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.

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.

Usutu virus: an emerging flavivirus in Europe.

Usutu virus (USUV) is an African mosquito-borne flavivirus belonging to the Japanese encephalitis virus serocomplex. USUV is closely related to Murray Valley encephalitis virus, Japanese encephalitis virus, and West Nile virus. USUV was discovered in South Africa in 1959. In Europe, the first true demonstration of circulation of USUV was reported in Austria in 2001 with a significant die-off of Eurasian blackbirds. In the subsequent years, USUV expanded to neighboring countries, including Italy, Germany, Spain, Hungary, Switzerland, Poland, England, Czech Republic, Greece, and Belgium, where it caused unusual mortality in birds. In 2009, the first two human cases of USUV infection in Europe have been reported in Italy, causing meningoencephalitis in immunocompromised patients. This review describes USUV in terms of its life cycle, USUV surveillance from Africa to Europe, human cases, its cellular tropism and pathogenesis, its genetic relationship with other flaviviruses, genetic diversity among USUV strains, its diagnosis, and a discussion of the potential future threat to Asian countries.

Flavivirus detection and differentiation by a microsphere array assay.

Flaviviruses of the Japanese encephalitis virus (JEV) serocomplex include major human and animal pathogens that have a propensity to spread and emerge in new geographic areas. Different genotypes or genetic lineages have been defined for many of these viruses, and they are distributed worldwide. Tools enabling rapid detection of new or emerging flaviviruses and differentiation of important subgroups have widespread application for arbovirus diagnosis and surveillance, and are crucial for detecting virus incursions, tracking virus emergence and for disease control. A microsphere suspension array assay was developed to identify JEV serocomplex flaviviruses of medical and veterinary importance. Assay performance was evaluated using representative virus strains as well as clinical and surveillance samples. The assay detected all JEV serocomplex viruses tested in this study with an apparent analytical sensitivity equal or better than the reference real-time or conventional RT-PCR assays and was able to identify mixed virus populations. The ability to identify mixed virus populations at a high analytical sensitivity would be pertinent in the Australian context when attempting to detect exotic JEV or West Nile virus (WNV), and differentiate from endemic Murray Valley encephalitis virus and WNV-Kunjin virus. The relatively low cost, the ability to identify mixed virus populations and the multiplex nature makes this assay valuable for a wide range of applications including diagnostic investigations, virus exclusions, and surveillance programs.

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.

Comparative genomic and phylogenetic analysis of the first Usutu virus isolate from a human patient presenting with neurological symptoms.

Usutu virus (USUV) is a mosquito-borne flavivirus, belonging to the Japanese encephalitis antigenic complex, that circulates among mosquitoes and birds. We describe and analyze the complete genome sequence of the first USUV strain isolated from an immunocompromised patient with neuroinvasive disease. This USUV isolate showed an overall nucleotide identity of 99% and 96%, respectively, with the genomes of isolates from Europe and Africa. Comparison of the human USUV complete polyprotein sequence with bird-derived strains, showed two unique amino acid substitutions. In particular, one substitution (S595G) was situated in the DIII domain of the viral Envelope protein that is recognized by flavivirus neutralizing antibodies. An additional amino acid substitution (D3425E) was identified in the RNA-dependent RNA polymerase (RdRp) domain of the NS5 protein. This substitution is remarkable since E3425 is highly conserved among the other USUV isolates that were not associated with human infection. However, a similar substitution was observed in Japanese encephalitis and in West Nile viruses isolated from humans. Phylogenetic analysis of the human USUV strain revealed a close relationship with an Italian strain isolated in 2009. Analysis of synonymous nucleotide substitutions (SNSs) among the different USUV genomes showed a specific evolutionary divergence among different countries. In addition, 15 SNSs were identified as unique in the human isolate. We also identified four specific nucleotide substitutions in the 5' and 3' untranslated regions (UTRs) in the human isolate that were not present in the other USUV sequences. Our analyses provide the basis for further experimental studies aimed at defining the effective role of these mutations in the USUV genome, their potential role in the development of viral variants pathogenic for humans and their evolution and dispersal out of Africa.

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.

Epizootic emergence of Usutu virus in wild and captive birds in Germany.

This study aimed to identify the causative agent of mass mortality in wild and captive birds in southwest Germany and to gather insights into the phylogenetic relationship and spatial distribution of the pathogen. Since June 2011, 223 dead birds were collected and tested for the presence of viral pathogens. Usutu virus (USUV) RNA was detected by real-time RT-PCR in 86 birds representing 6 species. The virus was isolated in cell culture from the heart of 18 Blackbirds (Turdus merula). USUV-specific antigen was demonstrated by immunohistochemistry in brain, heart, liver, and lung of infected Blackbirds. The complete polyprotein coding sequence was obtained by deep sequencing of liver and spleen samples of a dead Blackbird from Mannheim (BH65/11-02-03). Phylogenetic analysis of the German USUV strain BH65/11-02-03 revealed a close relationship with strain Vienna that caused mass mortality among birds in Austria in 2001. Wild birds from lowland river valleys in southwest Germany were mainly affected by USUV, but also birds kept in aviaries. Our data suggest that after the initial detection of USUV in German mosquitoes in 2010, the virus spread in 2011 and caused epizootics among wild and captive birds in southwest Germany. The data also indicate an increased risk of USUV infections in humans in Germany.

Feasibility of cross-protective vaccination against flaviviruses of the Japanese encephalitis serocomplex.

Serological cross-reactivity providing cross-protective immunity between antigenically related viruses is a cornerstone of vaccination. It was the immunological basis for the first human vaccine against smallpox introduced more than 200 years ago, and continues to underpin modern vaccine development as has recently been shown for human papillomavirus vaccines, which confer cross-protection against other oncogenic papillomavirus types not present in the vaccine. Here, we review the feasibility of cross-protective vaccination against an antigenic group of clinically important viruses belonging to the Japanese encephalitis serocomplex in the Flaviviridae family. We will discuss evidence suggesting that 'new generation' flavivirus vaccines may provide effective cross-protective immunity against heterologous Japanese encephalitis serocomplex viruses, and appraise potential risks associated with cross-reactive vaccine immunity. The review will also focus on the structural and mechanistic basis for cross-protective immunity among this group of flaviviruses, which is predominantly mediated by antibodies against a single viral surface protein.

Detection of Usutu-virus-specific IgG in blood donors from northern Italy.

We developed a novel enzyme-linked immunosorbent assay to detect the specific IgG response to Usutu virus (USUV) in humans, by evaluating 359 blood donors who were living in northeastern Italy. Our results demonstrate the presence of an anti-USUV response in 4 subjects with no history of other flavivirus infection.

Genotype v Japanese encephalitis virus is emerging.

Japanese encephalitis (JE) is a global public health issue that has spread widely to more than 20 countries in Asia and has extended its geographic range to the south Pacific region including Australia. JE has become the most important cause of viral encephalitis in the world. Japanese encephalitis viruses (JEV) are divided into five genotypes, based on the nucleotide sequence of the envelope (E) gene. The Muar strain, isolated from patient in Malaya in 1952, is the sole example of genotype V JEV. Here, the XZ0934 strain of JEV was isolated from Culex tritaeniorhynchus, collected in China. The complete nucleotide and amino acid sequence of XZ0934 strain have been determined. The nucleotide divergence ranged from 20.3% to 21.4% and amino acid divergence ranged from 8.4% to 10.0% when compared with the 62 known JEV isolates that belong to genotype I-IV. It reveals low similarity between XZ0934 and genotype I-IV JEVs. Phylogenetic analysis using both complete genome and structural gene nucleotide sequences demonstrates that XZ0934 belongs to genotype V. This, in turn, suggests that genotype V JEV is emerging in JEV endemic areas. Thus, increased surveillance and diagnosis of viral encephalitis caused by genotype V JEV is an issue of great concern to nations in which JEV is endemic.

Emergence of Usutu virus, an African mosquito-borne flavivirus of the Japanese encephalitis virus group, central Europe.

During late summer 2001 in Austria, a series of deaths in several species of birds occurred, similar to the beginning of the West Nile virus (WNV) epidemic in the United States. We necropsied the dead birds and examined them by various methods; pathologic and immunohistologic investigations suggested a WNV infection. Subsequently, the virus was isolated, identified, partially sequenced, and subjected to phylogenetic analysis. The isolates exhibited 97% identity to Usutu virus (USUV), a mosquito-borne Flavivirus of the Japanese encephalitis virus group; USUV has never previously been observed outside Africa nor associated with fatal disease in animals or humans. If established in central Europe, this virus may have considerable effects on avian populations; whether USUV has the potential to cause severe human disease is unknown.

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.