Prophylactic Administration of Gut Microbiome Metabolites Abrogated Microglial Activation and Subsequent Neuroinflammation in an Experimental Model of Japanese Encephalitis.

Short-chain fatty acids (SCFAs) are gut microbial metabolic derivatives produced during the fermentation of ingested complex carbohydrates. SCFAs have been widely regarded to have a potent anti-inflammatory and neuro-protective role and have implications in several disease conditions, such as, inflammatory bowel disease, type-2 diabetes, and neurodegenerative disorders. Japanese encephalitis virus (JEV), a neurotropic flavivirus, is associated with life threatening neuro-inflammation and neurological sequelae in infected hosts. In this study, we hypothesize that SCFAs have potential in mitigating JEV pathogenesis. Postnatal day 10 BALB/c mice were intraperitoneally injected with either a SCFA mixture (acetate, propionate, and butyrate) or PBS for a period of 7 days, followed by JEV infection. All mice were observed for onset and progression of symptoms. The brain tissue was collected upon reaching terminal illness for further analysis. SCFA-supplemented JEV-infected mice (SCFA + JEV) showed a delayed onset of symptoms, lower hindlimb clasping score, and decreased weight loss and increased survival by 3 days (p < 0.0001) upon infection as opposed to the PBS-treated JEV-infected animals (JEV). Significant downregulation of inflammatory cytokines TNF-α, MCP-1, IL-6, and IFN-Υ in the SCFA + JEV group relative to the JEV-infected control group was observed. Inflammatory mediators, phospho-NF-kB (P-NF-kB) and iba1, showed 2.08 ± 0.1 and 3.132 ± 0.43-fold upregulation in JEV versus 1.19 ± 0.11 and 1.31 ± 0.11-fold in the SCFA + JEV group, respectively. Tissue section analysis exhibited reduced glial activation (JEV group─42 ± 2.15 microglia/ROI; SCFA + JEV group─27.07 ± 1.8 microglia/ROI) in animals that received SCFA supplementation prior to infection as seen from the astrocytic and microglial morphometric analysis. Caspase-3 immunoblotting showed 4.08 ± 1.3-fold upregulation in JEV as compared to 1.03 ± 0.14-fold in the SCFA + JEV group and TUNEL assay showed a reduced cellular death post-JEV infection (JEV-6.4 ± 1.5 cells/ROI and SCFA + JEV-3.7 ± 0.73 cells/ROI). Our study critically contributes to the increasing evidence in support of SCFAs as an anti-inflammatory and neuro-protective agent, we further expand its scope as a potential supplementary intervention in JEV-mediated neuroinflammation.

Novel Flavivirus Attenuation Markers Identified in the Envelope Protein of Alfuy Virus.

Alfuy (ALFV) is an attenuated flavivirus related to the Murray Valley encephalitis virus (MVEV). We previously identified markers of attenuation in the envelope (E) protein of the prototype strain (ALFV3929), including the hinge region (E273-277) and lack of glycosylation at E154-156. To further determine the mechanisms of attenuation we assessed ALFV3929 binding to glycosaminoglycans (GAG), a known mechanism of flaviviruses attenuation. Indeed, ALFV3929 exhibited reduced binding to GAG-rich cells in the presence of heparin; however, low-passage ALFV isolates were relatively unaffected. Sequence comparisons between ALFV strains and structural modelling incriminated a positively-charged residue (K327) in ALFV3929 as a GAG-binding motif. Substitution of this residue to the corresponding uncharged residue in MVEV (L), using a previously described chimeric virus containing the prM & E genes of ALFV3929 in the backbone of MVEV (MVEV/ALFV-prME), confirmed a role for K327 in enhanced GAG binding. When the wild type residues at E327, E273-277 and E154-156 of ALFV3929 were replaced with the corresponding residues from virulent MVEV, it revealed each motif contributed to attenuation of ALFV3929, with the E327/E273-277 combination most dominant. These data demonstrate that attenuation of ALFV3929 is multifactorial and provide new insights for the rational design of attenuated flavivirus vaccines.

Determination of system level alterations in host transcriptome due to Zika virus (ZIKV) Infection in retinal pigment epithelium.

Previously, we reported that Zika virus (ZIKV) causes ocular complications such as chorioretinal atrophy, by infecting cells lining the blood-retinal barrier, including the retinal pigment epithelium (RPE). To understand the molecular basis of ZIKV-induced retinal pathology, we performed a meta-analysis of transcriptome profiles of ZIKV-infected human primary RPE and other cell types infected with either ZIKV or other related flaviviruses (Japanese encephalitis, West Nile, and Dengue). This led to identification of a unique ZIKV infection signature comprising 43 genes (35 upregulated and 8 downregulated). The major biological processes perturbed include SH3/SH2 adaptor activity, lipid and ceramide metabolism, and embryonic organ development. Further, a comparative analysis of some differentially regulated genes (ABCG1, SH2B3, SIX4, and TNFSF13B) revealed that ZIKV induced their expression relatively more than dengue virus did in RPE. Importantly, the pharmacological inhibition of ABCG1, a membrane transporter of cholesterol, resulted in reduced ZIKV infectivity. Interestingly, the ZIKV infection signature revealed the downregulation of ALDH5A1 and CHML, genes implicated in neurological (cognitive impairment, expressive language deficit, and mild ataxia) and ophthalmic (choroideremia) disorders, respectively. Collectively, our study revealed that ZIKV induces differential gene expression in RPE cells, and the identified genes/pathways (e.g., ABCG1) could potentially contribute to ZIKV-associated ocular pathologies.

Limited susceptibility of mice to Usutu virus (USUV) infection and induction of flavivirus cross-protective immunity.

Flaviviruses are RNA viruses that constitute a worrisome threat to global human and animal health. In Europe, West Nile virus (WNV) outbreaks have dramatically increased in number and severity in recent years, with dozens of human and horse deaths and a high avian mortality across the continent. Besides WNV, the only clinically relevant mosquito-borne flavivirus detected so far in Europe has been the Usutu virus (USUV), which after being reported for the first time in Austria in 2001, quickly spread across Europe, causing a considerable number of bird deaths and neurological disorders in a few immunocompromised patients. Even though USUV infects multiple avian species that develop antibodies, there is little information about USUV susceptibility, pathogenicity and cross-reactive immunity. Here, the susceptibility of suckling and adult mice to USUV infection and the induction of cross-protective immunity against WNV challenge have been addressed.

Viral Infection of the Central Nervous System and Neuroinflammation Precede Blood-Brain Barrier Disruption during Japanese Encephalitis Virus Infection.

UNLABELLED: Japanese encephalitis is an acute zoonotic, mosquito-borne disease caused by Japanese encephalitis virus (JEV). Japanese encephalitis is characterized by extensive inflammation in the central nervous system (CNS) and disruption of the blood-brain barrier (BBB). However, the pathogenic mechanisms contributing to the BBB disruption are not known. Here, using a mouse model of intravenous JEV infection, we show that virus titers increased exponentially in the brain from 2 to 5 days postinfection. This was accompanied by an early, dramatic increase in the level of inflammatory cytokines and chemokines in the brain. Enhancement of BBB permeability, however, was not observed until day 4, suggesting that viral entry and the onset of inflammation in the CNS occurred prior to BBB damage. In vitro studies revealed that direct infection with JEV could not induce changes in the permeability of brain microvascular endothelial cell monolayers. However, brain extracts derived from symptomatic JEV-infected mice, but not from mock-infected mice, induced significant permeability of the endothelial monolayer. Consistent with a role for inflammatory mediators in BBB disruption, the administration of gamma interferon-neutralizing antibody ameliorated the enhancement of BBB permeability in JEV-infected mice. Taken together, our data suggest that JEV enters the CNS, propagates in neurons, and induces the production of inflammatory cytokines and chemokines, which result in the disruption of the BBB. IMPORTANCE: Japanese encephalitis (JE) is the leading cause of viral encephalitis in Asia, resulting in 70,000 cases each year, in which approximately 20 to 30% of cases are fatal, and a high proportion of patients survive with serious neurological and psychiatric sequelae. Pathologically, JEV infection causes an acute encephalopathy accompanied by BBB dysfunction; however, the mechanism is not clear. Thus, understanding the mechanisms of BBB disruption in JEV infection is important. Our data demonstrate that JEV gains entry into the CNS prior to BBB disruption. Furthermore, it is not JEV infection per se, but the inflammatory cytokines/chemokines induced by JEV infection that inhibit the expression of TJ proteins and ultimately result in the enhancement of BBB permeability. Neutralization of gamma interferon (IFN-γ) ameliorated the enhancement of BBB permeability in JEV-infected mice, suggesting that IFN-γ could be a potential therapeutic target. This study would lead to identification of potential therapeutic avenues for the treatment of JEV infection.

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.

First cases of human Usutu virus neuroinvasive infection in Croatia, August-September 2013: clinical and laboratory features.

Few reports of human Usutu virus (USUV) infection have been reported to date. We describe the first three patients with USUV neuroinvasive infection in Zagreb and its surroundings from 30 August to 7 September 2013 during a West Nile virus (WNV) outbreak. Patients were aged 29, 56, and 61 years. The two older patients had several comorbidities (arterial hypertension, hyperlipidemia, and diabetes mellitus). All patients presented with meningitis and meningoencephalitis closely resembling WNV neuroinvasive disease. The main clinical features in all patients were headache, fever, nuchal rigidity, hand tremor, and hyperreflexia. Neuroimaging studies were normal and electroencephalography (EEG) revealed diffusely slow activity. The 29 years old, a previously healthy female patient, was deeply somnolent and disoriented for 4 days. Her recovery was slow and even 10 weeks after disease onset, she had memory and speech-fluency difficulties. The other two patients recovered promptly. USUV IgG antibodies were detected in all patients by ELISA with seroconversion documented in two of them. Titers of USUV-neutralizing antibodies were 10, 80, and 10, respectively. Because USUV and WNV share many clinical characteristics, USUV infection could be misdiagnosed as WNV. Testing for USUV should be considered in all suspected cases of meningoencephalitis, especially in areas where both viruses cocirculate.

The West Nile virus-like flavivirus Koutango is highly virulent in mice due to delayed viral clearance and the induction of a poor neutralizing antibody response.

UNLABELLED: The mosquito-borne West Nile virus (WNV) is responsible for outbreaks of viral encephalitis in humans, horses, and birds, with particularly virulent strains causing recent outbreaks of disease in eastern Europe, the Middle East, North America, and Australia. Previous studies have phylogenetically separated WNV strains into two main genetic lineages (I and II) containing virulent strains associated with neurological disease. Several WNV-like strains clustering outside these lineages have been identified and form an additional five proposed lineages. However, little is known about whether these strains have the potential to induce disease. In a comparative analysis with the highly virulent lineage I American strain (WNVNY99), the low-pathogenicity lineage II strain (B956), a benign Australian strain, Kunjin (WNVKUN), the African WNV-like Koutango virus (WNVKOU), and a WNV-like isolate from Sarawak, Malaysia (WNVSarawak), were assessed for neuroinvasive properties in a murine model and for their replication kinetics in vitro. While WNVNY99 replicated to the highest levels in vitro, in vivo mouse challenge revealed that WNVKOU was more virulent, with a shorter time to onset of neurological disease and higher morbidity. Histological analysis of WNVKOU- and WNVNY99-infected brain and spinal cords demonstrated more prominent meningoencephalitis and the presence of viral antigen in WNVKOU-infected mice. Enhanced virulence of WNVKOU also was associated with poor viral clearance in the periphery (sera and spleen), a skewed innate immune response, and poor neutralizing antibody development. These data demonstrate, for the first time, potent neuroinvasive and neurovirulent properties of a WNV-like virus outside lineages I and II. IMPORTANCE: In this study, we characterized the in vitro and in vivo properties of previously uncharacterized West Nile virus strains and West Nile-like viruses. We identified a West Nile-like virus, Koutango virus (WNVKOU), that was more virulent than a known virulent lineage I virus, WNVNY99. The enhanced virulence of WNVKOU was associated with poor viral clearance and the induction of a poor neutralizing antibody response. These findings provide new insights into the pathogenesis of West Nile virus.

Vector competence of Culex neavei (Diptera: Culicidae) for Usutu virus.

Usutu virus (USUV), a flavivirus belonging to the Japanese encephalitis serocomplex, was isolated for the first time from a Culex neavei mosquito in 1959 in South Africa. Despite multiple isolations of USUV from Cx. neavei in Africa, its vector competence remains unproven. Therefore, we infected Cx. neavei orally with the USUV reference strain and used reverse transcription-polymerase chain reaction and an indirect immunofluorescence assay to detect virus in bodies, legs, wings, and saliva of mosquitoes. We demonstrated the susceptibility of Cx. neavei mosquitoes for the USUV reference strain, its potential to be transmitted, and infection, dissemination, and transmission rates of 90.9%, 40.0%, and 81.3%, respectively. Also, we showed that infection rates are dependent on the virus titer of the blood meal. Given the bionomics of Cx. neavei, its role as enzootic vector for USUV in Africa in a mosquito-bird transmission cycle or as bridge vector for USUV transmission to humans is discussed.

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.

A single mutation in the Japanese encephalitis virus E protein (S123R) increases its growth rate in mouse neuroblastoma cells and its pathogenicity in mice.

We previously reported that the Japanese encephalitis virus (JEV) strain Mie/41/2002 has weak pathogenicity compared with the laboratory strain Beijing-1. To identify the determinants of its growth nature and pathogenicity, we produced intertypic viruses, rJEV(EB1-M41), rJEV(nEB1-M41) and rJEV(cEB1-M41), which contained the entire, the N-terminal, and the C-terminal half, respectively, of the Beijing-1 E region in the Mie/41/2002 background. The growth of rJEV(EB1-M41) in mouse neuroblastoma N18 cells and virulence in mice were similar to those of Beijing-1. rJEV(nEB1-M41) propagated in N18 cells to the same extent as did Beijing-1. Furthermore, we produced mutant viruses with single amino acid substitutions in the N-terminal half of the Mie/41/2002 E region. A Ser-123-Arg mutation in the Mie/41/2002 E protein exhibited significantly increased growth rate in N18 cells and virulence in mice. These results indicate that the position 123 in the E protein is responsible for determining the growth properties and pathogenicity of JEV.

Sub-fragments of the envelope gene are highly protective against the Japanese encephalitis virus lethal infection in DNA priming--protein boosting immunization strategies.

The envelope (E) gene of Japanese encephalitis virus (JEV) plays a major protective role against JEV infection. In order to locate the part of E gene that is responsible for this protection, an N-terminal fragment EA (nucleotide number 933-1877 bp of JEV genome) and a C-terminal fragment EB (nucleotide number 1851-2330 bp of JEV genome) from E gene were prepared. Both of these fragments were used in the form of recombinant proteins (rEA and rEB) and plasmid DNA (pEA, pM15EA and pEB) for immunizations. Recombinant EA protein (rEA) was previously found to be non-protective because it was expressed in an insoluble form. Plasmid EA (pEA) was also found to be non-protective unless it is preceded by a 15 mer signal peptide derived from the very C-terminal of the membrane gene (M) of JEV to form pM15EA plasmid indicating the importance of the signal peptide in the expression of EA immunogenicity. Although pM15EA and pEB are both immunogenic and protective against JEV lethal infection, the protection by both fragments however is not optimal. Even when pM15EA and pEB were used together for immunization, maximum protection as those induced by control vaccine was not achieved. However, if individual fragments (EA or EB) were used in a DNA priming-protein boosting or protein priming-DNA boosting strategy, high levels of protection were achieved by both fragments. This was especially true for EA fragment where the level of protection against JEV lethal infection was equal to that induced by commercially available vaccine alone. The protection correlated very well with the neutralizing antibody titers and the T helper cell involved in this process in mainly the Th1 type.

Zooprophylaxis or zoopotentiation: the outcome of introducing animals on vector transmission is highly dependent on the mosquito mortality while searching.

BACKGROUND: Zooprophylaxis, the diversion of disease carrying insects from humans to animals, may reduce transmission of diseases such as malaria. However, as the number of animals increases, improved availability of blood meals may increase mosquito survival, thereby countering the impact of diverting feeds. METHODS: Computer simulation was used to examine the effects of animals on the transmission of human diseases by mosquitoes. Three scenarios were modelled: (1) endemic transmission, where the animals cannot be infected, eg. malaria; (2) epidemic transmission, where the animals cannot be infected but humans remain susceptible, e.g. malaria; (3) epidemic disease, where both humans and animals can be infected, but develop sterile immunity, eg. Japanese encephalitis B. For each, the passive impact of animals as well as the use of animals as bait to attract mosquitoes to insecticide was examined. The computer programmes are available from the author. A teaching model accompanies this article. RESULTS: For endemic and epidemic malaria with significant searching-associated vector mortality, changing animal numbers and accessibility had little impact. Changing the accessibility of the humans had a much greater effect. For diseases with an animal amplification cycle, the most critical factor was the proximity of the animals to the mosquito breeding sites. CONCLUSION: Estimates of searching-associated vector mortality are essential before the effects of changing animal husbandry practices can be predicted. With realistic values of searching-associated vector mortality rates, zooprophylaxis may be ineffective. However, use of animals as bait to attract mosquitoes to insecticide is predicted to be a promising strategy.

A new strategy in design of +RNA virus infectious clones enabling their stable propagation in E. coli.

Infectious clone methodology is a valuable tool of modern experimental virology. However, its use is often constrained by the instability of infectious clone constructs during propagation in E. coli. To circumvent this problem, we have devised a strategy that could be suitable for design of +RNA virus molecular clones in general. An infectious clone is assembled as "infectious DNA," and expression of problem regions present in the viral cDNA is prevented during propagation in E. coli by insertion of short introns. To demonstrate the feasibility of this approach, a highly unstable Japanese encephalitis flavivirus infectious clone has been successfully converted into a remarkably stable infectious DNA construct with the specific infectivity of 10(6) pfu/microg in cell culture. The proposed strategy may be useful in the design of self-amplifying gene therapy vectors and development of new immunization methodologies, and could facilitate creation of molecular repositories of existing viral vaccines.

Hepatitis C virus nonstructural protein NS4B transforms NIH3T3 cells in cooperation with the Ha-ras oncogene.

Although the mechanism of carcinogenesis by hepatitis C virus (HCV) is not clearly known, core and NS3P protein have been shown to form tumors in specific cell lines. In this study, on the basis of the fact that the core and NS4B proteins of Kunjin virus translocate into the nucleus, we were prompted to investigate whether the HCV nonstructural protein NS4B has any function in tumor formation. First, we examined the location of the NS4B protein of HCV in transfected cells and then its oncogenic activity by transfection of NIH3T3 cells with the NS4B gene in the presence or absence of the Ha-ras gene. The NS4B protein was present only in the cytoplasm, particularly in the perinuclear region, different from the case of the Kunjun virus. The cells expressing HCV NS4B cooperatively with the Ha-ras gene showed loss of contact inhibition, morphological alterations, and anchorage-independent growth. These biological activities were confirmed by the transcription activation of the reporter gene from the AP1 promoter, by the NS4B protein in association with Ha-ras. Our results demonstrated that HCV NS4B protein in association with the Ha-ras gene played an important role in the malignant transformation of cells by HCV.

Loss of dimerisation of the nonstructural protein NS1 of Kunjin virus delays viral replication and reduces virulence in mice, but still allows secretion of NS1.

The flavivirus nonstructural protein NS1 has been implicated in viral RNA replication, although its precise role has not been identified. In its native state NS1 exists as a heat labile homodimer that is thought to be required for NS1 function and secretion. However, we have recently identified a cDNA clone of KUN virus (FLSD) that replicates efficiently in cell culture but produces and secretes NS1 in monomeric form. Sequence analysis of the NS1 gene in FLSD revealed a single amino acid substitution (proline(250) to leucine) when compared with the parental KUN virus. When site-directed mutagenesis was used to substitute leucine(250) with proline in FLSD to produce the clone 250pro, dimerisation was fully restored. Furthermore, time course experiments revealed that 250pro replicated in Vero cells significantly faster than FLSD and produced 100-fold more infectious virus early (12-24 h) in infection. This correlated with our observations that FLSD required approximately 10-fold more infectious virus than 250pro to produce disease in weanling mice after intraperitoneal inoculation. Taken together our results indicate that mutation from proline to leucine at residue 250 in KUN NS1 ablates dimer formation, slows virus replication, and reduces virulence in mice.

Completion of Kunjin virus RNA sequence and recovery of an infectious RNA transcribed from stably cloned full-length cDNA.

Completion of the Kunjin virus (KUN) RNA sequence showed that it is the longest flavivirus sequence reported (11,022 bases), commencing with a 5' noncoding region of 96 bases. The 3' noncoding sequence of 624 nucleotides included a unique insertion sequence of 46 bases adjacent to the stop codon, but otherwise it had properties similar to those of RNAs of closely related flaviviruses. A full-length KUN cDNA clone which could be stably propagated in Escherichia coli DH5 alpha was constructed; SP6 polymerase RNA transcripts from amplified cDNA were infectious when transfected into BHK-21 cells. A mutational change abolishing the BamHI restriction site at position 4049, leading to a conservative amino acid change of Arg-175 to Lys in the NS2A protein, was introduced into the cDNA during construction and was retained in the recovered virus. Extra terminal nucleotides introduced during cloning of the cDNA were shown to be present in the in vitro RNA transcripts but absent in the RNA of recovered virus. Although recovered virus differed from the parental KUN by a smaller plaque phenotype and delayed growth rate in BHK-21 cells and mice, it was very similar as assessed by several other criteria, such as peak titer during growth in cells, infectivity titer in cells and in mice, rate of adsorption and penetration in cells, replication at 39 degrees C, and neurovirulence after intraperitoneal injection in mice. The KUN stably cloned cDNA will provide a useful basis for future studies in defining and characterizing functional roles of all the gene products.