Dengue is a mosquito-borne flavivirus that is spreading at an unprecedented rate and has developed into a major health and economic burden in over 50 countries. Even though infected individuals develop potent and long-lasting serotype-specific neutralizing antibodies (Abs), the epitopes engaged by human neutralizing Abs have not been identified. Here, we demonstrate that the dengue virus (DENV)-specific serum Ab response in humans consists of a large fraction of cross-reactive, poorly neutralizing Abs and a small fraction of serotype-specific, potently inhibitory Abs. Although many mouse-generated, strongly neutralizing monoclonal antibodies (mAbs) recognize epitopes that are present on recombinant DENV envelope (E) proteins, unexpectedly, the majority of neutralizing Abs in human immune sera bound to intact virions but not to the ectodomain of purified soluble E proteins. These conclusions with polyclonal Abs were confirmed with newly generated human mAbs derived from DENV-immune individuals. Two of three strongly neutralizing human mAbs bound to E protein epitopes that were preserved on the virion but not on recombinant E (rE) protein. We propose that humans produce Abs that neutralize DENV infection by binding a complex, quaternary structure epitope that is expressed only when E proteins are assembled on a virus particle. Mapping studies indicate that this epitope has a footprint that spans adjacent E protein dimers and includes residues at the hinge between domains I and II of E protein. These results have significant implications for the DENV Ab and vaccine field. D engue viruses (DENVs) are emerging arboviruses and the causative agents of dengue fever and dengue hemorrhagic fever (DHF). The DENV complex consists of four distinct but related viruses, designated as serotypes (1, 2). A person infected with DENV develops an antibody (Ab) response that, to varying degrees, cross-reacts with all four serotypes. Despite the crossreactivity, Abs that are produced durably only prevent reinfection by the same homologous serotype. Serotype-specific neutralizing Abs can be detected 60 y after a primary infection, suggesting that Abs provide lifelong protection against the homologous serotype (3). People experiencing a secondary DENV infection with a different (heterologous) serotype face a greater risk for developing DHF. Ab-dependent enhancement by cross-reactive, weakly neutralizing Abs is the most widely suggested theory explaining the higher risk for DHF associated with secondary infection (4). The identity of DENV epitopes recognized by human Abs responsible for potent and long-term neutralization remains unknown. This is a significant knowledge gap impeding the current global effort to develop dengue vaccines that induce protective neutralizing Abs and not cross-reactive Abs with potential to enhance disease.The DENV envelope contains two integral membrane proteins designated envelope (E) and premembrane/membrane (prM/M) proteins. DENV E protein, which binds to cellular receptors and mediates viral fusion during...

Zika virus (ZIKV) is a re-emerging virus that has recently spread into dengue virus (DENV) endemic regions and cross-reactive antibodies (Abs) could potentially affect ZIKV pathogenesis. Using DENV-immune serum, it has been shown in vitro that antibody-dependent enhancement (ADE) of ZIKV infection can occur. Here we study the effects of pre-existing DENV immunity on ZIKV infection in vivo. We infect two cohorts of rhesus macaques with ZIKV; one cohort has been exposed to DENV 2.8 years earlier and a second control cohort is naïve to flaviviral infection. Our results, while confirming ADE in vitro, suggest that pre-existing DENV immunity does not result in more severe ZIKV disease. Rather our results show a reduction in the number of days of ZIKV viremia compared to naïve macaques and that the previous exposure to DENV may result in modulation of the immune response without resulting in enhancement of ZIKV pathogenesis.

Norwalk virus (NV) is a causative agent of acute epidemic nonbacterial gastroenteritis in humans. The inability to cultivate NV has required the use of molecular techniques to examine the genome organization and functions of the viral proteins. The function of the NV protein encoded by open reading frame 3 (ORF 3) has been unknown. In this paper, we report the characterization of the NV ORF 3 protein expressed in a cell-free translation system and in insect cells and show its association with recombinant virus-like particles (VLPs) and NV virions. Expression of the ORF 3 coding region in rabbit reticulocyte lysates resulted in the production of a single protein with an apparent molecular weight of 23,000 (23K protein), which is not modified by N-linked glycosylation. The ORF 3 protein was expressed in insect cells by using two different baculovirus recombinants; one recombinant contained the entire 3 end of the genome beginning with the ORF 2 coding sequences (ORFs 2؉3), and the second recombinant contained ORF 3 alone. Expression from the construct containing both ORF 2 and ORF 3 resulted in the expression of a single protein (23K protein) detected by Western blot analysis with ORF 3-specific peptide antisera. However, expression from a construct containing only the ORF 3 coding sequences resulted in the production of multiple forms of the ORF 3 protein ranging in size from 23,000 to 35,000. Indirect-immunofluorescence studies using an ORF 3 peptide antiserum showed that the ORF 3 protein is localized to the cytoplasm of infected insect cells. The 23K ORF 3 protein was consistently associated with recombinant VLPs purified from the media of insect cells infected with a baculovirus recombinant containing the entire 3 end of the NV genome. Western blot analysis of NV purified from the stools of NV-infected volunteers revealed the presence of a 35K protein as well as multiple highermolecular-weight bands specifically recognized by an ORF 3 peptide antiserum. These results indicate that the ORF 3 protein is a minor structural protein of the virion.

Norwalk virus (NV) is the prototype strain of a group of human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis. While these viruses do not grow in tissue culture cells or animal models, expression of the capsid protein in insect cells results in the self-assembly of recombinant NV virus-like particles (rNV VLPs) that are morphologically and antigenically similar to native NV. The X-ray structure of the rNV VLPs has revealed that the capsid protein folds into two principal domains: a shell (S) domain and a protruding (P) domain (B. V. V. Prasad, M. E. Hardy, T. Dokland, J. Bella, M. G. Rossmann, and M. K. Estes, Science 286:287-290, 1999). To investigate the structural requirements for the assembly of rNV VLPs, we performed mutational analyses of the capsid protein. We examined the ability of 10 deletion mutants of the capsid protein to assemble into VLPs in insect cell cultures. Deletion of the N-terminal 20 residues, suggested by the X-ray structure to be involved in a switching mechanism during assembly, did not affect the ability of the mutant capsid protein to self-assemble into 38-nm VLPs with a T‫3؍‬ icosahedral symmetry. Further deletions in the N-terminal region affected particle assembly. Deletions in the C-terminal regions of the P domain, involved in the interactions between the P and S domains, did not block the assembly process, but they affected the size and stability of the particles. Mutants carrying three internal deletion mutations in the P domain, involved in maintaining dimeric interactions, produced significantly larger 45-nm particles, albeit in low yields. The complete removal of the protruding domain resulted in the formation of smooth particles with a diameter that is slightly smaller than the 30-nm diameter expected from the rNV structure. These studies indicate that the shell domain of the NV capsid protein contains everything required to initiate the assembly of the capsid, whereas the entire protruding domain contributes to the increased stability of the capsid by adding intermolecular contacts between the dimeric subunits and may control the size of the capsid.Norwalk virus (NV) is the prototype strain of a group of noncultivable human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis (11,14). NV is a small, icosahedral, single-stranded, positive-stranded RNA virus whose capsid is formed by multiple copies of a single major structural protein (7,38). The NV genome is composed of three open reading frames (ORFs). The NV capsid protein is encoded by ORF2. Expression of the capsid protein in insect cells results in the self-assembly of recombinant NV empty virus-like particles (rNV VLPs) that are morphologically and antigenically similar to native NV (12, 27, 39).The three-dimensional structure of rNV VLPs was first determined by electron cryomicroscopy and computer imageprocessing techniques to a resolution of 22 Å (40). That study revealed that the rNV capsid has a diameter of 38.0 nm and exhibits Tϭ3 icosahedral symmetry, with a defi...

Venezuelan equine encephalitis virus (VEE) is an important equine and human pathogen of the Americas.In the adult mouse model, cDNA-derived, virulent V3000 inoculated subcutaneously (s.c.) causes high-titer peripheral replication followed by neuroinvasion and lethal encephalitis. A single change (G to A) at nucleotide 3 (nt 3) of the 5 untranslated region (UTR) of the V3000 genome resulted in a virus (V3043) that was avirulent in mice. The mechanism of attenuation by the V3043 mutation was studied in vivo and in vitro. Kinetic studies of virus spread in adult mice following s.c. inoculation showed that V3043 replication was reduced in peripheral organs compared to that of V3000, titers in serum also were lower, and V3043 was cleared more rapidly from the periphery than V3000. Because clearance of V3043 from serum began 1 to 2 days prior to clearance of V3000, we examined the involvement of alpha/beta interferon (IFN-␣/␤) activity in VEE pathogenesis. In IFN-␣/␤R ؊/؊ mice, the course of the wild-type disease was extremely rapid, with all animals dying within 48 h (average survival time of 30 h compared to 7.7 days in the wild-type mice). The mutant V3043 was as virulent as the wild type (100% mortality, average survival time of 30 h). Virus titers in serum, peripheral organs, and the brain were similar in V3000-and V3043-infected IFN-␣/␤R ؊/؊ mice at all time points up until the death of the animals. Consistent with the in vivo data, the mutant virus exhibited reduced growth in vitro in several cell types except in cells that lacked a functional IFN-␣/␤ pathway. In cells derived from IFN-␣/␤R ؊/؊ mice, the mutant virus showed no growth disadvantage compared to the wild-type virus, suggesting that IFN-␣/␤ plays a major role in the attenuation of V3043 compared to V3000. There were no differences in the induction of IFN-␣/␤ between V3000 and V3043, but the mutant virus was more sensitive than V3000 to the antiviral actions of IFN-␣/␤ in two separate in vitro assays, suggesting that the increased sensitivity to IFN-␣/␤ plays a major role in the in vivo attenuation of V3043.Venezuelan equine encephalitis virus (VEE) is a member of the Alphavirus genus in the Togaviridae family. The genome of this enveloped virus is a single-stranded, messenger-sense RNA molecule of approximately 11.5 kb (24), capped at the 5Ј end and polyadenylated at the 3Ј end. The genomic RNA encodes four nonstructural proteins (nsP1 through -4) and three structural proteins (capsid and two envelope glycoproteins, E1 and E2). The 5Ј untranslated region (UTR) in VEE is 45 nucleotides (nt) long, and although its sequence is not conserved among alphaviruses, the sequence predicts a stemloop structure that is conserved across the Alphavirus genus (55). It has been proposed that the complementary sequence at the 3Ј end of the minus strand also folds into a conserved secondary structure that may play a role as a promoter for the initiation of genome RNA synthesis from the minus-strand template (10,38,55). The nonstructural proteins are translated direct...