus (CHIKV) is a mosquito-borne febrile illness that is transmitted to humans through the bite of infected Aedes aegypti and Ae. albopictus mosquitoes.1 CHIKV belongs to the Alphavirus genus of the Togaviridae family whereas dengue viruses (DENVs) belong to genus Flavivirus of the family Flaviviridae. Both CHIKV and DENV are transmitted by the same mosquito vectors. The CHIKV was first isolated and characterized in humans and mosquitoes during an outbreak in Tanzania and Mozambique in 1955. 2 Afterwards, several outbreaks of CHIKV occurred and affected millions of people in eastern, southern, central Africa and Asia.3 CHIKV was introduced in the Americas in October 2013. As of April 2015, over 1,322,893 cases have been suspected and 30,309 confirmed to be CHIKV in the Americas. 4 It generally causes mild illness but sometimes can lead to severe and lifethreatening complications. The disease is characterized by an acute illness with fever, chills, headache, nausea, vomiting, joint pain with or without swelling, low back pain, and skin rash. DENV can progress to dengue hemorrhagic fever and dengue shock syndrome while CHIKV causes arthralgia, which may persist for months. 5 There is similarity in signs and symptoms of CHIK and DEN, which increases risks for misdiagnosis and underreporting of CHIKV infection in DEN-endemic areas. 5 The incubation period of CHIKV is usually 2-10 days, with constitutional symptoms lasting up to 7 days. The symptoms usually resolve within days to a few weeks; but in severe cases, these symptoms may last for months. Herein, we report a serological study of possible CHIKV infection with confirmation by 50% focal reduction neutralization test (FRNT 50 ) among febrile patients for the first time in N...