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Zika virus (ZIKV) has evolved from an overlooked mosquito-borne flavivirus into a global health threat due to its astonishing causal link to microcephaly and other disorders. ZIKV has been shown to infect neuronal progenitor cells of the fetal mouse brain, which is comparable to the first-trimester human fetal brain, and result in microcephaly. However, whether there are different effects between the contemporary ZIKV strain and its ancestral strain in the neonatal mouse brain, which is comparable with the second-trimester human fetal brain, is unclear. Here we adopted a mouse model which enables us to study the postnatal effect of ZIKV infection. We show that even 100 pfu of ZIKV can replicate and infect neurons and oligodendrocytes in most parts of the brain. Compared with the ancestral strain from Cambodia (CAM/2010), infection of the ZIKV strain from Venezuela (VEN/2016) leads to much more severe microcephaly, accompanied by more neuronal cell death, abolishment of oligodendrocyte development, and a more dramatic immune response. The serious brain damage caused by VEN/2016 infection would be helpful to elucidate why the American strain resulted in severe neurovirulence in infants and will provide clinical guidance for the diagnosis and treatment of infection by different ZIKV strains.

Zika virus (ZIKV) has become a global public health emergency due to its rapidly expanding range and its ability to cause severe congenital defects such as microcephaly. However, there are no FDA-approved therapies or vaccines against ZIKV infection. Through our screening of viral entry inhibitors, we found that chloroquine (CQ), a commonly used antimalarial and a FDA-approved drug that has also been repurposed against other pathogens, could significantly inhibit ZIKV infection in vitro, by blocking virus internalization. We also demonstrated that CQ attenuates ZIKV-associated morbidity and mortality in mice. Finally, we proved that CQ protects fetal mice from microcephaly caused by ZIKV infection. Our methodology of focusing on previously identified antivirals in screens for effectiveness against ZIKV proved to be a rapid and efficient means of discovering new ZIKV therapeutics. Selecting drugs that were previously FDA-approved, such as CQ, also improves the likelihood that they may more quickly reach stages of clinical testing and use by the public.

Zika virus (ZIKV) has spread rapidly in recent years and there is a need for antiviral treatments. Here, the authors develop an antiviral peptide, based on the stem region of ZIKV envelope protein, and show that it is safe in pregnant mice and inhibits ZIKV infection in pregnant mice and fetuses.

Japanese encephalitis virus (JEV) is a Flavivirus responsible for thousands of deaths every year for which there are no specific anti-virals. Here, Wang et al. report the cryo-EM structure of mature JEV at near-atomic resolution and identify structural elements that modulate stability and virulence.

Zika virus (ZIKV) has become a serious threat to global health since the outbreak in Brazil in 2015. Additional Chinese cases have continuously been reported since the first case of laboratory-confirmed ZIKV infection in China on 6 February 2016. Aedes aegypti is the most important vector for ZIKV. This study shows that two strains from China exhibit high levels of midgut infection and highly disseminated infection of salivary glands and ovaries. Both strains can transmit ZIKV to infant mice bitten by infectious mosquitoes. Moreover, the results provide the evidence of transovarial transmission of ZIKV in mosquitoes. The study indicates that the two Ae. aegypti strains are not only effective transmission vectors but also persistent survival hosts for ZIKV during unfavorable inter-epidemic periods. This function as a reservoir of infection has epidemiological implications that further enhance the risk of potential future outbreaks.Emerging Microbes & Infections (2017) 6, e23; doi:10.1038/emi.2017.8; published online 26 April 2017

Mosquitoes act as vectors of numerous pathogens that cause human diseases. Dengue virus (DENV) transmitted by mosquito, Aedes aegypti, is responsible for dengue fever epidemics worldwide with a serious impact on human health. Currently, disease control mainly relies on vector targeted intervention strategies. Therefore, it is imperative to understand the molecular mechanisms underlying the innate immune response of mosquitoes against pathogens. In the present study, the expression profiles of immunity-related genes in the midgut responding to DENV infection by feeding were analyzed by transcriptome and quantitative real-time PCR. The level of Antimicrobial peptides (AMPs) increased seven days post-infection (d.p.i.), which could be induced by the Toll immune pathway. The expression of reactive oxygen species (ROS) genes, including antioxidant genes, such as HPX7, HPX8A, HPX8B, HPX8C were induced at one d.p.i. and peaked again at ten d.p.i. in the midgut. Interestingly, down-regulation of the antioxidant gene HPX8C by RNA interference led to reduction in the virus titer in the mosquito, probably due to the elevated levels of ROS. Application of a ROS inhibitor and scavenger molecules further established the role of oxygen free radicals in the modulation of the immune response to DENV infection. Overall, our comparative transcriptome analyses provide valuable information about the regulation of immunity related genes in the transmission vector in response to DENV infection. It further allows us to identify novel molecular mechanisms underlying the host-virus interaction, which might aid in the development of novel strategies to control mosquito-borne diseases.

Animal models are critical to understand disease and to develop countermeasures for the ongoing epidemics of Zika virus (ZIKV). Here we report a non-human primate model using a 2016 contemporary clinical isolate of ZIKV. Upon subcutaneous inoculation, rhesus macaques developed fever and viremia, with robust excretion of ZIKV RNA in urine, saliva, and lacrimal fluid. Necropsy of two infected animals revealed that systematic infections involving central nervous system and visceral organs were established at the acute phrase. ZIKV initially targeted the intestinal tracts, spleen, and parotid glands, and retained in spleen and lymph nodes till 10 days post infection. ZIKV-specific immune responses were readily induced in all inoculated animals. The non-human primate model described here provides a valuable platform to study ZIKV pathogenesis and to evaluate vaccine and therapeutics.

Zika virus (ZIKV) has become a threat to global health since the outbreak in Brazil in 2015. Although ZIKV is generally considered an Aedes-transmitted pathogen, new evidence has shown that parts of the virus closely resemble Culex-transmitted viruses. Therefore, it is important to evaluate the competence of Culex species for ZIKV to understand their potential as vectors. In this study, female Culex pipiens quinquefasciatus were orally exposed to ZIKV. Mosquito midguts, salivary glands and ovaries were tested for ZIKV to measure infection and dissemination at 2, 4, 6, 8, 12, 16 and 18 days post exposure (pe). In addition, saliva was collected from mosquitoes after infection and infant mice were bitten by infected mosquitoes to measure the transmission ability of Cx. p. quinquefasciatus. The results showed that the peak time of virus appearance in the salivary glands was day 8 pe, with 90% infection rate and an estimated virus titer of 3.92±0.49 lg RNA copies/mL. Eight of the nine infant mice had positive brains after being bitten by infected mosquitoes, which meant that Cx. p. quinquefasciatus could be infected with and transmit ZIKV following oral infection. These laboratory results clearly demonstrate the potential role of Cx. p. quinquefasciatus as a vector of ZIKV in China. Because there are quite different vector management strategies required to control Aedes (Stegomyia) species and Cx. p. quinquefasciatus, an integrated approach may be required should a Zika epidemic occur.

ABSTRACT Glioblastoma (GBM) is the deadliest type of brain tumor, and glioma stem cells (GSCs) contribute to tumor recurrence and therapeutic resistance. Thus, an oncolytic virus targeting GSCs may be useful for improving GBM treatment. Because Zika virus (ZIKV) has an oncolytic tropism for infecting GSCs, we investigated the safety and efficacy of a live attenuated ZIKV vaccine candidate (ZIKV-LAV) for the treatment of human GBM in a GSC-derived orthotopic model. Intracerebral injection of ZIKV-LAV into mice caused no neurological symptoms or behavioral abnormalities. The neurovirulence of ZIKV-LAV was more attenuated than that of the licensed Japanese encephalitis virus LAV 14-14-2, underlining the superior safety of ZIKV-LAV for potential GBM treatment. Importantly, ZIKV-LAV significantly reduced intracerebral tumor growth and prolonged animal survival by selectively killing GSCs within the tumor. Mechanistically, ZIKV infection elicited antiviral immunity, inflammation, and GSC apoptosis. Together, these results further support the clinical development of ZIKV-LAV for GBM therapy. IMPORTANCE Glioblastoma (GBM), the deadliest type of brain tumor, is currently incurable because of its high recurrence rate after traditional treatments, including surgery to remove the main part of the tumor and radiation and chemotherapy to target residual tumor cells. These treatments fail mainly due to the presence of a cell subpopulation called glioma stem cells (GSCs), which are resistant to radiation and chemotherapy and capable of self-renewal and tumorigenicity. Because Zika virus (ZIKV) has an oncolytic tropism for infecting GSCs, we tested a live attenuated ZIKV vaccine candidate (ZIKV-LAV) for the treatment of human GBM in a human GSC-derived orthotopic model. Our results showed that ZIKV-LAV retained good efficacy against glioblastoma by selectively killing GSCs within the tumor. In addition, ZIKV-LAV exhibited an excellent safety profile upon intracerebral injection into the treated animals. The good balance between the safety of ZIKV-LAV and its efficacy against human GSCs suggests that it is a potential candidate for combination with the current treatment regimen for GBM therapy.

Dengue virus (DENV) is one of the most widespread arboviruses. The four DENV serotypes infect about 400 million people every year, causing 96 million clinical dengue cases, of which approximately 500'000 are severe and potentially life-threatening. The only licensed vaccine has a limited efficacy and is only recommended in regions with high endemicity. We previously reported that 2'-O-methyltransferase mutations in DENV-1 and DENV-2 block their capacity to inhibit type I IFNs and render the viruses attenuated in vivo, making them amenable as vaccine strains; here we apply this strategy to all four DENV serotypes to generate a tetravalent, non-chimeric live-attenuated dengue vaccine. 2'-O-methyltransferase mutants of all four serotypes are highly sensitive to type I IFN inhibition in human cells. The tetravalent formulation is attenuated and immunogenic in mice and cynomolgus macaques and elicits a response that protects from virus challenge. These results show the potential of 2'-O-methyltransferase mutant viruses as a safe, tetravalent, non-chimeric dengue vaccine.

Viral replicase recruitment and long-range RNA interactions are essential for RNA virus replication, yet the mechanism of their interplay remains elusive. Flaviviruses include numerous important human pathogens, e.g., dengue virus (DENV) and Zika virus (ZIKV). Here, we revealed a highly conserved, conformation-tunable cis-acting element named 5′-UAR-flanking stem (UFS) in the flavivirus genomic 5′ terminus. We demonstrated that the UFS was critical for efficient NS5 recruitment and viral RNA synthesis in different flaviviruses. Interestingly, stabilization of the DENV UFS impaired both genome cyclization and vRNA replication. Moreover, the UFS unwound in response to genome cyclization, leading to the decreased affinity of NS5 for the viral 5′ end. Thus, we propose that the UFS is switched by genome cyclization to regulate dynamic RdRp binding for vRNA replication. This study demonstrates that the UFS enables communication between flavivirus genome cyclization and RdRp recruitment, highlighting the presence of switch-like mechanisms among RNA viruses.

Heamagglutination is inhibited when antibodies are present because antibodies to the influenza virus will prevent attachment of the virus to red blood cells. The highest dilution of antibody that prevents hemagglutination is called the HI titer. Human monoclonal antibodies generated from single human B cells were tested to characterize their ability to inhibit hemagglutination against virus A/California/07/2009 (H1N1) and A/Brisbane/10/2007 (H3N2).

The human monoclonal antibodies generated from single human B cells were tested to characterize their ability to neutralize virus infectivity. The microneutralization assay is a highly sensitive and specific assay for detecting virus-specific neutralizing antibodies to influenza viruses. This protocol is to measure the ability of human monoclonal antibody to neutralize influenza virus by microneutralization assay.

Dengue used to be recognized as an imported and sporadic disease in China. Since June 2014, an unexpected large dengue outbreak has attacked Guangzhou, China, resulting in more than 40,000 cases. Among the 1,942 laboratory-confirmed hospitalized dengue cases, 121 were diagnosed as severe dengue according to the 2009 WHO guideline, and 2 patients finally died. Laboratory diagnosis and virus isolation demonstrated that the majority (96%) cases were caused by dengue virus serotype 1 (DENV-1), and the others by serotype 2 (DENV-2). 14 DENV strains were isolated from the sera of acute-phase dengue patients during this outbreak, and the complete envelope (E) gene of 12 DENV-1 strains and two DENV-2 strains were determined using RT-PCR assay. Phylogenetic analysis based on the E gene revealed the DENV-1 strains isolated during the outbreak belonged to genotype I and V, respectively. These isolates formed three clades. DENV-2 isolates were assigned to the same clade belonging to genotype cosmopolitan. These strains isolated in 2014 were closely related to the isolates obtained from the same province, Guangdong, in 2013. No amino acid mutations known to increase virulence were identified throughout the E protein of isolates in 2014. These results indicate that dengue is turning into endemic in Guangdong, China, and extensive seroepidemiological investigation and mosquito control measures are critically needed in the future.

In recent decades, the impact of dengue has increased both geographically and in intensity, and this disease is now a threat to approximately half of the world's population. An unexpected large outbreak of dengue fever was reported in Xishuangbanna Dai Autonomous Prefecture, Yunnan Province, China, in 2013. This was the first autochthonous outbreak with a significant proportion of severe dengue cases in mainland China in a decade. According to the 2009 World Health Organization guidelines, half of the 136 laboratory confirmed cases during the epidemic were severe dengue. The clinical presentation included severe haemorrhage (such as massive vaginal and gastrointestinal bleeding), severe plasma leakage (such as pleural effusion, ascites, or hypoproteinaemia), and organ involvement (such as myocarditis and lung impairment); 21 cases eventually deteriorated to shock. During this outbreak, all severe cases occurred in adults, among whom about 43% had co-morbid conditions. Nucleic acid detection and virus isolation confirmed dengue virus serotype 3 (DENV-3) to be the pathogenic agent of this outbreak. Phylogenetic analyses of envelope gene sequences showed that these DENV-3 isolates belonged to genotype II. This finding is of great importance to understand the circulation of DENV and predict the risk of severe disease in mainland China. Here, we provide a brief report of the epidemiology, clinical manifestations, and aetiology of this dengue fever outbreak, and characterize DENV strains isolated from clinical specimens.

RNA helicases and chaperones are the two major classes of RNA remodeling proteins, which function to remodel RNA structures and/or RNA-protein interactions, and are required for all aspects of RNA metabolism. Although some virus-encoded RNA helicases/chaperones have been predicted or identified, their RNA remodeling activities in vitro and functions in the viral life cycle remain largely elusive. Enteroviruses are a large group of positive-stranded RNA viruses in the Picornaviridae family, which includes numerous important human pathogens. Herein, we report that the nonstructural protein 2CATPase of enterovirus 71 (EV71), which is the major causative pathogen of hand-foot-and-mouth disease and has been regarded as the most important neurotropic enterovirus after poliovirus eradication, functions not only as an RNA helicase that 3'-to-5' unwinds RNA helices in an adenosine triphosphate (ATP)-dependent manner, but also as an RNA chaperone that destabilizes helices bidirectionally and facilitates strand annealing and complex RNA structure formation independently of ATP. We also determined that the helicase activity is based on the EV71 2CATPase middle domain, whereas the C-terminus is indispensable for its RNA chaperoning activity. By promoting RNA template recycling, 2CATPase facilitated EV71 RNA synthesis in vitro; when 2CATPase helicase activity was impaired, EV71 RNA replication and virion production were mostly abolished in cells, indicating that 2CATPase-mediated RNA remodeling plays a critical role in the enteroviral life cycle. Furthermore, the RNA helicase and chaperoning activities of 2CATPase are also conserved in coxsackie A virus 16 (CAV16), another important enterovirus. Altogether, our findings are the first to demonstrate the RNA helicase and chaperoning activities associated with enterovirus 2CATPase, and our study provides both in vitro and cellular evidence for their potential roles during viral RNA replication. These findings increase our understanding of enteroviruses and the two types of RNA remodeling activities.

Enterovirus 71 (EV71) is the most virulent pathogen among enteroviruses that cause hand, foot and mouth disease in children but rarely in adults. The mechanisms that determine the age-dependent susceptibility remain largely unclear. Here, we found that the paucity of invariant natural killer T (iNKT) cells together with immaturity of the immune system was related to the susceptibility of neonatal mice to EV71 infection. iNKT cells were crucial antiviral effector cells to protect young mice from EV71 infection before their adaptive immune systems were fully mature. EV71 infection led to activation of iNKT cells depending on signaling through TLR3 but not other TLRs. Surprisingly, iNKT cell activation during EV71 infection required TLR3 signaling in macrophages, but not in dendritic cells (DCs). Mechanistically, interleukin (IL)-12 and endogenous CD1d-restricted antigens were both required for full activation of iNKT cells. Furthermore, CD1d-deficiency led to dramatically increased viral loads in central nervous system and more severe disease in EV71-infected mice. Altogether, our results suggest that iNKT cells may be involved in controlling EV71 infection in children when their adaptive immune systems are not fully developed, and also imply that iNKT cells might be an intervention target for treating EV71-infected patients.

The dengue virus (DENV) envelope protein domain III (ED3) has been suggested to contain receptor recognition sites and the critical neutralizing epitopes. Up to date, relatively little work has been done on fine mapping of neutralizing epitopes on ED3 for DENV4. In this study, a novel mouse type-specific neutralizing antibody 1G6 against DENV4 was obtained with both prophylactic and therapeutic effects. The epitope was mapped to residues 387-390 of DENV4 envelope protein. Furthermore, site-directed mutagenesis assay identified two critical residues (T388 and H390). The epitope is variable among different DENV serotypes but is highly conserved among four DENV4 genotypes. Affinity measurement showed that naturally occurring variations in ED3 outside the epitope region did not alter the binding of mAb 1G6. These findings expand our understanding of the interactions between neutralizing antibodies and the DENV4 and may be valuable for rational design of DENV vaccines and antiviral drugs.

RNA replication of positive-strand (+)RNA viruses requires the protein-protein interactions among viral replicases and the association of viral replicases with intracellular membranes. Protein A from Wuhan nodavirus (WhNV), which closely associate with mitochondrial membranes, is the sole replicase required for viral RNA replication. Here, we studied the direct effects of mitochondrial membrane lipids (MMLs) on WhNV protein A activity in vitro. Our investigations revealed the self-interaction of WhNV protein A is accomplished via two different patterns (i.e., homotypic and heterotypic self-interactions via different interfaces). MMLs stimulated the protein A self-interaction, and this stimulation exhibited selectivity for specific phospholipids. Moreover, we found that specific phospholipids differently favor the two self-interaction patterns. Furthermore, manipulating specific phospholipid metabolism affected protein A self-interaction and the activity of protein A to replicate RNA in cells. Taken together, our findings reveal the direct effects of membrane lipids on a nodaviral RNA replicase.

Human adenovirus type 55 (HAdV-B55) represents a re-emerging human pathogen, and this adenovirus has been reported to cause outbreaks of acute respiratory diseases among military trainees and in school populations around the world. HAdV-B55 has been revealed to have evolved from homologous recombination between human adenovirus type 14 (HAdV-B14) and type 11 (HAdV-B11), but it presents different clinical manifestations from parental virus HAdV-B11. In the present paper, we report the distinct biological features of HAdV-B55 in comparison with the parental viruses HAdV-B11 and HAdV-B14 in cell cultures. The results showed that HAdV-B55 replicated well in various cells, similar to HAdV-B11 and HAdV-B14, but that its processing had a slower and milder cytopathic effect in the early stages of infection. Viral fitness analysis showed that HAdV-B55 exhibited higher levels of replication in respiratory cells than did either of its parents. Cytotoxicity and apoptosis analyses in A549 cells indicated that HAdV-B55 was less cytotoxic than HAdV-B11 and HAdV-B14 were and induced milder apoptosis. Finally, thermal sensitivity analysis revealed that HAdV-B55 exhibited lower thermostability than did either HAdV-B11 or HAdV-B14, which may limit the transmission of HAdV-B55 in humans. Together, the findings described here expand current knowledge about this re-emerging recombinant HAdV, shedding light on the pathogenesis of HAdV-B55.

The early diagnosis of West Nile virus (WNV) infection is important for successful clinical management and epidemiological control. The non-structural protein 1 (NS1) of flavivirus, a highly conserved and secreted glycoprotein, is abundant in the serum of flavivirus-infected patients and represents a useful early diagnostic marker. We developed a WNV-specific NS1 antigen-capture ELISA using two mouse monoclonal antibodies (MAbs) that recognised distinct epitopes of the NS1 protein of WNV as capture and detection antibodies. The antigen-capture ELISA displayed exclusive specificity to WNV without cross-reaction with other related members of the flavivirus family, including the dengue virus, yellow fever virus, Japanese encephalitis virus, and tick-borne encephalitis virus. Additionally, the specificity was presented as no false positive in normal (0/1003) and DENV-infected (0/107) human serum specimens. The detection limit of the antigen-capture ELISA was as low as 15 pg/ml of recombinant WNV NS1 protein (rWNV-NS1) and 6.1 plaque-forming units (PFU)/0.1 ml of WNV-infected culture supernatant. In mice infected with WNV, the NS1 protein was readily detected in serum as early as one day after WNV infection, prior to the development of clinical signs of the disease. The sensitivity of the NS1 capture ELISA (93.7%) was significantly higher (79.4%) than that of real-time reverse transcription polymerase chain reaction in 63 serum samples from WNV-infected mice (p = 0.035). This newly developed NS1 antigen-capture ELISA with high sensitivity and specificity could be used as an efficient method for the early diagnosis of WNV infection in animals or humans.

Knowledge of virulence and immunogenicity is important for development of live-attenuated dengue vaccines. We previously reported that an infectious clone-derived dengue type 4 virus (DENV-4) passaged in MRC-5 cells acquired a Glu345Lys (E-E345K) substitution in the E protein domain III (E-DIII). The same cloned DENV-4 was found to yield a single E-Glu327Gly (E-E327G) mutation after passage in FRhL cells and cause the loss of immunogenicity in rhesus monkeys. Here, we used site-directed mutagenesis to generate the E-E345K and E-E327G mutants from DENV-4 and DENV-4Δ30 infectious clones and propagated in Vero or MRC-5 cells. The E-E345K mutations were consistently presented in viruses recovered from MRC-5 cells, but not Vero cells. Recombinant E-DIII proteins of E345K and E327G increased heparin binding correlated with the reduced infectivity by heparin treatment in cell cultures. Different from the E-E327G mutant viruses to lose the immunogencity in rhesus monkeys, the E-E345K mutant viruses were able to induce neutralizing antibodies in rhesus monkeys with an almost a 10-fold lower level of viremia as compared to the wild type virus. Monkeys immunized with the E-E345K mutant virus were completely protected with no detectable viremia after live virus challenges with the wild type DENV-4. These results suggest that the E-E345K mutant virus propagated in MRC-5 cells may have potential for the use in live-attenuated DENV vaccine development.

Human enterovirus 71 (HEV71) has emerged as the leading cause of viral encephalitis in children in most Asian countries. The roles of host miRNAs in the neurological pathogenesis of HEV71 infection remain unknown. In the present study, comprehensive miRNA expression profiling in HEV71-infected human neuroblastoma SH-SY5Y cells was performed using the Affymetrix Gene Chip microarray assay and was validated using real-time RT-PCR. Among the 69 differentially expressed miRNAs, miR-1246 was specifically induced by HEV71 infection in human neuroblastoma cells, but inhibition of miR-1246 failed to affect HEV71 replication. Parallel mRNA and microRNA profiling based on the 35 K Human Genome Array identified 182 differentially regulated genes. Target prediction of miR-1246 and network modeling revealed 14 potential target genes involved in cell death and cell signaling. Finally, a combined analysis of the results from mRNA profiling and miR-1246 target predication led to the identification of disc-large homolog 3 (DLG3), which is associated with neurological disorders, for further validation. Sequence alignment and luciferase reporter assay showed that miR-1246 directly bound with the 3'-UTR of DLG3 gene. Down-regulation of miR-1246 induced significant changes in DLG3 expression levels in HEV71-infected SHSY5Y cells. Together, these results suggested that miR-1246 might play a role in neurological pathogenesis of HEV71 by regulating DLG3 gene in infected cells. These findings provide new information on the miRNA and mRNA profiles of HEV71-infected neuroblastoma cells. The biological significance of miR-1246 and DLG3 during the course of HEV71 infection deserves further investigation.

The worldwide expansion of four serotypes of dengue virus (DENV) poses great risk to global public health. Several vaccine candidates are under development. However, none is yet available for humans. In the present study, a novel strategy to produce tetravalent DENV vaccine based on envelope protein domain III (EDIII) was proposed. Tandem EDIIIs of two serotypes (type 1-2 and type 3-4) of DENV connected by a Gly-Ser linker ((Gly4Ser)3) were expressed in E. coli, respectively. Then, the two bivalent recombinant EDIIIs were equally mixed to form the tetravalent vaccine candidate MixBiEDIII, and used to immunize BALB/c mice. The results showed that specific IgG and neutralizing antibodies against all four serotypes of DENV were successfully induced in the MixBiEDIII employing Freund adjuvant immunized mice. Furthermore, in the suckling mouse model, sera from mice immunized with MixBiEDIII provided significant protection against four serotypes of DENV challenge. Our data demonstrated that MixBiEDIII, as a novel form of subunit vaccine candidates, might have the potential to be further developed as a tetravalent dengue vaccine in the near future.

Human enterovirus type 71 (EV71) is the major pathogen of hand-foot-and-mouth disease (HFMD) and has been associated with severe neurological disease and even death in infants and young children. The pathogenesis of EV71 infection in the human central nervous system remains unclear. In this study, human whole genome microarray was employed to perform transcriptome profiling in SH-SY5Y human neuroblastoma cells infected with EV71. The results indicated that EV71 infection lead to altered expression of 161 human mRNAs, including 74 up-regulated genes and 87 down-regulated genes. Bioinformatics analysis indicated the possible roles of the differentially regulated mRNAs in selected pathways, including cell cycle/proliferation, apoptosis, and cytokine/chemokine responses. Finally, the microarray results were validated using real-time RT-PCR with high identity. Overall, our results provided fundamental information regarding the host response to EV71 infection in human neuroblastoma cells, and this finding will help explain the pathogenesis of EV71 infection and virus-host interaction.

Dengue virus is transmitted by Aedes mosquitoes and infects at least 100 million people every year. Progressive urbanization in Asia and South-Central America and the geographic expansion of Aedes mosquito habitats have accelerated the global spread of dengue, resulting in a continuously increasing number of cases. A cost-effective, safe vaccine conferring protection with ideally a single injection could stop dengue transmission. Current vaccine candidates require several booster injections or do not provide protection against all four serotypes. Here we demonstrate that dengue virus mutants lacking 2'-O-methyltransferase activity are highly sensitive to type I IFN inhibition. The mutant viruses are attenuated in mice and rhesus monkeys and elicit a strong adaptive immune response. Monkeys immunized with a single dose of 2'-O-methyltransferase mutant virus showed 100% sero-conversion even when a dose as low as 1,000 plaque forming units was administrated. Animals were fully protected against a homologous challenge. Furthermore, mosquitoes feeding on blood containing the mutant virus were not infected, whereas those feeding on blood containing wild-type virus were infected and thus able to transmit it. These results show the potential of 2'-O-methyltransferase mutant virus as a safe, rationally designed dengue vaccine that restrains itself due to the increased susceptibility to the host's innate immune response.

The emerging human enterovirus 71 (EV71) represents a growing threat to public health, and no vaccine or specific antiviral is currently available. Human intravenous immunoglobulin (IVIG) is clinical used in treating severe EV71 infections. However, the discovery of antibody dependent enhancement (ADE) of EV71 infection illustrates the complex roles of antibody in controlling EV71 infection. In this study, to identify the distinct role of each IgG subclass on neutralization and enhancement of EV71 infection, different lots of pharmaceutical IVIG preparations manufactured from Chinese donors were used for IgG subclass fractionation by pH gradient elution with the protein A-conjugated affinity column. The neutralization and ADE capacities on EV71 infection of each purified IgG subclass were then assayed, respectively. The neutralizing activity of human IVIG is mainly mediated by IgG1 subclass and to less extent by IgG2 subclass. Interestingly, IgG3 fraction did not have neutralizing activity but enhanced EV71 infection in vitro. These results revealed the different roles of human IgG subclasses on EV71 infection, which is of critical importance for the rational design of immunotherapy and vaccines against severe EV71 diseases.

Chikungunya virus (CHIKV), a member of the Alphavirus genus, is an important human emerging/re-emerging pathogen. Currently, there are no effective antiviral drugs or vaccines against CHIKV infection. Herein, we construct an infectious clone of CHIKV and an eGFP reporter CHIKV (eGFP-CHIKV) with an isolated strain (assigned to Asian lineage) from CHIKV-infected patients. The eGFP-CHIKV reporter virus allows for direct visualization of viral replication through the levels of eGFP expression. Using a known CHIKV inhibitor, ribavirin, we confirmed that the eGFP-CHIKV reporter virus could be used to identify inhibitors against CHIKV. Importantly, we developed a novel and reliable eGFP-CHIKV reporter virus-based neutralization assay that could be used for rapid screening neutralizing antibodies against CHIKV.

Flaviviruses are a group of human pathogenic, enveloped RNA viruses that includes dengue (DENV), yellow fever (YFV), West Nile (WNV), and Japanese encephalitis (JEV) viruses. Cross-reactive antibodies against Flavivirus have been described, but most of them are generally weakly neutralizing. In this study, a novel monoclonal antibody, designated mAb 2A10G6, was determined to have broad cross-reactivity with DENV 1-4, YFV, WNV, JEV, and TBEV. Phage-display biopanning and structure modeling mapped 2A10G6 to a new epitope within the highly conserved flavivirus fusion loop peptide, the (98)DRXW(101) motif. Moreover, in vitro and in vivo experiments demonstrated that 2A10G6 potently neutralizes DENV 1-4, YFV, and WNV and confers protection from lethal challenge with DENV 1-4 and WNV in murine model. Furthermore, functional studies revealed that 2A10G6 blocks infection at a step after viral attachment. These results define a novel broadly flavivirus cross-reactive mAb with highly neutralizing activity that can be further developed as a therapeutic agent against severe flavivirus infections in humans.

Human Enterovirus 71 (EV71) has emerged as one of the predominant causative agents of hand, foot and mouth disease (HFMD) with global impact. Despite the inactivated vaccine being licensed, other vaccine candidates based on advanced technology platforms are under development. In this report, we rationally designed and constructed two DNA-launched live attenuated vaccine candidates (pDL-EV71) under the control of specific promoters. In vitro and in vivo transfection with pDL-EV71 driven by the CMV promoter successfully yielded fully infectious EV71. More importantly, the administration of pDL-EV71 did not cause clinical symptoms following intracranial or intramuscular inoculation in neonatal and IFNα/βR-/- mice, demonstrating its safety profile. Moreover, a single-dose or two-dose immunization with pDL-EV71 elicited robust neutralizing antibodies against EV71 as well as an antigen-specific cellular response in mice. A single-dose immunization with 10 μg of pDL-EV71 conferred complete protection against lethal EV71 infection in neonates born to immunized maternal mice. Overall, our present results demonstrate that pDL-EV71 is a safe and effective vaccine candidate against EV71 for further development. [ABSTRACT FROM AUTHOR]

Guaico Culex virus (GCXV) is a newly identified segmented Jingmenvirus from Culex spp. mosquitoes in Central and South America. The genome of GCXV is composed of four or five single-stranded positive RNA segments. However, the infection kinetics and transmission capability of GCXV in mosquitoes remain unknown. In this study, we used reverse genetics to rescue two GCXVs (4S and 5S) that contained four and five RNA segments, respectively, in C6/36 cells. Further in vitro characterization revealed that the two GCXVs exhibited comparable replication kinetics, protein expression and viral titers. Importantly, GCXV RNAs were detected in the bodies, salivary glands, midguts and ovaries of Culex quinquefasciatus at 4-10 days after oral infection. In addition, two GCXVs can colonize Cx. quinquefasciatus eggs, resulting in positive rates of 15%-35% for the second gonotrophic cycle. In conclusion, our results demonstrated that GCXVs with four or five RNA segments can be detected in Cx. quinquefasciatus eggs during the first and second gonotrophic cycles after oral infection. [ABSTRACT FROM AUTHOR]