P3 Strain Research Articles

Increased secretion of bacterial pyomelanin caused by light accelerates corrosion of low alloy steel

Microorganisms in the waterline zone can secrete pigments to avoid damage caused by ultraviolet radiation, some of which have corrosive effects. In this work, we found that the secretion of pyomelanin by P3 strain of Pseudoalteromonas lipolytica significantly increases under strong lighting conditions, accelerating the corrosion of the material. Molecular mechanisms indicate that strong light, as a stressful environmental factor, enhances the expression of melanin secretion-related genes to prevent bacteria from being damaged by ultraviolet radiation. Therefore, this work proposes a new corrosion mechanism in the waterline zone, pigment-producing microorganisms are also involved in the waterline corrosion process.

Japanese encephalitis virus inhibits superinfection of Zika virus in cells by the NS2B protein.

Superinfection exclusion (SIE) is a phenomenon in which a preexisting infection prevents a secondary infection. SIE has been described for several flaviviruses, such as West Nile virus vs Nhumirim virus and Dengue virus vs yellow fever virus. Zika virus (ZIKV) is an emerging flavivirus posing threats to human health. The SIE between ZIKV and Japanese encephalitis virus (JEV) is investigated in this study. Our results demonstrate for the first time that JEV inhibits ZIKV infection in both mammalian and mosquito cells, whether co-infects or subsequently infects after ZIKV. The exclusion effect happens at the stage of ZIKV RNA replication. Further studies show that the expression of JEV NS2B protein is sufficient to inhibit the replication of ZIKV, and the outer membrane region of NS2B (46-103 aa) is responsible for this SIE. JEV infection and NS2B expression also inhibit the infection of the vesicular stomatitis virus. In summary, our study characterized a SIE caused by JEV NS2B. This may have potential applications in the prevention and treatment of ZIKV or other RNA viruses.IMPORTANCEThe reemerged Zika virus (ZIKV) has caused severe symptoms in humans and poses a continuous threat to public health. New vaccines or antiviral agents need to be developed to cope with possible future pandemics. In this study, we found that infection of Japanese encephalitis virus (JEV) or expression of NS2B protein well inhibited the replication of ZIKV. It is worth noting that both the P3 strain and vaccine strain SA14-14-2 of JEV exhibited significant inhibitory effects on ZIKV. Additionally, the JEV NS2B protein also had an inhibitory effect on vesicular stomatitis virus infection, suggesting that it may be a broad-spectrum antiviral factor. These findings provide a new way of thinking about the prevention and treatment of ZIKV.

High-throughput sequencing-based Detection of Japanese encephalitis virus and its effect on micro ribonucleic acid

it was to explore the mechanism of Japanese encephalitis virus (JEV) and micro ribonucleic acid (miRNA) under high-throughput sequencing. 20 experimental mice, with good growth status and no disease infection, were selected. The cells used in the experiment included mouse microglial cell line (BV2), mouse neuroblastoma cell line (NA), and mouse brain endothelial cell line (bEnd.3). JEV titration was performed with JEV-infected cells, ribonucleic acid (RNA) in the cells was extracted, and finally the miRNA high-throughput sequencing data was analyzed. Agarose gel electrophoresis showed that the 28S and 18S electrophoresis bands were bright. Among the miRNAs detected in mouse brain tissues, 2986 were down-regulated and 1251 were up-regulated. Among miRNAs detected in NA cells, 4238 the decreasing expression and 2356 were expressed increasingly. In reducing miRNA expression, 1 multiplicity of infection (MOI) of P3 strain infection was more significant than 0.1 MOI. 10 miRNAs with significantly decreasing expression were miR-466d-3p, miR-381–3p, miR-540–3p, miR-466a-3p, miR-467a-3p, miR-574–5p, miR-199a-5p, miR-467a-5p, miR-674–5p, and miR-376b-3p. These were all obviously down-regulated in JEV-infected BV2, NA, and bEnd.3 neurons. High-throughput sequencing of JEV-infected mouse brain tissues and mouse neuronal cells found that JEV infection led to down-regulation of overall miRNA expression in host cells.

Exploring Diversity of Bacterial Spot Associated Xanthomonas Population of Pepper in Southwest Florida.

Bacterial spot caused by Xanthomonas spp. is a significant disease that challenges pepper growers worldwide and is particularly severe in a hot and humid environment. Understanding the pathogen's population biology is critical for sustainable disease management. The goal of this study was to characterize the species, race, and bactericide sensitivity of bacterial spot-associated Xanthomonas collected from pepper in Florida. A survey of pepper production fields in southwest Florida between 2019 and 2021-covering two counties, eight farms, and two transplant facilities-resulted in the isolation of 542 Xanthomonas euvesicatoria and 35 Xanthomonas perforans strains. Four races were identified on pepper, of which most strains were race P1 (42%), race P6 (26%), race P3 (24%), and less common was race P4 (8%). All X. perforans strains were characterized as race P1 and showed a compatible reaction on tomato. Sixty-two and 96% of strains were sensitive to copper sulfate and streptomycin, respectively. One farm that did not use copper to manage the disease contained only copper-sensitive strains and was the only farm with race P3 strains. Strains were assayed for starch hydrolysis activity of which a third of X. euvesicatoria strains were strongly amylolytic, a characteristic not typically observed in X. euvesicatoria. All X. perforans strains produced bacteriocins against X. euvesicatoria in vitro. The Xanthomonas population causing bacterial spot on pepper in southwest Florida is diverse and dynamic; thus, regular monitoring provides pertinent information to plant breeders and growers for designing disease management strategies.

Method validation for quantitative analyzing aflatoxin productivity in Aspergillus sp. isolated from soybean paste

Non-aflatoxigenic Aspergillus oryzae and aflatoxigenic A. flavus cannot be clearly identified by partial sequencing of the internal transcribed spacer (ITS) and 18S ribosomal ribonucleic acid (18S rRNA) regions. This study aimed to compare the accuracy among three aflatoxin detection methods using ultra-performance liquid chromatography (UPLC), high-performance liquid chromatography (HPLC), and an enzyme-linked immunosorbent assay (ELISA) kit and to select the non-aflatoxigenic Aspergillus sp. isolated from soybean paste. All analytical methods were suitable according to the international standards of Codex Alimentarius FAO-WHO (CODEX) or the Ministry of Food and Drug Safety (MFDS). UPLC exhibited the best of limit of detection (LOD) and limit of quantification (LOQ). Based on UPLC, HPLC, and the ELISA kit assay, the P5 and P7 strains isolated from soybean paste had 1,663.49, 1,468.12, and ⟩20 μg/kg and 1,470.08, 1,056.73, and ⟩20 μg/kg, respectively, detected and re-identified as A. flavus. In contrast, the P3 and P4 strains (A. oryzae), which were detected below the MFDS standards in all assays, were confirmed as non-aflatoxigenic fungi. Among the methods evaluated for quantitative analysis of aflatoxin, UPLC and HPLC are superior in terms of accuracy, and the ELISA kit rapidly detects low concentrations of aflatoxin. Furthermore, this study demonstrates that any Aspergillus sp. isolated for use as a fermentation starter should be analyzed for potential aflatoxin production using UPLC and HPLC for accurate quantitative analysis or ELISA for the rapid detection of low-level concentrations of aflatoxin.

Protective Immune Responses Induced by an mRNA-LNP Vaccine Encoding prM-E Proteins against Japanese Encephalitis Virus Infection.

Japanese encephalitis virus (JEV) is an important zoonotic pathogen, which causes central nervous system symptoms in humans and reproductive disorders in swine. It has led to severe impacts on human health and the swine industry; however, there is no medicine available for treating yet. Therefore, vaccination is the best preventive measure for this disease. In the study, a modified mRNA vaccine expressing the prM and E proteins of the JEV P3 strain was manufactured, and a mouse model was used to assess its efficacy. The mRNA encoding prM and E proteins showed a high level of protein expression in vitro and were encapsulated into a lipid nanoparticle (LNP). Effective neutralizing antibodies and CD8+ T-lymphocytes-mediated immune responses were observed in vaccinated mice. Furthermore, the modified mRNA can protect mice from a lethal challenge with JEV and reduce neuroinflammation caused by JEV. This study provides a new option for the JE vaccine and lays a foundation for the subsequent development of a more efficient and safer JEV mRNA vaccine.

Precise localization and dynamic distribution of Japanese encephalitis virus in the rain nuclei of infected mice.

Japanese encephalitis virus (JEV) is a pathogen that causes severe vector-borne zoonotic diseases, thereby posing a serious threat to human health. Although JEV is potentially neurotropic, its pathogenesis and distribution in the host have not been fully elucidated. In this study, an infected mouse model was established using a highly virulent P3 strain of JEV. Immunohistochemistry and in situ hybridization, combined with anatomical imaging of the mouse brain, were used to dynamically localize the virus and construct three-dimensional (3D) images. Consequently, onset of mild clinical signs occurred in some mice at 3.5 d post JEV infection, while most mice displayed typical neurological signs at 6 d post-infection (dpi). Moreover, brain pathology revealed typical changes associated with non-suppurative encephalitis, which lasted up to 8 d. The earliest detection of viral antigen was achieved at 3 dpi in the thalamus and medulla oblongata. At 6 dpi, the positive viral antigen signals were mainly distributed in the cerebral cortex, olfactory area, basal ganglia, thalamus, and brainstem regions in mice. At 8 dpi, the antigen signals gradually decreased, and the localization of JEV tended to concentrate in the cerebrum and thalamus, while no viral antigen was detected in the brain at 21 dpi. In this model, the viral antigen was first expressed in the reticular thalamic nucleus (Rt), and the virus content is relatively stable. The expression of the viral antigen in the hippocampal CA2 region, the anterior olfactory nucleus, and the deep mesencephalic nucleus was high and persistent. The 3D images showed that viral signals were mostly concentrated in the parietal cortex, occipital lobe, and hippocampus, near the mid-sagittal plane. In the early stages of infection in mice, a large number of viral antigens were detected in denatured and necrotic neurons, suggesting that JEV directly causes neuronal damage. From the time of its entry, JEV is widely distributed in the central nervous system thereby causing extensive damage.

Complete protection for mice conferred by a DNA vaccine based on the Japanese encephalitis virus P3 strain used to prepare the inactivated vaccine in China

BackgroundThe incidence of Japanese encephalitis (JE) has been dramatically reduced in China after sufficient vaccine coverage. The live-attenuated Japanese encephalitis virus (JEV) vaccine SA14–14-2 is believed to have strongly contribute to this decrease. Another vaccine that seems to have decreased in importance is an inactivated vaccine based on the JEV P3 strain, which is considered to be modifiable, such as being transformed into a DNA vaccine to improve its immunogenicity.MethodsIn this study, the protective efficacy induced by the Japanese encephalitis DNA vaccine candidate pV-JP3ME encoding the premembrane (prM) and envelope (E) proteins of the P3 strain was assessed in BALB/c mice. The prM/E genes of the JEV P3 strain were subcloned into the vector pVAX1 (pV) to construct pV-JP3ME.ResultsThe plasmid DNA was immunized into BALB/c mice, and high titers of IgG antibody and neutralizing antibody (nAb) against JEV were detected. The key cytokines in splenocytes were secreted upon stimulation with JEV antigens. Finally, complete protective efficacy was generated after challenge with the JEV P3 strain in the mice.ConclusionsThe DNA vaccine pV-JP3ME based on the JEV P3 strain in this study can induce specific humoral immune and cytokine responses and provide complete protection against JEV in mice.

SARS-CoV-2 growth, furin-cleavage-site adaptation and neutralization using serum from acutely infected hospitalized COVID-19 patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), emerged at the end of 2019 and by mid-June 2020 the virus had spread to at least 215 countries, caused more than 8 000 000 confirmed infections and over 450 000 deaths, and overwhelmed healthcare systems worldwide. Like severe acute respiratory syndrome coronavirus (SARS-CoV), which emerged in 2002 and caused a similar disease, SARS-CoV-2 is a betacoronavirus. Both viruses use human angiotensin-converting enzyme 2 (hACE2) as a receptor to enter cells. However, the SARS-CoV-2 spike (S) glycoprotein has a novel insertion that generates a putative furin cleavage signal and this has been postulated to expand the host range. Two low-passage (P) strains of SARS-CoV-2 (Wash1 : P4 and Munich : P1) were cultured twice in Vero E6 cells and characterized virologically. Sanger and MinION sequencing demonstrated significant deletions in the furin cleavage signal of Wash1 : P6 and minor variants in the Munich : P3 strain. Cleavage of the S glycoprotein in SARS-CoV-2-infected Vero E6 cell lysates was inefficient even when an intact furin cleavage signal was present. Indirect immunofluorescence demonstrated that the S glycoprotein reached the cell surface. Since the S protein is a major antigenic target for the development of neutralizing antibodies, we investigated the development of neutralizing antibody titres in serial serum samples obtained from COVID-19 human patients. These were comparable regardless of the presence of an intact or deleted furin cleavage signal. These studies illustrate the need to characterize virus stocks meticulously prior to performing either in vitro or in vivo pathogenesis studies.

Bioavailability of Zn from layered double hydroxides: The effects of plant growth-promoting rhizobacteria (PGPR)

There is widespread interest in developing layered double hydroxides (LDH) in agricultural applications to supply essential nutrients for plants. The present study sought to gain a better understanding of the specific effects of Zn-Mg-Fe(III)-LDH intercalated with nitrate (LDH-N) and phosphate (LDH-P) anions in the absence and presence of plant growth-promoting rhizobacteria (PGPR) on Zn phyto-availability, using sand culture experiments. The LDH were synthesized and their characteristics were investigated by XRD patterns, FT-IR spectra, and elemental analysis. In the first experiment, predetermined amounts of LDH-N or LDH-P mixed with 500 g sand quartz to provide 0, 5, 15, 25 and 50 mg Zn kg−1 sand. In the second study, the potential of three pseudomonas strains, i.e., pseudomonas sp. A5 (P1), p. putida P19 (P2) and P. fluorescens P52 (P3) (high Zn-solubilizing bacteria) on kinetic release and plant availability of Zn from LDH were investigated in a batch and a pot experiment. The LDH appreciably improved the Zn content of plants, irrespective of the sources applied. However, the concentration and uptake of Zn in root and shoot of maize plants grown in the LDH-N-sand system were higher than those grown in the LDH-P-sand culture. Results of the second experiment showed that, in the LDH-N treatments, all the three pseudomonas strains increased Zn content of roots and shoots. While in the LDH-P treatments, none of the strains had a significant effect on Zn concentrations of root and shoot as compared to the control. According to the results of the batch study, the ability of bacteria in cumulative Zn released increased by increasing the time of incubation. Among three strains, the highest cumulative Zn release from LDH was recorded in the presence of P2, followed by P1 and P3 strains. This study suggested that LDH-N and LDH-P are effective sources for the supply of Zn and improving the growth of maize under Zn deficiency conditions and also highlighted the potential of PGPR to increase Zn availability.

Enhanced biosynthesis of phenazine-1-carboxamide by Pseudomonas chlororaphis strains using statistical experimental designs.

Phenazine-1-carboxamide (PCN) is one of the major biocontrol agents produced by plant growth-promoting rhizosphere (PGPR) pseudomonads including Pseudomonas chlororaphis. In this study, a combined strategy of genetic modification and statistical experimental designs was applied to obtain mutants of P. chlororaphis strains with high-yield PCN production. To achieve this, the lon gene was knocked out in wild-type P. chlororaphis HT66 and the breeding mutant P3 strain with a non-scar deletion strategy. The resulting HT66Δlon and P3Δlon mutants produced a significantly higher PCN production in shake-flask cultures which was 5- and 9-folds greater than their native counterparts. The potential ability of strain P3Δlon for PCN production was further optimized by statistical designs. A two-level Plackett-Burman (PB) experimental design with six variables was employed to scrutinize medium components that significantly influence PCN production. Notably, glycerol, tryptone, and soy peptone were identified to be the most significant factors (p < 0.05). Response surface methodology (RSM) based on the central composite design (CCD) was adopted to determine these factors optimal levels and their interactive effects between culture components for PCN production. The predicted maximum PCN production was 9002mg/L, whereas an actual PCN production of 9174mg/L was recorded in the validation experiments using the optimal medium containing glycerol 37.08mL/L, tryptone 20.00g/L, and soy peptone 25.03g/L, which was nearly threefolds higher than without optimization and 20-folds higher than the wild-type strain. In conclusion, the results revealed that P. chlororaphis display a high potential for industrial-scale production for phenazine biopesticides.

Myeloid-Derived Suppressor Cells Inhibit T Follicular Helper Cell Immune Response in Japanese Encephalitis Virus Infection.

Resolution of viral infections requires activation of innate cells to initiate and maintain adaptive immune responses. In this study, we examined Japanese encephalitis virus (JEV) infection leading to acute encephalopathy depending on suppression of the adaptive immune responses mediated by innate cells. Infection with P3 strains of JEV enhanced myeloid-derived suppressor cell (MDSC) populations, and the survival rate of JEV-infected mice improved after MDSC depletion. Mechanically, P3-induced MDSCs suppressed CD4+ T cell immune responses, especially responses of T follicular helper (Tfh) cells, leading to decreased splenic B cells (CD19+) and blood plasma cells (CD19+CD138+) and reduced levels of total IgM and JEV-specific neutralizing Abs. Upon depleting P3-induced MDSCs in vivo, the Tfh cell population, B cells, plasma cells, and Ab production recovered. These findings provide unique insights regarding MDSC functions in mediating immune suppression via inhibiting Tfh cell responses and further impairing humoral immunity, which facilitate the progression of infection.

Antibacterial and antibiofilm potential of leaves extracts of Mirabilis jalapa L. and Ajuga bracteosa wall. against Pseudomonas aeruginosa

In the Present study the antibacterial and antibiofilm potential of Mirabilis jalapa and Ajuga bracteosa crude methanolic extracts of leaves against P1, P2 and P3 clinical strains of Pseudomonas aeruginosa was explored. Antibacterial activity was investigated by agar well diffusion method and the antibiofilm potential was determined by Anti-Pellicle assay and exopolysaccharide inhibition (EI) by Congo red (CR) assay. In antibacterial assay, M. jalapa extract showed good antibacterial activity against P. aeruginosa with maximum 13.5 mm inhibition zone (ZI) against P2 strain and minimum 12.8 mm ZI against P3 strain of P. aeruginosa as compared to A. bracteosa which showed a maximum 13.4 mm ZI and a minimum of 12.7 mm against both P2 and P3 strains of P. aeruginosa, respectively. Both M. Jalapa and A. bracteosa extracts showed moderate (+++) and weak (++++) effect on pellicle inhibition against the tested strains of P. aeruginosa. In CR assay, a moderate antibiofilm effect was recorded on EI for both plants. Based on the results it was concluded that methanol extracts of M. jalapa and A. bracteosa possessed good antibacterial activity and moderate antibiofilm potential against P. aeruginosa. Keywords: Agar well diffusion assay; Ajuga bracteosa; Mirabilis jalapa; Natural products; Opportunistic pathogen http://dx.doi.org/10.19045/bspab.2017.60062

Differential modulation of avian β-defensin and Toll-like receptor expression in chickens infected with infectious bronchitis virus.

The host innate immune response either clears invading viruses or allows the adaptive immune system to establish an effective antiviral response. In this study, both pathogenic (passage 3, P3) and attenuated (P110) infectious bronchitis virus (IBV) strains were used to study the immune responses of chicken to IBV infection. Expression of avian β-defensins (AvBDs) and Toll-like receptors (TLRs) in 16 tissues of chicken were compared at 7 days PI. The results showed that P3 infection upregulated the expression of AvBDs, including AvBD2, 4, 5, 6, 9, and 12, while P110 infection downregulated the expression of AvBDs, including AvBD3, 4, 5, 6, and 9 in most tissues. Meanwhile, the expression level of several TLRs showed a general trend of upregulation in the tissues of P3-infected chickens, while they were downregulated in the tissues of P110-infected chickens. The result suggested that compared with the P110 strain, the P3 strain induced a more pronounced host innate immune response. Furthermore, we observed that recombinant AvBDs (including 2, 6, and 12) demonstrated obvious anti-viral activity against IBV in vitro. Our findings contribute to the proposal that IBV infection induces an increase in the messenger RNA (mRNA) expression of some AvBDs and TLRs, which suggests that AvBDs may play significant roles in the resistance of chickens to IBV replication.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-015-6786-8) contains supplementary material, which is available to authorized users.

Japanese Encephalitis, Tibet, China

Japanese Encephalitis, Tibet, China

Study on the protective efficacy of SA14-14-2 attenuated Japanese encephalitis against different JE virus isolates circulating in China

Prior to 1976 only Japanese encephalitis virus (JEV) genotype III could be detected in China. Recently, numerous genotype I JEV strains have been isolated from JE patients, mosquitoes and pigs while genotype III strains remain present. Two kinds of JEV vaccines are currently used in China for the prevention disease: the JE live attenuated vaccine (LAV) SA14-14-2 virus and the inactivated P3 strain (IPV) vaccine. The SA14-14-2 and P3 viral strains were isolated in the year of 1953 and 1949 respectively and both belonged to the JEV genotype III. In order to evaluate the protective efficacy of both vaccines against the JEV genotype I isolates we conducted vaccination-challenge protection assays in mice. These data demonstrated that both LAV (≥234pfu virus) and IPV (1:5 dilution) vaccines effectively conferred protection against all 16 isolates tested following intraperitoneal (i.p.) challenge. However, when vaccinated mice were challenged via intracerebral (i.c.) injection, ≥60% LAV vaccinated animals were protected against challenge with most JEV isolates but only ≤40% protection was observed following vaccination with IPV. These results indicated that JE vaccines used in China still protected effectively against both JEV genotypes now prevalent in China and that the LAV formulation conferred higher levels of protection compared to the protection conferred by IPV.

Using population dynamics analysis by DGGE to design the bacterial consortium isolated from mangrove sediments for biodegradation of PAHs

There are many PAH-degrading bacteria in mangrove sediments and in order to explore their degradation potential, surface sediment samples were collected from a mangrove area in Fugong, Longhai, Fujian Province of China. A total of 53 strains of PAH-degrading bacteria were isolated from the mangrove sediments, consisting of 14 strains of phenanthrene (Phe), 13 strains of pyrene (Pyr), 13 strains of benzo[a]pyrene (Bap) and 13 strains of mixed PAH (Phe+Pyr+Bap)-degrading bacteria. All of the individual colonies were identified by 16S rDNA sequencing. Based on the information of bacterial PCR-DGGE profiles obtained during enrichment batch culture, Phe, Pyr, Bap and mixed PAH-degrading consortia consisted of F1, F2, F3, F4 and F15 strains, B1, B3, B6, B7 and B13 strains, P1, P2, P3, P5 and P7 strains, M1, M2, M4, M12 and M13 strains, respectively. In addition, the degradation ability of these consortia was also determined. The results showed that both Phe and mixed PAH-degrading consortia had the highest ability to degrade the Phe in a liquid medium, with more than 91% being degraded in 3 days. But the biodegradation percentages of Pyr by Pyr-degrading consortium and Bap by Bap-degrading consortium were relatively lower than that of the Phe-degrading consortium. These results suggested that a higher degradation of PAHs depended on both the bacterial consortium present and the type of PAH compound. Moreover, using the bacterial community structure analysis method, where the consortia consist of different PAH-degrading bacteria, the information from the PCR-DGGE profiles could be used in the bioremediation of PAHs in the future.

Identification of IAA-producing endophytic bacteria from micropropagated Echinacea plants using 16S rRNA sequencing

The presence of latent bacteria is a serious problem in plant tissue cultures. While endophytes are generally beneficial to plants in situ, they may affect culture growth under the modified conditions in vitro. The present study was undertaken to identify and characterize endophytic bacteria associated with the medicinal plant Echinacea in tissue culture. Based on classical microbiological tests and 16S rRNA analyses, it was found that endophytic bacteria associated with aseptically micropropagated Echinacea plantlets are representatives of several genera, Acinetobacter, Bacillus, Pseudomonas, Wautersia (Ralstonia) and Stenotrophomonas. Based on TLC and HPLC analyses, we found that Pseudomonas stutzeri P3 strain produces plant hormone, auxin (indole-3-acetic acid, IAA). Antibiotic resistance was also assessed as a virulence factor. The majority of endophytic bacteria were resistant to the antibiotic kanamycin, but susceptible to chloramphenicol. Recommendations for propagating Echinaceain vitro cultures involve the addition of chloramphenicol, tetracycline, and ampicillin, antibiotics that cause no side effects on these plant species.

Comparison of nucleotide and deduced amino acid sequences of E gene of the newly isolated Japanese encephalitis virus strains and inactivated vaccine strain P3

To analyze the difference of nucleotides and deduced amino acids sequences E gene between the newly isolated Japanese encephalitis (JE) virus strains from mosquitoes or patients and P3 strain. The E gene sequences of corresponding strains of JE virus were obtained from GenBank. Computer analyze of nucleic acid data and deduced amino acid sequence were accomplished using the Clustal X (1.8), DNASTAR, GENEDOC (3.2) programs. The result showed that compared with the Fujian strains and P3 strain the nucleotide sequence homology was up to 98.3%, and the amino acid sequence homology was up to 98.2%, respectively. Compared with the Shanghai strains and P3 strain, the nucleotide sequence differences were 12%, and the amino acid sequence homology was up to 98.2%, respectively. Compared with P3 strain, there were nineteen amino acid variations in E gene of all the newly isolated strains. Between P3 and all the newly isolated JE virus strains, there are three common variations at E-129, E-222, E-366. And two common variations E-160 and E-487 were found only in Fujian strains, common variations at E-129, E-222, E-227, E-366 in Shanghai strains. There are some differences between P3 strain and JE viruses which were isolated from mosquitoes belonging to genotype I in Shanghai and from patients belonging to genotype III from Fujian province. But these variations are not in the important locations affecting the biological characteristic of the viruses.

Immunogenicity of a Japanese encephalitis DNA vaccine candidate in cynomolgus monkeys

A Japanese encephalitis (JE) vaccine candidate encoding JE virus premembrane (prM) and envelope (E) genes, designated pNJEME, was evaluated for safety and immunogenicity in non-human primate, cynomolgus monkeys. pNJEME was constructed using a vector (pNGVL4a) designed to address some of the safety concerns of DNA vaccine. In two different experiments, two immunizations with 300 μg of pNJEME by intramuscular (i.m.) injection, and 3 μg of pNJEME using a gene gun, and three immunizations by i.m. injection with 500 μg of pNJEME were performed. All the three protocols induced low to high levels of neutralizing antibody, indicating an ability of pNJEME to induce neutralizing antibody in monkeys with a wide individual variation in response to pNJEME. In one experiment designed to compare the DNA vaccine with a commercial inactivated JE vaccine, three immunizations by i.m. inoculation with 300 μg of pNJEME or by gene gun administration with 3 μg of pNJEME induced similar levels of neutralizing antibody to those induced by three immunizations with a human dose of the inactivated vaccine in most monkeys. After intranasal challenge with the Beijing P3 or JaTH160 strain of JE virus, pNJEME-immunized monkeys showed anamnestic neutralizing antibody responses, indicating that pNJEME induced memory B cells which were responsive to infection with JE virus. No systemic and local reactions were observed in any monkeys after i.m. or gene gun inoculations with plasmid DNAs.