Simultaneous detection of Zika, chikungunya, dengue, yellow fever, West Nile, and Japanese encephalitis viruses by a two-tube multiplex real-time RT-PCR assay.

Due to the concurrent prevalence and increasing risk of coinfection of the clinically important Arboviruses, timely and accurate differential diagnosis is important for clinical management and the epidemiological investigation. A two-tube multiplex real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for the simultaneous detection of Zika virus (ZIKV), chikungunya virus (CHIKV), dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), and Japanese encephalitis virus (JEV) was developed and optimized with high specificity and sensitivity. The detection limit for all the six viruses could reach as low as five genome equivalent copies and 2.8 × 10-3 tissue culture infectious doses (TCID50 ) for ZIKV, YFV, CHIKV and 2.8 × 10-2 TCID50  for JEV per reaction, with high accuracy and precision (R2 > 0.99). The coefficient of variation of intra-assay and inter-assay for our quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was low, and the obtained positive rates ad Ct values of this assay were comparable with singleplex commercial kits. Moreover, the multiplex qRT-PCR assay was able to detect possible co-infections without competitive inhibition of target viral genomes. In conclusion, our rapid, sensitive, cost-effective multiplex qRT-PCR will be of great use for differential diagnosis in a clinical setting and epidemiological investigation during surveillance.

Nucleoside Analogs with Selective Antiviral Activity against Dengue Fever and Japanese Encephalitis Viruses.

Dengue virus (DENV) and Japanese encephalitis virus (JEV) are important arthropod-borne viruses from the Flaviviridae family. DENV is a global public health problem with significant social and economic impacts, especially in tropical and subtropical areas. JEV is a neurotropic arbovirus endemic to east and southeast Asia. There are no U.S. FDA-approved antiviral drugs available to treat or to prevent DENV and JEV infections, leaving nearly one-third of the world's population at risk for infection. Therefore, it is crucial to discover potent antiviral agents against these viruses. Nucleoside analogs, as a class, are widely used for the treatment of viral infections. In this study, we discovered nucleoside analogs that possess potent and selective anti-JEV and anti-DENV activities across all serotypes in cell-based assay systems. Both viruses were susceptible to sugar-substituted 2'-C-methyl analogs with either cytosine or 7-deaza-7-fluoro-adenine nucleobases. Mouse studies confirmed the anti-DENV activity of these nucleoside analogs. Molecular models were assembled for DENV serotype 2 (DENV-2) and JEV RNA-dependent RNA polymerase replication complexes bound to nucleotide inhibitors. These models show similarities between JEV and DENV-2, which recognize the same nucleotide inhibitors. Collectively, our findings provide promising compounds and a structural rationale for the development of direct-acting antiviral agents with dual activity against JEV and DENV infections.

Computational Prediction of Usutu Virus E Protein B Cell and T Cell Epitopes for Potential Vaccine Development.

Usutu virus (family Flaviviridae), once confined to Africa, has emerged in Europe a decade ago. The virus has been spreading throughout Europe at a greater pace mostly affecting avian species. While most bird species remain asymptomatic carriers of this virus, few bird species are highly susceptible. Lately, Usutu virus (USUV) infections in humans were reported sporadically with severe neuroinvasive symptoms like meningoencephalitis. As so much is unknown about this virus, which potentially may cause severe diseases in humans, there is a need for more studies of this virus. In this study, we have used computational tools to predict potential B cell and T cell epitopes of USUV envelope (E) protein. We found that amino acids between positions 68 and 84 could be a potential B cell epitope, while amino acids between positions 53 and 69 could be a potential major histocompatibility complex (MHC) class I- and class II-restricted T cell epitope. By homology 3D modeling of USUV E protein, we found that the predicted B cell epitope was predominantly located in the coil region, while T cell epitope was located in the beta-strand region of the E protein. Additionally, the potential MHC class I T cell epitope (LAEVRSYCYL) was predicted to bind to nearly 24 human leucocyte antigens (HLAs) (IC50 ≤5000 nm) covering nearly 86.44% of the Black population and 96.90% of the Caucasoid population. Further in vivo studies are needed to validate the predicted epitopes.

Widespread activity of multiple lineages of Usutu virus, western Europe, 2016.

In the summer of 2016, Belgium, France, Germany and the Netherlands reported widespread Usutu virus (USUV) activity based on live and dead bird surveillance. The causative USUV strains represented four lineages, of which two putative novel lineages were most likely recently introduced into Germany and spread to other western European countries. The spatial extent of the outbreak area corresponded with R0 values > 1. The occurrence of the outbreak, the largest USUV epizootic registered so far in Europe, allowed us to gain insight in how a recently introduced arbovirus with potential public health implications can spread and become a resident pathogen in a naïve environment. Understanding the ecological and epidemiological factors that drive the emergence or re-emergence of USUV is critical to develop and implement timely surveillance strategies for adequate preventive and control measures. Public health authorities, blood transfusion services and clinicians in countries where USUV was detected should be aware of the risk of possible USUV infection in humans, including in patients with unexplained encephalitis or other neurological impairments, especially during late summer when mosquito densities peak.

Perturbation in the conserved methyltransferase-polymerase interface of flavivirus NS5 differentially affects polymerase initiation and elongation.

UNLABELLED: The flavivirus NS5 is a natural fusion of a methyltransferase (MTase) and an RNA-dependent RNA polymerase (RdRP). Analogous to DNA-dependent RNA polymerases, the NS5 polymerase initiates RNA synthesis through a de novo mechanism and then makes a transition to a processive elongation phase. However, whether and how the MTase affects polymerase activities through intramolecular interactions remain elusive. By solving the crystal structure of the Japanese encephalitis virus (JEV) NS5, we recently identified an MTase-RdRP interface containing a set of six hydrophobic residues highly conserved among flaviviruses. To dissect the functional relevance of this interface, we made a series of JEV NS5 constructs with mutations of these hydrophobic residues and/or with the N-terminal first 261 residues and other residues up to the first 303 residues deleted. Compared to the wild-type (WT) NS5, full-length NS5 variants exhibited consistent up- or downregulation of the initiation activities in two types of polymerase assays. Five representative full-length NS5 constructs were then tested in an elongation assay, from which the apparent single-nucleotide incorporation rate constant was estimated. Interestingly, two constructs exhibited different elongation kinetics from the WT NS5, with an effect rather opposite to what was observed at initiation. Moreover, constructs with MTase and/or the linker region (residues 266 to 275) removed still retained polymerase activities, albeit at overall lower levels. However, further removal of the N-terminal extension (residues 276 to 303) abolished regular template-directed synthesis. Together, our data showed that the MTase-RdRP interface is relevant in both polymerase initiation and elongation, likely with different regulation mechanisms in these two major phases of RNA synthesis. IMPORTANCE: The flavivirus NS5 is very unique in having a methyltransferase (MTase) placed on the immediate N terminus of its RNA-dependent RNA polymerase (RdRP). We recently solved the crystal structure of the full-length NS5, which revealed a conserved interface between MTase and RdRP. Building on this discovery, here we carried out in vitro polymerase assays to address the functional relevance of the interface interactions. By explicitly probing polymerase initiation and elongation activities, we found that perturbation in the MTase-RdRP interface had different impacts on different phases of synthesis, suggesting that the roles and contribution of the interface interactions may change upon phase transitions. By comparing the N-terminal-truncated enzymes with the full-length NS5, we collected data to indicate the indispensability to regular polymerase activities of a region that was functionally unclarified previously. Taken together, we provide biochemical evidence and mechanistic insights for the cross talk between the two enzyme modules of flavivirus NS5.

Structure-based mutational analysis of several sites in the E protein: implications for understanding the entry mechanism of Japanese encephalitis virus.

UNLABELLED: Japanese encephalitis virus (JEV), which causes viral encephalitis in humans, is a serious risk to global public health. The JEV envelope protein mediates the viral entry pathway, including receptor-binding and low-pH-triggered membrane fusion. Utilizing mutagenesis of a JEV infectious cDNA clone, mutations were introduced into the potential receptor-binding motif or into residues critical for membrane fusion in the envelope protein to systematically investigate the JEV entry mechanism. We conducted experiments evaluating infectious particle, recombinant viral particle, and virus-like particle production and found that most mutations impaired virus production. Subcellular fractionation confirmed that five mutations--in I0, ij, BC, and FG and the R9A substitution-impaired virus assembly, and the assembled virus particles of another five mutations--in kl and the E373A, F407A, L221S, and W217A substitutions--were not released into the secretory pathway. Next, we examined the entry activity of six mutations yielding infectious virus. The results showed N154 and the DE loop are not the only or major receptor-binding motifs for JEV entry into BHK-21 cells; four residues, H144, H319, T410, and Q258, participating in the domain I (DI)-DIII interaction or zippering reaction are important to maintain the efficiency of viral membrane fusion. By continuous passaging of mutants, adaptive mutations from negatively charged amino acids to positively charged or neutral amino acids, such as E138K and D389G, were selected and could restore the viral entry activity. IMPORTANCE: Recently, there has been much interest in the entry mechanism of flaviviruses into host cells, including the viral entry pathway and membrane fusion mechanism. Our study provides strong evidence for the critical role of several residues in the envelope protein in the assembly, release, and entry of JEV, which also contributes to our understanding of the flaviviral entry mechanism. Furthermore, we demonstrate that the H144A, H319A, T410A, and Q258A mutants exhibit attenuated fusion competence, which may be used to develop novel vaccine candidates for flaviviruses.

Lack of usutu virus RNA in cerebrospinal fluid of patients with encephalitis of unknown etiology, Tuscany, Italy.

Usutu virus (USUV) is an African mosquito-borne flavivirus associated with human neurological disorders in Europe. Recently, USUV introduction in Europe has been traced back to Eurasian blackbirds deaths in the Tuscany region of Italy in 1996. Ninety-six cerebrospinal fluid (CSF) samples from patients with encephalitis of unknown etiology diagnosed in 2010-2013 were screened to determine whether USUV circulates in humans in Tuscany. Using real-time polymerase chain reaction, no positive patient was found. USUV does not seem to cause neuroinvasive disorders in humans in Tuscany.

Replacement of pr gene with Japanese encephalitis virus pr using reverse genetics reduces antibody-dependent enhancement of dengue virus 2 infection.

Severe dengue is more likely found during secondary heterologous dengue virus (DENV) infection or primary infection of infants born to dengue-immune mothers and led to the hypothesis of antibody-dependent enhancement (ADE). It has been reported that pre-membrane (prM)-reactive antibodies do not efficiently neutralize DENV infection but instead potently promote ADE infection. Meanwhile, these enhancing anti-prM antibodies mainly react with the precursor (pr) peptide. To evaluate the effect of pr gene substitution on neutralization and ADE of DENV infection, a novel chimeric dengue virus (JEVpr/DENV2) was rationally constructed by replacing the DENV pr gene with Japanese encephalitis virus (JEV) pr gene, based on the full-length infectious complementary DNA (cDNA) clone of DENV2 ZS01/01. We found that chimeric JEVpr/DENV2 showed reduced virulence and good immunogenicity. In addition, anti-JEVpr/DENV2 sera showed broad cross-reactivity and efficient neutralizing activity with all four DENV serotypes and immature DENV2 (ImDENV2). Most importantly, compared with anti-DENV2 sera, anti-JEVpr/DENV2 sera showed significantly reduced enhancing activity of DENV infection in K562 cells. These results suggest that the ADE activities could be reduced by replacing the DENV pr gene with JEV pr gene. These findings may help us better understand the pathogenesis of DENV infection and provide a reference for the development of a vaccine against DENV.

A novel approach to propagate flavivirus infectious cDNA clones in bacteria by introducing tandem repeat sequences upstream of virus genome.

Despite tremendous efforts to improve the methodology for constructing flavivirus infectious cDNAs, the manipulation of flavivirus cDNAs remains a difficult task in bacteria. Here, we successfully propagated DNA-launched type 2 dengue virus (DENV2) and Japanese encephalitis virus (JEV) infectious cDNAs by introducing seven repeats of the tetracycline-response element (7×TRE) and a minimal cytomegalovirus (CMVmin) promoter upstream of the viral genome. Insertion of the 7×TRE-CMVmin sequence upstream of the DENV2 or JEV genome decreased the cryptic E. coli promoter (ECP) activity of the viral genome in bacteria, as measured using fusion constructs containing DENV2 or JEV segments and the reporter gene Renilla luciferase in an empty vector. The growth kinetics of recombinant viruses derived from DNA-launched DENV2 and JEV infectious cDNAs were similar to those of parental viruses. Similarly, RNA-launched DENV2 infectious cDNAs were generated by inserting 7×TRE-CMVmin, five repeats of the GAL4 upstream activating sequence, or five repeats of BamHI linkers upstream of the DENV2 genome. All three tandem repeat sequences decreased the ECP activity of the DENV2 genome in bacteria. Notably, 7×TRE-CMVmin stabilized RNA-launched JEV infectious cDNAs and reduced the ECP activity of the JEV genome in bacteria. The growth kinetics of recombinant viruses derived from RNA-launched DENV2 and JEV infectious cDNAs displayed patterns similar to those of the parental viruses. These results support a novel methodology for constructing flavivirus infectious cDNAs, which will facilitate research in virology, viral pathogenesis and vaccine development of flaviviruses and other RNA viruses.

Evaluation of chimeric DNA vaccines consisting of premembrane and envelope genes of Japanese encephalitis and dengue viruses as a strategy for reducing induction of dengue virus infection-enhancing antibody response.

Neutralizing antibodies induced by dengue virus (DENV) infection show viral infection-enhancing activities at sub-neutralizing doses. On the other hand, preimmunity against Japanese encephalitis virus (JEV), a congener of DENV, does not increase the severity of DENV infection. Several studies have demonstrated that neutralizing epitopes in the genus Flavivirus are mainly located in domain III (DIII) of the envelope (E) protein. In this study, chimeric premembrane and envelope (prM-E) gene-based expression plasmids of JEV and DENV1 with DIII substitution of each virus were constructed for use as DNA vaccines and their immunogenicity evaluated. Sera from C3H/He and ICR mice immunized with a chimeric gene containing DENV1 DIII on a JEV prM-E gene backbone showed high neutralizing antibody titers with less DENV infection-enhancing activity. Our results confirm the applicability of this approach as a new dengue vaccine development strategy.

Usutu virus, Italy, 1996.

Retrospective analysis of archived tissue samples from bird deaths in the Tuscany region of Italy in 1996 identified Usutu virus. Partial sequencing confirmed identity with the 2001 Vienna strain and provided evidence for a much earlier introduction of this virus into Europe than previously assumed.

Comparative full length genome sequence analysis of Usutu virus isolates from Africa.

BACKGROUND: Usutu virus (USUV), a flavivirus belonging to the Japanese encephalitis serocomplex, was identified in South Africa in 1959 and reported for the first time in Europe in 2001. To date, full length genome sequences have been available only for the reference strain from South Africa and a single isolate from each of Austria, Hungary, and Italy. METHODS: We sequenced four USUV isolates from Senegal and the Central African Republic (CAR) between 1974 and 2007 and compared the sequence data to USUV strains from Austria, Hungary, Italy, and South Africa using a Bayesian Markov chain Monte Carlo method. We further clarified the taxonomic status of a USUV strain isolated in CAR in 1969 and proposed earlier as a subtype of USUV due to an asymetric serological cross-reactivity with USUV reference strain. RESULTS: A comparison of the four newly obtained USUV sequences with those from SouthAfrica_1959, Vienna_2001, Budapest_2005, and Italy_2009 revealed that they are all 96-99% and 99% similar at the nucleotide and amino acid levels, respectively. The phylogenetic relationships between these sequences indicated that a strain isolated in Senegal in 1993 is most closely related to the USUV strains detected in Europe. Analysis of a strain isolated from a human in CAR in 1981 (CAR_1981) revealed the presence of specific amino acid substitutions and a deletion in the 3' noncoding region. This is the first fully sequenced human USUV isolate.The putative USUV subtype, CAR_1969, was 81% and 94% identical at the nucleotide and amino acid levels, respectively, compared to the other USUV strains. Our phylogenetic analyses support the serological identification of CAR_1969 as a subtype of USUV. CONCLUSIONS: In this study, we investigate the genetic diversity of USUV in Africa and the phylogenetic relationship of isolates from Africa and Europe for the first time. The results suggest a low genetic diversity within USUV, the existence of a distinct USUV subtype strain, and support the hypothesis that USUV was introduced to Europe from Africa. Further sequencing and analysis of USUV isolates from other African countries would contribute to a better understanding of its genetic diversity and geographic distribution.

Molecular characterization of Japanese encephalitis viruses circulating in pigs and mosquitoes on pig farms in the Chinese province of Henan.

Since the first Chinese case report of Japanese encephalitis, Japanese encephalitis virus (JEV) has circulated in China for at least 60 years. Even though pigs play a critical role in the JEV transmission cycle information on the prevalence of JEV in pigs has not been investigated in China. As the central Chinese province of Henan has the largest human population in China, a history of serious JEV and is the largest pig producing province it was chosen for this study. We have found that currently natural infection with JEV in pigs and mosquitoes is prevalent and both genotypes 1 and 3 co-circulate in pigs and mosquitoes in central China. Phylogenetic analysis showed that all of the newly obtained pig-derived JEV isolates are more closely related to isolates from the 1950s to 1960s than to those recently isolated from humans and mosquitoes. Further analyses based on all the previous reported Chinese isolates indicates that presently genotype 3 JEV is the predominant genotype in pigs but genotype 1 JEV is emerging and spreading rapidly in recent years. Our study provides information for understanding the current epidemiology of JEV in China and suggests possible measures applicable to the further control of JEV.

Evaluation of the genotype I Japanese encephalitis virus as a stable viral vector for foreign gene expression.

Manipulation of the flavivirus genome to accommodate and express a heterologous gene of interest has become an attractive approach for gene delivery and the development of viral-vectored vaccines. However, due to the inherent genetic instability of the flavivirus genomes, the construction of recombinant viruses carrying a foreign gene could be problematic and heavily resistant. In this study, the possibility of the Japanese encephalitis virus (JEV) as a stable flavivirus vector for the expression of a foreign gene was assessed using reverse genetics. The full-length cDNA genome of genotype I (GI) JEV inherently possessed excellent stability and manipulability in a bacterial host, while mutations and deletions accumulated in the cDNA genomes of genotype Ⅲ (GⅢ) JEV strains. Using the GI JEV as backbones, we generate a panel of recombinant viruses expressing various foreign genes. All recombinant viruses exhibited excellent genetic stability and efficiently express foreign genes for at least ten serial passages in vitro. In application, a convenient, rapid and reliable image-based assay for neutralizing antibody testing and antiviral drug discovery was established with a mCherry-reporter recombinant virus (rBJ-mCherry). Meanwhile, the recombinant viruses expressing the antigens of the African swine fever virus (ASFV) or Classical swine fever virus (CSFV) could effectively induce antibody responses to the JEV vector and foreign antigens in a mouse vaccination model. Therefore, GI JEV strains could serve as viral vectors accommodating the expression of large foreign genes.

Novel reverse genetics of genotype I and III Japanese encephalitis viruses assembled through transformation associated recombination in yeast: The reporter viruses expressing a green fluorescent protein for the antiviral screening assay.

Japanese encephalitis virus (JEV) belongs to a zoonotic flavivirus and is the main cause of viral encephalitis in humans throughout Asia. During the past two decades, both genotypes I (G) and III (GIII) JEVs co-existed in many Asian countries, and the prevalent strains have shifted from GIII to GI. Since all licensed JE vaccines are derived from GIII strains and no specific treatment is available, the development of novel vaccines and specific antiviral drugs against both genotypes is urgently required. To solve the unstable issue of the JEV infectious cDNA clone and establish a reliable antiviral screening assay, we established the yeast artificial chromosome (YAC)-based reverse genetics systems for GI and GIII JEV strains through transformation-associated recombination (TAR) technology in yeast. The YAC-based infectious clones of GI and GIII JEV exhibited high genetic stability both in yeast and E. coli. Using these reverse genetics systems, recombinant EGFP-reporter viruses were generated and remained stable for at least 10 passages in vitro. An image-based antiviral assay for JEV was developed with the EGFP-reporter viruses, and two drugs were identified to have a broad-spectrum inhibitory effect on GI and GIII JEV replication, which provide potential new therapeutic for the treatment of multiple genotypes JEV infection.

Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States.

In late summer 1999, an outbreak of human encephalitis occurred in the northeastern United States that was concurrent with extensive mortality in crows (Corvus species) as well as the deaths of several exotic birds at a zoological park in the same area. Complete genome sequencing of a flavivirus isolated from the brain of a dead Chilean flamingo (Phoenicopterus chilensis), together with partial sequence analysis of envelope glycoprotein (E-glycoprotein) genes amplified from several other species including mosquitoes and two fatal human cases, revealed that West Nile (WN) virus circulated in natural transmission cycles and was responsible for the human disease. Antigenic mapping with E-glycoprotein-specific monoclonal antibodies and E-glycoprotein phylogenetic analysis confirmed these viruses as WN. This North American WN virus was most closely related to a WN virus isolated from a dead goose in Israel in 1998.

Neutralization Potency of Sera from Vietnamese Patients with Japanese Encephalitis (JE) against Genotypes I and V JE Viruses.

Japanese encephalitis virus (JEV) is classified into 5 genotypes (GI, GII, GIII, GIV, and GV), and the GI and GIII strains are the most widely distributed in JE endemic areas. In recent years, GV JEV has been detected in China and Korea, suggesting that GV JEV may invade other JE endemic areas, including Vietnam, and that more attention should be paid to the JEV strains circulating in these areas. In this study, we investigated the neutralization ability of the sera collected from 22 Vietnamese patients with JE who lived in northern Vietnam against the GI and GV JEV strains. In most cases, the ratios of the titer against GV to that against GI (GV:GI) were equal to or less than 1:4. However, the titer against GV JEV was equivalent (1:1) to that against GI JEV in only a few cases, and no serum had a ratio higher than 1:1. Thus, our results did not show convincing evidence that GV JEV was emerging in northern Vietnam in 2014.

Survival of mice immunized with monoclonal antibodies against glycoprotein E of Japanese encephalitis virus before or after infection with Japanese encephalitis, West Nile, and Dengue viruses.

In the present study, the effect of monoclonal antibodies (MAbs) against glycoprotein E (gE) of Japanese encephalitis virus (JEV) strain 733913 administered 1 day before or 2 days after intracerebral (i.c.) challenge with West Nile virus (WNV) strain 68856 or Dengue virus (DENV-2) strain P23085, was studied in mice. Furthermore, two JEV strains belonging to group II (strains 641686 and 691004) that have lost reactivity against virus-specific MAbs were also used in passive immunization experiments. MAbs as ascitic fluids were administered intraperitoneally (i.p.) in mice. Hemagglutination-inhibition- (HAI) positive JEV-specific (Hs-3) MAbs given 2 days after the virus infection showed reduced mortality along with increased survival of mice challenged with WNV or with DENV-2. Also the HAI-positive flavivirus cross-reactive (Hx) MAbs produced a marginal increase in the survival of mice challenged with both JEV strains 641686 and 691004 belonging to the group II. As the MAbs reacting with HAI-positive JEV-specific (Hs) and HAI-negative JEV-specific (NHs) epitopes were neutralizing and protective in mice against JEV strain 733913 challenge, the results indicated presence of the cross-protection phenomenon that might be occurring in some of the localities endemic for the three closely related flaviviruses.