The hypothesis that quiescent CD4+ T lymphocytes carrying proviral DNA provide a reservoir for human immunodeficiency virus-type 1 (HIV-1) in patients on highly active antiretroviral therapy (HAART) was examined. In a study of 22 patients successfully treated with HAART for up to 30 months, replication-competent virus was routinely recovered from resting CD4+ T lymphocytes. The frequency of resting CD4+ T cells harboring latent HIV-1 was low, 0.2 to 16.4 per 10(6) cells, and, in cross-sectional analysis, did not decrease with increasing time on therapy. The recovered viruses generally did not show mutations associated with resistance to the relevant antiretroviral drugs. This reservoir of nonevolving latent virus in resting CD4+ T cells should be considered in deciding whether to terminate treatment in patients who respond to HAART.

Combination therapy for HIV-1 infection can reduce plasma virus to undetectable levels, indicating that prolonged treatment might eradicate the infection. However, HIV-1 can persist in a latent form in resting CD4+ T cells. We measured the decay rate of this latent reservoir in 34 treated adults whose plasma virus levels were undetectable. The mean half-life of the latent reservoir was very long (43.9 months). If the latent reservoir consists of only 1 x 10(5) cells, eradication could take as long as 60 years. Thus, latent infection of resting CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective anti-retroviral therapy.

Cellular mRNA of higher eukaryotes and many viral RNA are methylated at the N-7 and 2′-O positions of the 5′ guanosine cap by specific nuclear and cytoplasmic methyltransferases (MTases), respectively. Whereas N-7 methylation is essential for RNA translation and stability 1, the function of 2′-O methylation has remained uncertain since its discovery 35 years ago 2-4. Here, we show that a West Nile virus (WNV) mutant (E218A) that lacks 2′-O MTase activity was attenuated in wild type primary cells and mice but was pathogenic in the absence of type I interferon (IFN) signaling. 2′-O methylation of viral RNA did not affect IFN induction in WNV-infected fibroblasts but instead modulated the antiviral effects of IFN-induced proteins with tetratricopeptide repeats (IFIT), which are interferon-stimulated genes (ISG) implicated in regulation of protein translation. Poxvirus and coronavirus mutants that lacked 2′-O MTase activity similarly showed enhanced sensitivity to the antiviral actions of IFN and specifically, IFIT proteins. Our results demonstrate that the 2′-O methylation of the 5′ cap of viral RNA functions to subvert innate host antiviral responses through escape of IFIT-mediated suppression, and suggest an evolutionary explanation for 2′-O methylation of cellular mRNA: to distinguish self from non-self RNA. Differential methylation of cytoplasmic RNA likely serves as a paradigm for pattern recognition and restriction of propagation of foreign viral RNA in host cells.

The recent rapid spread of Zika virus and its unexpected linkage to birth defects and an autoimmune-neurological syndrome has generated worldwide concern. Zika virus is a flavivirus like dengue, yellow fever and West Nile viruses. We present the 3.8Å resolution structure of mature Zika virus determined by cryo-electron microscopy. The structure of Zika virus is similar to other known flavivirus structures except for the ~10 amino acids that surround the Asn154 glycosylation site found in each of the 180 envelope glycoproteins that make up the icosahedral shell. The carbohydrate moiety associated with this residue, recognizable in the cryo-EM electron density, may function as an attachment site of the virus to host cells. This region varies not only among Zika virus strains but also in other flaviviruses and suggests that changes in this region influence virus transmission and disease.

Zika virus (ZIKV) has recently emerged as an explosive pandemic associated with severe neuropathology in newborns and adults1. There are no ZIKV-specific treatments or preventatives; thus, development of a safe and effective vaccine is a high priority. Messenger RNA (mRNA) has emerged as a versatile and highly effective platform to deliver vaccine antigens and therapeutic proteins2,3. Here, we demonstrate that a single low-dose intradermal immunization with lipid nanoparticle-encapsulated nucleoside-modified mRNA (mRNA-LNP) encoding the pre-membrane and envelope (prM-E) glycoproteins of a 2013 ZIKV outbreak strain elicited potent and durable neutralizing antibody responses in mice and non-human primates. Immunization with 30 μg of nucleoside-modified ZIKV mRNA-LNPs protected mice from ZIKV challenges at 2 weeks or 5 months post-vaccination, and a single dose of 50 μg was sufficient to protect non-human primates from a challenge at 5 weeks post-vaccination. These data demonstrate that nucleoside-modified mRNA-LNPs elicit rapid and durable protective immunity and thus represent a new and promising vaccine candidate for the global fight against ZIKV.

SUMMARY The emergence of ZIKV infection has prompted a global effort to develop safe and effective vaccines. We engineered a lipid nanoparticle (LNP) encapsulated modified mRNA vaccine encoding wild-type or variant ZIKV structural genes and tested immunogenicity and protection in mice. Two doses of modified mRNA LNPs encoding prM-E genes that produced virus-like particles resulted in high neutralizing antibody titers (~ 1/100,000) that protected against ZIKV infection and conferred sterilizing immunity. To offset a theoretical concern of ZIKV vaccines inducing antibodies that cross-react with the related Dengue virus (DENV), we designed modified prM-E RNA encoding mutations destroying the conserved fusion-loop epitope in the E protein. This variant protected against ZIKV and diminished production of antibodies enhancing DENV infection in cells or mice. A modified mRNA vaccine can prevent ZIKV disease and be adapted to reduce the risk of sensitizing individuals to subsequent exposure to DENV, should this become a clinically-relevant concern.

F laviviruses are single-stranded RNA viruses vectored principally by arthropods that cause severe illnesses in humans. The extensive global spread and epidemic transmission of flaviviruses during the last seven decades has been remarkable. The mosquito-borne dengue viruses (DENV) infect an estimated 400 million humans each year; more than a quarter of the world's population lives in areas where DENV is now endemic 1 . By comparison, only sporadic DENV epidemics were documented before the Second World War 2 . The introductions of West Nile (WNV) and Zika (ZIKV) viruses into the Western Hemisphere was followed by rapid geographical spread, large numbers of human infections and considerable morbidity 3,4 . Ongoing yellow fever virus (YFV) transmission and its encroachment on urban environments, despite the existence of an effective vaccine, poses a serious public health challenge 5-7 . Other flaviviruses present ongoing health risks or are beginning to emerge in different parts of the world, including Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV) and Usutu virus (USUV).The epidemic potential of flaviviruses reflects many factors related to the unique characteristics of their insect vectors, the consequences of poorly planned urbanization that creates ideal arthropod breeding habitats, the geographical expansion of vectors, changing environmental conditions and extensive global travel 8,9 . Beyond arthropods and humans, flaviviruses are also known to infect a wide array of animal species and can be important veterinary pathogens that threaten economically important domesticated animals 10-14 . These vertebrate animal hosts may constitute important stable reservoirs and contribute to defining conditions that support the introduction of new viral species and transmission among humans 15 . The continued threat of flavivirus emergence and re-emergence highlights a need for a detailed fundamental understanding of the biology of these viruses, the immune responses that can contain them and the possible countermeasures that can blunt their impact on public health should new outbreaks occur. Flavivirus structure and replicationFlaviviruses are small (~50 nm) spherical virus particles that incorporate a single genomic RNA of positive-sense polarity encoding three structural and seven non-structural proteins (Fig. 1a). Our knowledge of the biology of flaviviruses has advanced considerably with the availability of high-resolution structures of viral structural proteins and of virions at different stages of the replication cycle or in complex with antibodies or host factors 16 . Crystal structures of the enzymatic non-structural proteins also have been solved, accelerating advances in an understanding of virus replication and pathogenesis [17][18][19] and enabling structure-guided drug discovery, as reviewed elsewhere 20 .Virion structure and morphogenesis. Flaviviruses are assembled using three viral structural proteins (C, prM and E), a host lipid envelope and the viral genomic RNA. The structure of ...

SUMMARY Zika virus (ZIKV) infection during pregnancy has emerged as a global public health problem because of its ability to cause severe congenital disease. Here, we developed six mouse monoclonal antibodies (mAbs) against ZIKV including four (ZV-48, ZV-54, ZV-64, and ZV-67) that were ZIKV-specific and neutralized infection of African, Asian, and American strains to varying degrees. X-ray crystallographic and competition binding analyses of Fab fragments and scFvs defined three spatially distinct epitopes in DIII of the envelope protein corresponding to the lateral ridge (ZV-54 and ZV-67), C–C′ loop (ZV-48 and ZV-64), and ABDE sheet (ZV-2) regions. In vivo passive transfer studies revealed protective activity of DIII-lateral ridge specific neutralizing mAbs in a mouse model of ZIKV infection. Our results suggest that DIII is targeted by multiple type-specific antibodies with distinct neutralizing activity, which provides a path for developing prophylactic antibodies for use in pregnancy or designing epitope-specific vaccines against ZIKV.