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Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi =... May 2024Objective To prepare monoclonal antibodies against the envelope protein extracellular domain (Eecto) of Zika virus (ZIKV) in mice. Methods A prokaryotic expression...
Objective To prepare monoclonal antibodies against the envelope protein extracellular domain (Eecto) of Zika virus (ZIKV) in mice. Methods A prokaryotic expression plasmid, pET28a-ZIKV-Eecto of ZIKV Eecto, was constructed, transformed into Escherichia coli BL21 and induced by isopropyl β-D-thiogalactoside (IPTG). The recombinant Eecto protein was expressed in the form of inclusion bodies, and purified proteins were obtained through denaturation, renaturation and ultrafiltration. After three rounds of immunization with the Eecto protein, the serum of BALB/c mice was obtained and the titer of polyclonal antibodies in serum was determined. The reactivity of polyclonal antibodies was analyzed with Western blotting and immunofluorescence assay in HEK293T cells expressing the ZIKV prME. Spleen cells from mice with higher antibody titers were prepared and fused with SP2/0 myeloma cells. The hybridoma cells secreting antibodies were screened through the limited dilution method, and the ascites containing antibody were harvested for titer measurement and subclass analysis. The Eecto from the envelope proteins of Japanese encephalitis virus (JEV), Yellow fever virus (YFV), Dengue virus (DENV1-4), and Tick borne encephalitis virus (TBEV) were coated and used to analyze the cross-reactivity of ZIKV monoclonal antibodies by ELISA. Further specificity analysis was conducted on antibodies with high titers and strong specificity. Results The plasmid pET28a-ZIKV-Eecto was successfully constructed. The purified Eecto protein was obtained with good immunogenicity. Four monoclonal antibodies were prepared and screened, namely 1D6, 4F11, 4H7, and 4F8. Among them, 1D6, 4H7, and 4F8 are IgG (K) type antibodies, and 4F11 is an IgM (K) antibody. The ascitic fluid titer of 1D6 was higher than 1:10. Antibodies 1D6 and 4H7 are ZIKV-specific and showed no cross-reactivity with other Flaviviruses. Conclusion The mice monoclonal antibodies against ZIKV-Eecto are produced successfully, which will provide experimental materials for the establishment of ZIKV detection methods and the study of its pathogenesis.
Topics: Animals; Zika Virus; Mice, Inbred BALB C; Antibodies, Monoclonal; Viral Envelope Proteins; Mice; Humans; HEK293 Cells; Female; Antibodies, Viral; Protein Domains; Enzyme-Linked Immunosorbent Assay
PubMed: 38790101
DOI: No ID Found -
The Journal of General Virology May 2024target their replication on membranous structures derived from the ER, where both viral and host proteins play crucial structural and functional roles. Here, we have...
target their replication on membranous structures derived from the ER, where both viral and host proteins play crucial structural and functional roles. Here, we have characterized the involvement of the ER-associated degradation (ERAD) pathway core E3 ligase complex (SEL1L-HRD1) regulator proteins in the replication of Japanese encephalitis virus (JEV). Through high-resolution immunofluorescence imaging of JEV-infected HeLa cells, we observe that the virus replication complexes marked by NS1 strongly colocalize with the ERAD adapter SEL1L, lectin OS9, ER-membrane shuttle factor HERPUD1, E3 ubiquitin ligase HRD1 and rhomboid superfamily member DERLIN1. NS5 positive structures also show strong overlap with SEL1L. While these effectors show significant transcriptional upregulation, their protein levels remain largely stable in infected cells. siRNA mediated depletion of OS9, SEL1L, HERPUD1 and HRD1 significantly inhibit viral RNA replication and titres, with SEL1L depletion showing the maximum attenuation of replication. By performing protein translation arrest experiments, we show that SEL1L, and OS9 are stabilised upon JEV infection. Overall results from this study suggest that these ERAD effector proteins are crucial host-factors for JEV replication.
Topics: Humans; Virus Replication; Encephalitis Virus, Japanese; Endoplasmic Reticulum-Associated Degradation; Ubiquitin-Protein Ligases; HeLa Cells; Membrane Proteins; Host-Pathogen Interactions; Endoplasmic Reticulum; Proteins; Antigens, Differentiation
PubMed: 38787366
DOI: 10.1099/jgv.0.001995 -
Veterinary Sciences May 2024The Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, has a wide host range, extending from pigs and ardeid birds to opportunistic dead-end hosts, such as...
The Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, has a wide host range, extending from pigs and ardeid birds to opportunistic dead-end hosts, such as humans and horses. However, JEV encephalitis infections in aquatic mammals are rare, with only two cases in seals reported to date. Here, we report a lethal case of JEV and co-infection in an aquarium-housed harbor seal in Japan. We isolated JEV from the brain of the dead seal and characterized its phylogeny and pathogenicity in mice. The virus isolate from the seal was classified as genotype GIb, which aligns with recent Japanese human and mosquito isolates as well as other seal viruses detected in China and Korea, and does not exhibit a unique sequence trait distinct from that of human and mosquito strains. We demonstrated that the seal isolate is pathogenic to mice and causes neuronal symptoms. These data suggest that seals should be considered a susceptible dead-end host for circulating JEV in natural settings.
PubMed: 38787188
DOI: 10.3390/vetsci11050215 -
Tropical Medicine and Infectious Disease May 2024Japanese encephalitis virus (JEV) has a positive-sense single-stranded RNA genome and belongs to the genus of the family . Persistent JEV infection was previously shown...
Japanese encephalitis virus (JEV) has a positive-sense single-stranded RNA genome and belongs to the genus of the family . Persistent JEV infection was previously shown in pig blood cells, which act as a natural reservoir of this virus. We aimed to determine the pathogenicity factors involved in persistent JEV infection by analyzing the pathogenicity and genome sequences of a virus isolated from a persistent infection model. We established persistent JEV infections in cells by inoculating mouse fetus primary cell cultures with the Beijing-1 strain of JEV and then performing repeated infected cell passages, harvesting viruses after each passage while monitoring the plaque size over 100 generations. The virus growth rate was compared among Vero, C6/36, and Neuro-2a cells. The pathogenicity was examined in female ICR mice at several ages. Additionally, we determined the whole-genome sequences. The 134th Beijing-1-derived persistent virus (ME134) grew in Vero cells at a similar rate to the parent strain but did not grow well in C6/36 or Neuro-2a cells. No differences were observed in pathogenicity after intracerebral inoculation in mice of different ages, but the survival time was extended in older mice. Mutations in the persistent virus genomes were found across all regions but were mainly focused in the NS3, NS4b, and 3'NCR regions, with a 34-base-pair deletion found in the variable region. The short deletion in the 3'NCR region appeared to be responsible for the reduced pathogenicity and growth efficiency.
PubMed: 38787050
DOI: 10.3390/tropicalmed9050117 -
International Immunopharmacology Jun 2024
Corrigendum to "Intravenous immunoglobulin alleviates Japanese encephalitis virus-induced peripheral neuropathy by inhibiting the ASM/ceramide pathway" [Int. Immunopharmacol. 133 (2024) 112083].
PubMed: 38762402
DOI: 10.1016/j.intimp.2024.112270 -
Veterinaria Italiana Dec 2023Japanese encephalitis virus (JEV) is a zoonotic arbovirus that causes abortion, stillbirth, and congenital defects in pigs, and epidemic encephalitis in humans....
Japanese encephalitis virus (JEV) is a zoonotic arbovirus that causes abortion, stillbirth, and congenital defects in pigs, and epidemic encephalitis in humans. Currently, there is scarcity of information on JEV infection in pigs in Nigeria. Since the Culex tritaeniorhynchus vector of JEV is present in Nigeria and considering recent anecdotal reports of abortions and birth of weak piglets in some pig farms in southwestern Nigeria, there is a need for studies on the presence of the virus and its true burden among pig populations in the country. Serum samples (n=368) obtained from farm-reared pigs in four States of southwestern Nigeria were screened for JEV-specific IgG antibodies using a commercial ELISA kit. An overall JEV seropositivity of 35.1% (95% CI: 30.18 - 39.93%) was obtained, with detectable antibodies in pigs of all age groups, breeds, sex, and locations. Our results suggest natural exposure of these unvaccinated intensively reared pigs to JEV circulating silently in the swine population with significant association of the seropositivity with location (state/community in which the pig farms exist) and breed of the pigs studied. This first report of detection of anti-JEV antibodies in pigs in Nigeria indicates that JEV circulated among these pigs and underscores the need for active surveillance for JEV in humans, pigs, and mosquitoes to provide valuable epidemiological data for the design of effective control strategies against the virus, thus forestalling potential future outbreaks of the infection.
Topics: Animals; Nigeria; Swine; Encephalitis Virus, Japanese; Encephalitis, Japanese; Swine Diseases; Female; Seroepidemiologic Studies; Male; Antibodies, Viral
PubMed: 38756025
DOI: 10.12834/VetIt.3021.20221.2 -
Emerging Microbes & Infections Dec 2024Reverse genetic systems are mainly used to rescue recombinant viral strains in cell culture. These tools have also been used to generate, by inoculating infectious...
Reverse genetic systems are mainly used to rescue recombinant viral strains in cell culture. These tools have also been used to generate, by inoculating infectious clones, viral strains directly in living animals. We previously developed the "Infectious Subgenomic Amplicons" (ISA) method, which enables the rescue of single-stranded positive sense RNA viruses by transfecting overlapping subgenomic DNA fragments. Here, we provide proof-of-concept for direct generation of infectious particles following the inoculation of subgenomic amplicons. First, we rescued a strain of tick-borne encephalitis virus in mice to transpose the ISA method . Subgenomic DNA fragments were amplified using a 3-fragment reverse genetics system and inoculated intramuscularly. Almost all animals were infected when quantities of DNA inoculated were at least 20 µg. We then optimized our procedure in order to increase the animal infection rate. This was achieved by adding an electroporation step and/or using a simplified 2- fragment reverse genetics system. Under optimal conditions, a large majority of animals were infected with doses of 20 ng of DNA. Finally, we demonstrated the versatility of this method by applying it to Japanese encephalitis and Chikungunya viruses. This method provides an efficient strategy for rescue of arboviruses. Furthermore, in the context of the development of DNA-launched live attenuated vaccines, this new approach may facilitate the generation of attenuated strains . It also enables to deliver a substance free of any vector DNA, which seems to be an important criterion for the development of human vaccines.
Topics: Animals; Mice; Encephalitis Viruses, Tick-Borne; Reverse Genetics; Arboviruses; Chikungunya virus; Encephalitis Virus, Japanese; DNA, Viral; Encephalitis, Tick-Borne; Female; Genome, Viral; Chikungunya Fever; Humans
PubMed: 38742328
DOI: 10.1080/22221751.2024.2356140 -
Brain and Nerve = Shinkei Kenkyu No... May 2024Fisher syndrome is recognized as a variant of Guillain-Barré syndrome, encompassing acute onset immune-mediated neuropathies marked by the classical triad of ataxia,... (Review)
Review
Fisher syndrome is recognized as a variant of Guillain-Barré syndrome, encompassing acute onset immune-mediated neuropathies marked by the classical triad of ataxia, areflexia, and ophthalmoplegia. Generally, Fisher syndrome follows a self-limited course with a good prognosis. Ophthalmoplegia, typically bilateral, progresses to complete external ophthalmoplegia within 1-2 weeks. Ataxia, often very severe, may cause an inability to walk without support despite normal strength. Fisher syndrome is also frequently concomitant with additional clinical features, including ptosis, internal ophthalmoplegia, facial nerve palsy, sensory deficits, and bulbar palsy. The confirmation of an antecedent infection is often established. Among the ganglioside antibodies, anti-GQ1b antibodies exhibit positivity in over 80% of patients. The syndrome manifests in three distinct types: a partial subtype exhibiting only a subset of the triad symptoms, Bickerstaff's brainstem encephalitis marked by impaired consciousness and pyramidal tract signs, and an overlapping subtype with Guillain-Barré syndrome, characterized by weakness in the extremities.
Topics: Humans; Miller Fisher Syndrome; Gangliosides; Prognosis; Guillain-Barre Syndrome
PubMed: 38741489
DOI: 10.11477/mf.1416202636 -
Parasites & Vectors May 2024Japanese encephalitis virus (JEV) is an emerging mosquito-borne Orthoflavivirus that poses a significant public health risk in many temperate and tropical regions in...
BACKGROUND
Japanese encephalitis virus (JEV) is an emerging mosquito-borne Orthoflavivirus that poses a significant public health risk in many temperate and tropical regions in Asia. Since the climate in some endemic countries is similar to temperate climates observed in Europe, understanding the role of specific mosquito species in the transmission of JEV is essential for predicting and effectively controlling the potential for the introduction and establishment of JEV in Europe.
METHODS
This study aimed to investigate the vector competence of colonized Culex pipiens biotype molestus mosquitoes for JEV. The mosquitoes were initially collected from the field in southern Sweden. The mosquitoes were offered a blood meal containing the Nakayama strain of JEV (genotype III), and infection rates, dissemination rates, and transmission rates were evaluated at 14, 21, and 28 days post-feeding.
RESULTS
The study revealed that colonized Swedish Cx. pipiens are susceptible to JEV infection, with a stable infection rate of around 10% at all timepoints. However, the virus was only detected in the legs of one mosquito at 21 days post-feeding, and no mosquito saliva contained JEV.
CONCLUSIONS
Overall, this research shows that Swedish Cx. pipiens can become infected with JEV, and emphasizes the importance of further understanding of the thresholds and barriers for JEV dissemination in mosquitoes.
Topics: Animals; Culex; Encephalitis Virus, Japanese; Sweden; Mosquito Vectors; Encephalitis, Japanese; Female; Saliva; Humans
PubMed: 38741172
DOI: 10.1186/s13071-024-06269-7 -
Acta Tropica Jul 2024Japanese encephalitis (JE) is a mosquito-borne disease with a spatial distribution that is linked to geo-environmental factors. The spatial distribution of JE cases and...
Japanese encephalitis (JE) is a mosquito-borne disease with a spatial distribution that is linked to geo-environmental factors. The spatial distribution of JE cases and correlated geo-environmental factors were investigated in two critical counties in southern and northern China. Based on maps, enhanced thematic mapper (ETM) remote sensing datasets from Landsat and spatial datasets of JE cases, spatial distribution and spatial cluster analyses of JE cases at the village scale were performed by using the standard deviational ellipse and Ripleys K-function. Global and regional spatial cluster analyses of JE cases were also performed by using Moran's index. Regression analysis was used to analyze the relationships between geo-environmental characteristics and the risk of JE cases. At the study sites, the JE cases were not spatially clustered at the village or district (global) level, whereas there was a spatial cluster at the district (local) level. Diversity-related features for JE patients at the district and village levels were detected at two sites. In the southern counties, the distance of a village from a road was related to the village-level JE risk (OR: 0.530, 95 CI: 0.297-0.947, P = 0.032), and the number of township-level JE cases was linked to the distance of the district center from the road (R =-0.467, P = 0.025) and road length (R = 0.516, P = 0.012) in the administrative area. In northern China, the modified normalized difference water index (MNDWI) in the 5 km buffer around the village was related to village-level JE risk (OR: 0.702, 95% CI: 0.524-0.940, P = 0.018), and the number of township-level JE cases was related to the MNDWI in the administrative region (R =-0.522, P = 0.038). This study elucidates the spatial distribution patterns of JE cases and risk, as well as correlated geo-environmental features, at various spatial scales. This study will significantly assist the JE control efforts of the local Centers for Disease Control and Prevention (CDC), which is the base-level CDC, particularly concerning the allocation of medicine and medical staff, the development of immunological plans, and the allocation of pesticides and other control measures for the mosquito vectors of JE.
Topics: China; Humans; Encephalitis, Japanese; Spatial Analysis; Cluster Analysis; Female; Male; Child; Adult; Adolescent; Middle Aged; Young Adult; Child, Preschool; Infant; Aged; Environment; Topography, Medical
PubMed: 38729328
DOI: 10.1016/j.actatropica.2024.107246