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Hepatitis A virus (HAV) live-attenuated vaccine H2 strain has been approved for clinical use for decades with ideal safety profiles in nonhuman primate models and humans. Recently, type I interferon (IFN) receptor-deficient mice were shown to be susceptible to HAV infection. Herein, we sought to determine the infection and replication dynamics of the H2 in Ifnar

Continued uncontrolled transmission of SARS-CoV-2 in many parts of the world is creating conditions for substantial evolutionary changes to the virus

The sudden emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) has caused global panic in 2003, and the risk of SARS-CoV outbreak still exists. However, no specific antiviral drug or vaccine is available; thus, the development of therapeutic antibodies against SARS-CoV is needed. In this study, a nanobody phage-displayed library was constructed from peripheral blood mononuclear cells of alpacas immunized with the recombinant receptor-binding domain (RBD) of SARS-CoV. Four positive clones were selected after four rounds of bio-panning and subjected to recombinant expression in E. coli. Further biological identification demonstrated that one of the nanobodies, S14, showed high affinity to SARS-CoV RBD and potent neutralization activity at the picomole level against SARS-CoV pseudovirus. A competitive inhibition assay showed that S14 blocked the binding of SARS-CoV RBD to either soluble or cell-expressed angiotensin-converting enzyme 2 (ACE2). In summary, we developed a novel nanobody targeting SARS-CoV RBD, which might be useful for the development of therapeutics against SARS.

The association of Zika virus (ZIKV) infection with microcephaly has raised alarm worldwide. Their causal link has been confirmed in different animal models infected by ZIKV. However, the molecular mechanisms underlying ZIKV pathogenesis are far from clear. Hence, we performed global gene expression analysis of ZIKV-infected mouse brains to unveil the biological and molecular networks underpinning microcephaly. We found significant dysregulation of the sub-networks associated with brain development, immune response, cell death, microglial cell activation, and autophagy amongst others. We provided detailed analysis of the related complicated gene networks and the links between them. Additionally, we analyzed the signaling pathways that were likely to be involved. This report provides systemic insights into not only the pathogenesis, but also a path to the development of prophylactic and therapeutic strategies against ZIKV infection.

West Nile virus (WNV) is an important neurotropic flavivirus that is widely distributed globally. WNV strain XJ11129 was first isolated in Xinjiang, China, and its genetic and biological characteristics remain largely unknown. In this study, phylogenetic and sequence analyses revealed that XJ11129 belongs to lineage 1a and shares high genetic identity with the highly pathogenic strain NY99. Then, the full-length genomic cDNA of XJ11129 was amplified and assembled using a modified Gibson assembly (GA) method. The virus (named rXJ11129) was successfully rescued in days following this method. Compared with other wild-type WNV isolates, rXJ11129 exhibited virulence indistinguishable from that of the NY99 strain in vivo. In summary, the genomic and virulence phenotypes of rXJ11129 were characterized in vivo and in vitro, and these data will improve the understanding of the spread and pathogenesis of this reemerging virus. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12250-020-00246-x) contains supplementary material, which is available to authorized users.

Zika virus (ZIKV) and Japanese encephalitis virus (JEV) are closely related flaviviruses, ZIKV circulates in the population that has been JEV vaccinated in Southeast Asian countries. This alerts that a pre-existing immunity to JEV would impact ZIKV infection and/or pathogenesis. Herein we showed that the pre-existing immunity to JEV SA14-14-2 vaccination provided an ample protection against non-lethal or lethal dose of ZIKV infection in mice. This was in sharp contrast to the passive immunization of JEV antibodies, which failed to affect ZIKV infection or pathogenesis in mice, albeit these antibodies exhibited cross-reactivity and antibody dependent enhancement (ADE) of ZIKV infection in vitro. Furthermore, we determined that JEV vaccine-elicited CD8+ T cells were required to mediate the heterotypic protection of ZIKV infection, which cross-reacted to ZIKV E and NS5 antigens (E294-302 and NS52839-2848). Adoptive transfer of these CD8+ T cells could partially protect the mice from ZIKV challenge. Therefore, although short of epidemiological evidence, these results suggested that cross-reactive CD8+ T cells activated by JEV vaccination could protect potential ZIKV infection in human populations.

Background The clinical management of coronavirus disease 2019 (COVID-19) is dependent on understanding the underlying factors that contribute to the disease severity. In the absence of effective antiviral therapies, other host immunomodulatory therapies such as targeting inflammatory response are currently being used without clear evidence of their effectiveness. Because inflammation is an essential component of host antiviral mechanisms, therapies targeting inflammation may adversely affect viral clearance and disease outcome. Objective To understand whether the persistent presence of the virus is a key determinant in the disease severity during COVID-19 and to determine whether the viral reactivation in some patients is associated with infectious viral particles. Methods The data for patients were available including the onset of the disease, duration of viral persistence, measurements of inflammatory markers such as IL-6 and C-reactive protein, chest imaging, disease symptoms, and their durations among others. Follow-up tests were performed to determine whether the viral negative status persists after their recovery. Results Our data show that patients with persistent viral presence (>16 days) have more severe disease outcomes including extensive lung involvement and requirement of respiratory support. Two patients who died of COVID-19 were virus-positive at the time of their death. Four patients demonstrated virus-positive status on the follow-up tests, and these patient samples were sent to viral culture facility where virus culture could not be established. Conclusions These data suggest that viral persistence is the key determining factor of the disease severity. Therapies that may impair the viral clearance may impair the host recovery from COVID-19.

The COVID-19 pandemic urgently needs therapeutic and prophylactic interventions. Here, we report the rapid identification of SARS-CoV-2-neutralizing antibodies by high-throughput single-cell RNA and VDJ sequencing of antigen-enriched B cells from 60 convalescent patients. From 8,558 antigen-binding IgG1+ clonotypes, 14 potent neutralizing antibodies were identified, with the most potent one, BD-368-2, exhibiting an IC50 of 1.2 and 15 ng/mL against pseudotyped and authentic SARS-CoV-2, respectively. BD-368-2 also displayed strong therapeutic and prophylactic efficacy in SARS-CoV-2-infected hACE2-transgenic mice. Additionally, the 3.8 A cryo-EM structure of a neutralizing antibody in complex with the spike-ectodomain trimer revealed the antibody's epitope overlaps with the ACE2 binding site. Moreover, we demonstrated that SARS-CoV-2-neutralizing antibodies could be directly selected based on similarities of their predicted CDR3H structures to those of SARS-CoV-neutralizing antibodies. Altogether, we showed that human neutralizing antibodies could be efficiently discovered by high-throughput single B cell sequencing in response to pandemic infectious diseases.

Summary The World Health Organization has declared SARS-CoV-2 virus outbreak a world-wide pandemic. However, there is very limited understanding on the immune responses, especially adaptive immune responses to SARS-CoV-2 infection. Here, we collected blood from COVID-19 patients who have recently become virus-free and therefore were discharged, and detected SARS-CoV-2-specific humoral and cellular immunity in 8 newly discharged patients. Follow-up analysis on another cohort of 6 patients 2 weeks post discharge also revealed high titers of IgG antibodies. In all 14 patients tested, 13 displayed serum neutralizing activities in a pseudotype entry assay. Notably, there was a strong correlation between neutralization antibody titers and the numbers of virus-specific T cells. Our work provides a basis for further analysis of protective immunity to SARS-CoV-2, and understanding the pathogenesis of COVID-19, especially in the severe cases. It has also implications in developing an effective vaccine to SARS-CoV-2 infection., Highlights 1. SARS-CoV-2-specific antibodies are detected in COVID-19 convalescent subjects. 2. Most COVID-19 convalescent individuals have detectable neutralizing antibodies. 3. Cellular immune responses to SARS-CoV-2 are found in COVID-19 convalescent subjects 4. Neutralization antibody titers correlate with the numbers of virus-specific T cells., In blood samples from COVID-19 convalescent subjects, Ni et al. have detected SARS-CoV-2-specific humoral and cellular immunity. Most subjects display serum neutralizing activities, which correlate with the numbers of virus-specific T cells.

Background: Chikungunya virus (CHIKV), a mosquito-borne alphavirus, has become a global public health concern. The lack of effective vaccines and antiviral agents are largely attributed to the elusive infection and dissemination dynamics in vivo. Methods: In this study, we designed and developed a novel, replication-competent, CHIKV reporter virus (CHIKV-iRFP) encoding a near infrared fluorescent protein (iRFP) for in vivo imaging. Findings: In vitro and in vivo characterization demonstrated that CHIKV-iRFP retained similar replication and virulence phenotype to the parental CHIKV. Neonatal BABL/c mice and IFNAR-/- A129 mice displayed highly susceptive to CHIKV-iRFP infection. Following intracranial (i.c.) inoculation, CHIKV-iRFP efficiently replicated and disseminated into whole body, resulting in rapid death in a age-dependent manner. Remarkably, upon i.c. inoculation, CHIKV-iRFP readily disseminated from footpad to the head and whole skeleton, with a specific tropism for bone marrow. Interpretation: Taken together, this novel reporter virus provides a powerful tool to visualize CHIKV replication and to test the efficacy of vaccines and antiviral therapeutics in a noninvasive and real-time manner. Funding: The National Key Research and Development Program of China (2018YFA0507201), the National Natural Science Foundation of China (No. 81572003). Declaration of Interest: The authors declare no conflict of interest. Ethical Approval: The experimental protocols were approved by the Animal Experiment Committee of Beijing Institute of Microbiology and Epidemiology, Beijing, China.

Zika virus (ZIKV) has evolved into a global health threat because of its causal link to congenital Zika syndrome. Different case reports indicate that ZIKV infection of pregnant women may cause a spectrum of abnormalities in children. Here we evaluated a large cohort of children with perinatal exposure to ZIKV and identified a spectrum of neurodevelopmental abnormalities in the group. In addition, we show that infection of the mouse neonatal brain by a contemporary ZIKV strain instead of an Asian ancestral strain can cause microcephaly and various abnormal neurological functions. These include defects in social interaction and depression, impaired learning and memory, in addition to severe motor defects, which are present in adult mice as well as in our cohort of children. Importantly, although mouse brains infected later after birth do not have apparent abnormal brain structure, those mice still show significant impairments of visual cortical functions, circuit organization and experience-dependent plasticity. Thus, our study suggests that special attention should be paid to all children born to ZIKV infected mothers for screening of abnormal behaviors and sensory function during childhood. Funding Statement: This work was supported by the Strategic Priority Research Program and Innovation Program of the Chinese Academy of Sciences (No. XDB32020100, QYZDJ-SSW-SMC007), the National Natural Science Foundation of China (NSFC) (31730108, 31430037, U170220023, 81772176, 31670883, 91542201, 81590765, and 31500145), Beijing Municipal Science & Technology Commission (Z181100001518001), the Interdisciplinary Research Funds of Beijing Normal University, the CAMS Initiative Innovative Medicine (No. 2016-I2M-1-005), Major Project for "Significant New Drugs Innovation and Development" (2015ZX09102023), NIH R01 AI140718, AI069120, AI056154 and AI078389 502 grants, MOST (China, 2016YFD0500304). C.L. was supported by the PUMC Youth Fund (No. 3332016125). Declaration of Interests: The authors confirm that there are no known conflicts of interest associated with this publication. Ethics Approval Statement: The experimental procedures on mice were performed under biosafety level 2 (BSL-2) at Beijing Institute of Microbiology and Epidemiology with Institutional Biosafety Committee approval. The mouse care and experimentation were approved by the Animal Care and Use Committees of the Institutional Animal Care and Use Committee at Institute of Genetics and Developmental Biology (IGDB), Chinese Academy of Sciences, Beijing Institute of Microbiology and Epidemiology and the State Key Laboratory of Cognitive Neuroscience & Learning at Beijing Normal University. For the infant cohort, the study protocol was approved by the institutional review boards at Fundacao Oswaldo Cruz (Fiocruz) and the University of California, Los Angeles. All mothers provided written informed consent for themselves and for their children.

Dengue is an acute, emerging, infectious disease transmitted by Aedes mosquitoes that has become a serious global public health problem. The DEN2-FJ10 and DEN2-FJ11 strains of the dengue 2 virus were originally isolated from the serum of a patient with dengue fever in Fujian Province, China, in 1999. Our data provide the first assessment of the vector competence of Aedes mosquitoes with respect to the DEN2-FJ10 and DEN2-FJ11 strains of the dengue virus. There were significant differences in the replication rates of these two viral strains in Aedes albopictus and Aedes aegypti (P0.05); replication of the DEN2-FJ10 strain was greater in Ae. aegypti than in Ae. albopictus 5 days post infection whereas replication of the DEN2-FJ11 was greater in Ae. albopictus than in Ae. aegypti 7 days post infection. The replicative ability of the DEN2-FJ11 strain was greater than that of the DEN2-FJ10 strain in infected Ae. albopictus. In infected Ae. aegypti, rapid proliferation of the DEN2-FJ10 strain occurred earlier than in the DEN2-FJ11 strain. There were no significant differences in the midgut and salivary gland infection rates of Ae. albopictus and Ae. aegypti with respect to either viral strain. Although the DEN2-FJ10 and DEN2-FJ11 strains differ in their virulence to neonatal rats, there was no significant difference in the ability of either Ae. albopictus or Ae. aegypti to transmit the DEN2-FJ10 and DEN2-FJ10 strains of the dengue 2 virus (P0.05). In summary, our results indicate that Ae. albopictus and Ae. aegypti mosquitoes are moderately competent vectors of the DEN2-FJ10 and DEN2-FJ11 strains of the dengue virus and provide the first evidence of the effect of these two viral strains on the vector competence of mosquitoes in China.

The rapid spread and potential link with birth defects have made Zika virus (ZIKV) a global public health problem. The virus was discovered 70years ago, yet the knowledge about its genomic structure and the genetic variations associated with current ZIKV explosive epidemics remains not fully understood. In this review, the genome organization, especially conserved terminal structures of ZIKV genome were characterized and compared with other mosquito-borne flaviviruses. It is suggested that major viral proteins of ZIKV share high structural and functional similarity with other known flaviviruses as shown by sequence comparison and prediction of functional motifs in viral proteins. Phylogenetic analysis demonstrated that all ZIKV strains circulating in the America form a unique clade within the Asian lineage. Furthermore, we identified a series of conserved amino acid residues that differentiate the Asian strains including the current circulating American strains from the ancient African strains. Overall, our findings provide an overview of ZIKV genome characterization and evolutionary dynamics in the Americas and point out critical clues for future virological and epidemiological studies.

Zika virus (ZIKV), a mosquito-borne flavivirus, is a current global public health concern. The flavivirus envelope (E) glycoprotein is responsible for virus entry and represents a major target of neutralizing antibodies for other flaviviruses. Here, we report the structures of ZIKV E protein at 2.0 Å and in complex with a flavivirus broadly neutralizing murine antibody 2A10G6 at 3.0 Å. ZIKV-E resembles all the known flavivirus E structures but contains a unique, positively charged patch adjacent to the fusion loop region of the juxtaposed monomer, which may influence host attachment. The ZIKV-E-2A10G6 complex structure reveals antibody recognition of a highly conserved fusion loop. 2A10G6 binds to ZIKV-E with high affinity in vitro and neutralizes currently circulating ZIKV strains in vitro and in mice. The E protein fusion loop epitope represents a potential candidate for therapeutic antibodies against ZIKV.

West Nile virus (WNV) is a neurotropic human pathogen that has caused increasing infected cases over recent years. There is currently no licensed vaccine or effective drug for prevention and treatment of WNV infection in humans. To facilitate antiviral drug discovery and neutralizing antibody detection, a WNV cDNA clone containing a luciferase reporter gene was constructed through incorporating Gaussia luciferase (Gluc) gene within the capsid-coding region of WNV genome. Transfection of BHK-21 cells with the cDNA clone-derived RNA generated luciferase reporter WNV (WNV-Gluc) and the stable WNV-Gluc with high titers (>10(7)PFU/ml) was obtained through plaque purification. Luciferase activity was used to effectively quantify the viral production of WNV-Gluc. Using the reporter virus WNV-Gluc, we developed a luciferase based assay in a 12-well format for evaluating neutralizing antibodies. The reporter virus could be a powerful tool for epidemiological investigation of WNV, vaccine evaluation, antiviral drug screening, and the study of WNV replication and pathogenesis.

Hand-foot-and-mouth disease (HFMD) remains a major health concern in the Asia-Pacific regions, and its major causative agents include human enterovirus 71 (EV71) and coxsackievirus A16. A desirable vaccine against HFMD would be multivalent and able to elicit protective responses against multiple HFMD causative agents. Previously, we have demonstrated that a thermostable recombinant EV71 vaccine candidate can be produced by the insertion of a foreign peptide into the BC loop of VP1 without affecting viral replication. Here we present crystal structures of two different naturally occurring empty particles, one from a clinical C4 strain EV71 and the other from its recombinant virus containing an insertion in the VP1 BC loop. Crystal structure analysis demonstrated that the inserted foreign peptide is well exposed on the particle surface without significant structural changes in the capsid. Importantly, such insertions do not seem to affect the virus uncoating process as illustrated by the conformational similarity between an uncoating intermediate of another recombinant virus and that of EV71. Especially, at least 18 residues from the N terminus of VP1 are transiently externalized. Altogether, our study provides insights into vaccine development against HFMD.

Nonstructural protein-1 (NS1) of the Japanese encephalitis virus (JEV) is an immunogenic protein that is a potential candidate for the development of vaccines and diagnostic reagents. NS1 is known to be more specific than the E protein in serological testing of flavivirus infections. However, NS1 exhibits cross-reactivity among flaviviruses even within the same genus and more so within a serocomplex. However, the cross-reactive epitopes on JEV NS1 are poorly characterized. The present study describes the full mapping of a linear B-cell epitope that is common and specific to the JEV serocomplex of Flaviviridae. We generated an NS1-specific monoclonal antibody that cross-reacts with the West Nile virus (WNV) NS1 protein by immunizing mice with recombinant JEV NS1. For epitope mapping, 51 partially overlapping peptides spanning the entire NS1 protein were expressed with a glutathione S-transferase (GST) tag and screened using monoclonal antibodies. Two linear epitope-containing peptides were identified using enzyme-linked immunosorbent assay (ELISA). By sequentially removing amino acid residues from the carboxy and amino terminal of peptides, we successfully identified the smallest unit of the linear epitope required to react with the monoclonal antibody. The linear epitope was located in amino acids residues ²²⁷ETHTLW²³². Furthermore, results of the sequence alignment revealed that the epitope was highly conserved among JEV strains. Notably, the epitope is highly conserved among viruses of the JEV serocomplex. Furthermore, the homologous regions on NS1 proteins from dengue viruses showed no cross-reactivity with the monoclonal antibodies. The epitope was recognized by antisera against the WNV but not against the dengue virus. This novel JEV serocomplex-specific linear B-cell epitope of NS1 would be helpful in the development of new vaccines and diagnostic assays.

The live attenuated Japanese encephalitis (JE) vaccine SA14-14-2 was licensed decades ago and now approved for clinical use in most JE endemic countries. Large-scale clinical trials demonstrate ideal safety and efficacy profile of this Chinese vaccine. The SA14-14-2 vaccine was derived from a virulent strain SA14 after hundreds of serial passaging in cells and animals, concern about virulence reversion remains exist. In the present study, to study the in vitro and in vivo genetic and attenuation stability of the vaccine virus, SA14-14-2 was serially passaged in Vero cells and mouse brain followed by sequence comparison and attenuation phenotype analysis. The results showed that no significant mutation was acquired after serial passaging in Vero cells except a single Ser66Leu mutation within capsid protein, which had no effect on viral virulence in mice. Importantly, serial passaging of SA14-14-2 in suckling mouse brain resulted in emergence of adaptive mutations and increased virulence in mice. Population and plaque-purified clone consensus sequence analysis showed four adaptive mutations in envelope (E) protein, F107L, K138E, T226R and I270T, sequentially occurred and become predominant during serial passaging in suckling mouse brain. Especially, these adaptive mutations were close related with the enhanced neurovirulence and neuroinvasiveness in mice. Our results provide experimental evidence of highly genetic and attenuation stability of SA14-14-2 following passaging in Vero cells, and reveal the potential virulence reversion during passaging in mouse brain in association with critical adaptive mutations in E protein. These findings are important for quality control and evaluation of live JE vaccines and will help understand the attenuation mechanism of flavivirus vaccine.

Tick-borne encephalitis virus (TBEV) represents one of the most dangerous human pathogens circulating in Europe and East Asia. No effective treatment for TBEV infection currently exists, and vaccination is the primary preventive measure. Although several inactivated vaccines have been licensed, the development of novel vaccines against TBEV remains a high priority in disease-endemic countries. In the present study, a live chimeric recombinant TBEV (ChinTBEV) was created by substituting the major structural genes of TBEV for the corresponding regions of Japanese encephalitis virus (JEV) live vaccine strain SA14-14-2. The resulting chimera had a small-plaque phenotype, replicated efficiently in both mammalian and mosquito cells. The preliminary data from in vitro passaging indicated the potential for stability of ChinTBEV. ChinTBEV also exhibited significantly attenuated neuroinvasiveness in mice upon either intraperitoneal or subcutaneous inoculation in comparison with its parental TBEV. Importantly, a single immunisation with ChinTBEV elicited TBEV-specific IgG and neutralising antibody responses in a dose-dependent manner, providing significant protection against lethal TBEV challenge in mice. Taken together, the results of this proof-of-concept study indicate that ChinTBEV can be further developed as a potential vaccine candidate against TBEV infection. Moreover, the construction of this type of flavivirus chimera using a JEV vaccine strain as the genetic backbone represents a universal vaccine approach.

Human coxsackievirus A16 (CA16) infection results in hand, foot, and mouth disease (HFMD) along with other severe neurological diseases in children and poses an important public health threat in Asian countries. During an HFMD epidemic in 2009 in Guangdong, China, two CA16 strains (GD09/119 and GD09/24) were isolated and characterized. Although both strains were similar in plaque morphology and growth properties in vitro, the two isolates exhibited distinct pathogenicity in neonatal mice upon intraperitoneal or intracranial injection. Complete genome sequences of both CA16 strains were determined, and the possible virulence determinants were analyzed and predicted. Phylogenetic analysis revealed that these CA16 isolates from Guangdong belonged to the B1b genotype and were closely related to other recent CA16 strains isolated in mainland China. Similarity and bootscanning analyses of these CA16 strains detected homologous recombination with the EV71 prototype strain BrCr in the non-structural gene regions and the 3'-untranslated regions. Together, the phenotypic and genomic characterizations of the two clinical CA16 isolates circulating in China were compared in detail, and the potential amino acid residues responsible for CA16 virulence in mice were predicted. These findings will help explain the evolutionary relationship of the CA16 strains circulating in China, warranting future studies investigating enterovirus virulence.

RNA interference (RNAi) functions as a potent antiviral immunity in plants and invertebrates; however, whether RNAi plays antiviral roles in mammals remains unclear. Here, using human enterovirus 71 (HEV71) as a model, we showed HEV71 3A protein as an authentic viral suppressor of RNAi during viral infection. When the 3A-mediated RNAi suppression was impaired, the mutant HEV71 readily triggered the production of abundant HEV71-derived small RNAs with canonical siRNA properties in cells and mice. These virus-derived siRNAs were produced from viral dsRNA replicative intermediates in a Dicer-dependent manner and loaded into AGO, and they were fully active in degrading cognate viral RNAs. Recombinant HEV71 deficient in 3A-mediated RNAi suppression was significantly restricted in human somatic cells and mice, whereas Dicer deficiency rescued HEV71 infection independently of type I interferon response. Thus, RNAi can function as an antiviral immunity, which is induced and suppressed by a human virus, in mammals.

Human adenovirus type 14 (HAdV-B14) was first reported in 1955 from the Netherlands and since then had been associated with outbreaks of febrile respiratory illness (FRI). In China, sporadic HAdV-B14 infections were first identified in 2010, in Guangzhou and Beijing. In 2012, an outbreak of FRI occurred in Beijing and the etiological agent was determined to be HAdV-B14. We present a complete HAdV-B14 genome sequence isolated from this recent FRI outbreak. Virus in 30 throat swab samples was detected using polymerase chain reaction assays, and confirmed by sequencing of the fiber, hexon and penton genes. Comparative genomics and phylogenetic analysis showed that the newly isolated HAdV-B14 (HAdV-B14 CHN) shared highest sequence homology with a 2006 isolate from the United States and clustered closely with other HAdV-B14 strains. It is expected that data from the present study will help in devising better protocols for virus surveillance, and in developing preventative measures., Highlights • Isolation of HAdV-B14 was performed from the outbreak of febrile respiratory illness. • Full genome sequence of HAdV-B14 CHN strain has been reported. • Several nucleotide substitutions were reported in the HAdV-B14 CHN genome.

Human Enterovirus 71 (EV71) is recognized as the leading causative agent of hand-foot-and-mouth disease (HFMD) in the Asia-Pacific region in recent years. There are still no approved antiviral drugs or vaccines against EV71 infection yet. In this study, we have developed an advanced platform for production of the virus-like particles (VLPs) for EV71 in Saccharomyces Cerevisiae by co-expressing P1 and 3CD genes of EV71. These VLPs exhibited similar morphology and protein composition as EV71 empty particles produced from EV71-infected cells. Immunization with VLPs in mice elicited robust neutralization antibodies against EV71 and potent cellular immune response. In vivo challenge experiments showed that the immune sera induced by VLP conferred protection in neonate mice against lethal EV71 challenge. Together, our study indicated that VLP from yeast is another potential vaccine candidate against EV71 infection.

While self-replicating, non-infectious subgenomic flavivirus replicons have been described, most of them are RNA transcripts under the control of an Sp6 or T7 promoter. In this study, using West Nile virus (WNV) as a model, a series of DNA-based reporter replicons under the control of a minimal cytomegalovirus (CMV) immediate-early promoter were constructed, and functional analysis showed that these reporter replicons replicate efficiently in mammalian cells. When the DNA-based WNV replicon was used to immunize mice, NS1-specific IgG antibodies and anti-WNV neutralizing antibodies were both induced. Additionally, immunization with this DNA-based WNV replicon induced high levels of lymphocyte proliferation and enhanced the secretion of IFN-γ. These results suggest that this type of DNA-based replicon can induce humoral and cellular immune responses in mice, indicating that this type of DNA-based replicon may serve as a useful platform for vaccine development and protein expression.

Japanese encephalitis virus (JEV) and West Nile virus (WNV) are closely related mosquito-borne flaviviruses that cause severe encephalitic diseases with global impact. Cross protection among JEV and WNV has been previously described, and most cross reactive epitopes were identified within the domain II of E protein (EDII). In this study, the E protein domain III (EDIII) of JEV was successfully expressed in Escherichia coli, purified by a Ni-NTA column and characterized by Western blotting assay. Competitive inhibition assay showed that this recombinant JEV EDIII blocks the entry of JEV into BHK-21 cells. Mice immunized with the recombinant JEV EDIII developed high IgG and neutralizing antibodies titers against JEV. Most importantly, antibodies induced by JEV EDIII could neutralize WNV in vitro and partially protected mice against lethal WNV challenge. These results demonstrate that immunization with JEV EDIII induces cross-protective immunity against WNV infection, indicating a possible role of EDIII for the cross-protection among flavivirus.

Hemagglutinin (HA) is an important influenza virus surface antigen that is highly topical in influenza research. In the present study, the genes encoding the HA1 and HA2 proteins from the 2009 pandemic influenza virus H1N1 (A/California/04/2009(H1N1)) were cloned into a prokaryotic expression plasmid pCold-TF, and soluble fusion proteins containing H1N1 HA1 and HA2 were produced by transformed Escherichia coli. Western blot assays were used to examine the immunoreactivity of the recombinant proteins using polyclonal and monoclonal antibodies derived against the whole virus A/California/04/2009(H1N1). Recombinant protein immunoreactivity was also analyzed qualitatively by ELISA and hemagglutination inhibition using human serum samples. These results will aid future immunological and serological studies of the 2009 pandemic H1N1 virus HA.

Research highlights ▶ The D–R mathematical model predicts future trends in the spread of human H1N1. ▶ The D–R algorithm may have broader application to other human viral diseases. ▶ D–R modeling will help design surveillance programs for the spread of viral diseases. ▶ The predictive value of the D–R model is superior to the grey prediction model., Incidences of H1N1 viral infections in Mainland China are collected by the Ministry of Health, the People's Republic of China. The number of confirmed cases and the timing of these outbreaks from May 13 to July 22, 2009 were obtained and subjected to a novel mathematical model to simulate the infection profile (time vs number). The model was predicated upon the grey prediction theory which allows assignment of future trends using limited numbers of data points. During the period of our analysis, the number of confirmed H1N1 cases in Mainland China increased from 1 to 1772. The efficiency of our model to simulate these data points was evaluated using Sum of squares of error (SSE), Relative standard error (RSE), Mean absolute deviation (MAD) and Average relative error (ARE). Results from these analyses were compared to similar calculations based upon the grey prediction algorithm. Using our equation, defined herein as equation D–R, results showed that SSE = 6742.00, RSE = 10.69, MAD = 7.07, ARE = 2.47% were all consistent with the D–R algorithm performing well in the estimation of future trends of H1N1 cases in Mainland China. Calculations using the grey theory had no predictive value [ARE for GM(1,1) = −104.63%]. To validate this algorithm, we performed a second analysis using new data obtained from cases reported to the WHO and CDC in the US between April 26 and June 8, 2009. In like manner, the model was equally predictive. The success of the D–R mathematical model suggests that it may have broader application to other viral infections among the human population in China and may be modified for application to other regions of the world.

Summary Zika virus (ZIKV) directly infects neural progenitors and impairs their proliferation. How ZIKV interacts with the host molecular machinery to impact neurogenesis in vivo is not well understood. Here, by systematically introducing individual proteins encoded by ZIKV into the embryonic mouse cortex, we show that expression of ZIKV-NS2A, but not Dengue virus (DENV)-NS2A, leads to reduced proliferation and premature differentiation of radial glial cells and aberrant positioning of newborn neurons. Mechanistically, in vitro mapping of protein-interactomes and biochemical analysis suggest interactions between ZIKA-NS2A and multiple adherens junction complex (AJ) components. Functionally, ZIKV-NS2A, but not DENV-NS2A, destabilizes the AJ complex, resulting in impaired AJ formation and aberrant radial glial fiber scaffolding in the embryonic mouse cortex. Similarly, ZIKA-NS2A, but not DENV-NS2A, reduces radial glial cell proliferation and causes AJ deficits in human forebrain organoids. Together, our results reveal pathogenic mechanisms underlying ZIKV infection in the developing mammalian brain.

Zika virus (ZIKV) persists in the semen of male patients, a first for flavivirus infection. Here, we demonstrate that ZIKV can induce inflammation in the testis and epididymidis, but not in the prostate or seminal vesicle, and can lead to damaged testes after 60 days post-infection in mice. ZIKV induces innate immune responses in Leydig, Sertoli, and epididymal epithelial cells, resulting in the production of pro-inflammatory cytokines/chemokines. However, ZIKV does not induce a rapid and abundant cytokine production in peritubular cell and spermatogonia, suggesting that these cells are vulnerable for ZIKV infection and could be the potential repositories for ZIKV. Our study demonstrates a correlation between ZIKV and testis infection/damage and suggests that ZIKV infection, under certain circumstances, can eventually lead to male infertility.