Identification and characterization of two Bacillus anthracis bacteriophages.

Anthrax is an acute infectious zoonotic disease caused by Bacillus anthracis, a bacterium that is considered a potential biological warfare agent. Bacillus bacteriophages shape the composition and evolution of bacterial communities in nature and therefore have important roles in the ecosystem community. B. anthracis phages are not only used in etiological diagnostics but also have promising prospects in clinical therapeutics or for disinfection in anthrax outbreaks. In this study, two temperate B. anthracis phages, vB_BanS_A16R1 (A16R1) and vB_BanS_A16R4 (A16R4), were isolated and showed siphovirus-like morphological characteristics. Genome sequencing showed that the genomes of phages A16R1 and A16R4 are 36,569 bp and 40,059 bp in length, respectively. A16R1 belongs to the genus Wbetavirus, while A16R4 belongs to the genus Hubeivirus and is the first phage of that genus found to lyse B. anthracis. Because these two phages can comparatively specifically lyse B. anthracis, they could be used as alternative diagnostic tools for identification of B. anthracis infections.

Novel Strategy for Rapidly and Safely Distinguishing Bacillus anthracis and Bacillus cereus by Use of Peptide Mass Fingerprints Based on Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

The objective of this study was to construct a rapid, high-throughput, and biosafety-compatible screening method for Bacillus anthracis and Bacillus cereus based on matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). MALDI-TOF MS coupled to ClinProTools was used to discover MALDI-TOF MS biomarker peaks and generate a classification model based on a genetic algorithm (GA) to differentiate between different Bacillus anthracis and Bacillus cereus isolates. Thirty Bacillus anthracis and 19 Bacillus cereus strains were used to construct and analyze the model, and 40 Bacillus strains were used for validation. For the GA screening model, the cross-validation values, which reflect the ability of the model to handle variability among the test spectra, and the recognition capability values, which reflect the model's ability to correctly identify its component spectra, were all 100%. This model contained 10 biomarker peaks (m/z 3,339.9, 3,396.3, 3,682.4, 5,476.7, 6,610.6, 6,680.1, 7,365.3, 7,792.4, 9,475.8, and 10,934.1) used to correctly identify 28 Bacillus anthracis and 12 Bacillus cereus isolates from 40 Bacillus isolates, with a sensitivity and specificity of 100%. With the obvious advantages of being rapid, highly accurate, and highly sensitive and having a low cost and high throughput, MALDI-TOF MS ClinProTools is a powerful and reliable tool for screening Bacillus anthracis and Bacillus cereus strains.

Genetic diversity of Bacillus anthracis Ames lineage strains in China.

BACKGROUND: Anthrax is an endemic disease that persists in the rural regions of China. The global genetic population structure of B.anthracis has also been defined by the canonical single-nucleotide polymorphisms (canSNP) and multiple-locus variable-number tandem repeat analysis (MLVA). Five canSNP lineages were found in China, and the A.Br.Ames lineage has been the second predominant group in recent years in China. The objective of this study was to reveal genetic diversity of the Ames lineage strains by MLVA. METHODS: Two molecular typing methods, canSNP and MLVA with 15markers were used to study the genetic relationship among the Ames lineage strains. The outbreak information associated with these strains was also collected and investigated. RESULTS: From 2007 to 2018, a total of 21 human anthrax infection outbreaks (68 patients) associated with B. anthracis Ames lineage strains were reported in China. Ames lineage strain-associated human anthrax is mainly distributed in the northern part of China, including the provinces of Inner Mongolia, Liaoning, Gansu, and Xinjiang. In the study, a total of 30 Ames lineage strains were included and 10 MLVA15 genotypes were identified. These strains were mainly found in northeast China, Inner Mongolia and Liaoning. In recent years, the Ames lineage strains were isolated in the two provinces every year. The 18 Ames lineage strains isolated from Inner Mongolia were divided into eight MLVA15 genotypes. From 2010 to 2015, there were continuous reports of outbreaks in Keyouzhongqi County, Inner Mongolia, and the strains that were isolated annually in succession belonged to the MLVA15-30 genotype. CONCLUSIONS: The Ames lineage strains are widely distributed in northern China. Their genetic diversity can be illustrated by the results of the MLVA. The genetic characteristics of the Ames lineage strains from outbreaks in different provinces varied. In some areas, human anthrax outbreaks occurred annually in succession, and these related strains grouped together. These observations indicate that the local environment was persistently contaminated with B. anthracis spores, vaccination of livestock should become the fundamental control measure in the areas.

Epidemiology of Human Anthrax in China, 1955-2014.

Using national surveillance data for 120,111 human anthrax cases recorded during 1955-2014, we analyzed the temporal, seasonal, geographic, and demographic distribution of this disease in China. After 1978, incidence decreased until 2013, when it reached a low of 0.014 cases/100,000 population. The case-fatality rate, cumulatively 3.6% during the study period, has also decreased since 1990. Cases occurred throughout the year, peaking in August. Geographic distribution decreased overall from west to east, but the cumulative number of affected counties increased during 2005-2014. The disease has shifted from industrial to agricultural workers; 86.7% of cases occurred in farmers and herdsmen. Most (97.7%) reported cases were the cutaneous form. Although progress has been made in reducing incidence, this study highlights areas that need improvement. Adequate laboratory diagnosis is lacking; only 7.6% of cases received laboratory confirmation. Geographic expansion of the disease indicates that livestock control programs will be essential in eradicating anthrax.

Niche modeling predictions of the potential distribution of Marmota himalayana, the host animal of plague in Yushu County of Qinghai.

BACKGROUND: After the earthquake on 14, April 2010 at Yushu in China, a plague epidemic hosted by Himalayan marmot (Marmota himalayana) became a major public health concern during the reconstruction period. A rapid assessment of the distribution of Himalayan marmot in the area was urgent. The aims of this study were to analyze the relationship between environmental factors and the distribution of burrow systems of the marmot and to predict the distribution of marmots. METHODS: Two types of marmot burrows (hibernation and temporary) in Yushu County were investigated from June to September in 2011. The location of every burrow was recorded with a global positioning system receiver. An ecological niche model was used to determine the relationship between the burrow occurrence data and environmental variables, such as land surface temperature (LST) in winter and summer, normalized difference vegetation index (NDVI) in winter and summer, elevation, and soil type. The predictive accuracies of the models were assessed by the area under the curve of the receiving operator curve. RESULTS: The models for hibernation and temporary burrows both performed well. The contribution orders of the variables were LST in winter and soil type, NDVI in winter and elevation for the hibernation burrow model, and LST in summer, NDVI in summer, soil type and elevation in the temporary burrow model. There were non-linear relationships between the probability of burrow presence and LST, NDVI and elevation. LST of 14 and 23 °C, NDVI of 0.22 and 0.60, and 4100 m were inflection points. A substantially higher probability of burrow presence was observed in swamp soil and dark felty soil than in other soil types. The potential area for hibernation burrows was 5696 km(2) (37.7% of Yushu County), and the area for temporary burrows was 7711 km(2) (51.0% of Yushu County). CONCLUSIONS: The results suggested that marmots preferred warm areas with relatively low altitudes and good vegetation conditions in Yushu County. Based on these results, the present research is useful in understanding the niche selection and distribution pattern of marmots in this region.

Epidemiological Characteristics of Human Anthrax - China, 2018-2021.

Introduction: The epidemic of human anthrax is at a low level in China in recent years, but the reported incidence increased in 2021. In order to understand the current landscape of research and knowledge about anthrax in China, the epidemiological characteristics of anthrax in humans from 2018 to 2021 were analyzed and the prevention and control suggestions were proposed. Methods: Surveillance data of anthrax in humans and livestock, together with human outbreaks data during 2018-2021, were collected and analyzed by descriptive statistics methods. The number and proportion of outbreaks, cases and deaths by provincial-level administrative divisions (PLADs), clinical types, and contributing factors were calculated. Results: A total of 1,244 cases of human anthrax and 53 outbreaks were reported from 2018 to 2021 in China. While the incidence of anthrax declined from 2018 to 2020, it increased in 2021. The regions of anthrax were mainly located in the west and the northeast PLADs of China, though cases were reported in some central and eastern PLADs in 2021. Young and middle-aged men involved in animal husbandry were found to be at a higher risk of anthrax. All the reported outbreaks were associated with the exposure of infected livestock. A total of 296 livestock anthrax cases were reported. Conclusions: The increased incidence and wider geographical distribution of human anthrax in 2021 were found to be the result of inadequate supervision of diseased animals as well as updated diagnostic criteria. As such, the monitoring of risk factors and emergency preparation procedures should be strengthened at the national level. In addition, it is also critical to strengthen health education for high-risk occupational groups and strengthen professional training for local clinicians. Finally, more measures should be carried out to strengthen anthrax surveillance in livestock husbandry.

Genetic source tracking of human plague cases in Inner Mongolia-Beijing, 2019.

On 12 November 2019, one couple from the Sonid Left Qi (County) in the Inner Mongolia Autonomous Region was diagnosed with pneumonic plague in Beijing. The wife acquired the infection from her husband. Thereafter, two bubonic plague cases were identified in Inner Mongolia on November 16th and 24th. In this study, genome-wide single nucleotide polymorphism (SNP) analysis was used to identify the phylogenetic relationship of Yersinia pestis strains isolated in Inner Mongolia. Strains isolated from reservoirs in 2018 and 2019 in Inner Mongolia, together with the strain isolated from Patient C, were further clustered into 2.MED3m, and two novel lineages (2.MED3q, 2.MED3r) in the 2.MED3 population. According to the analysis of PCR-based molecular subtyping methods, such as the MLVA 14 scheme and seven SNP allele sequencing, Patients A/B and D were classified as 2.MED3m. In addition, strains from rodents living near the patients' residences were clustered into the same lineage as patients. Such observations indicated that human plague cases originated from local reservoirs. Corresponding phylogenetic analysis also indicated that rodent plague strains in different areas in Inner Mongolia belong to different epizootics rather than being caused by spreading from the same epizootic in Meriones unguiculatus in 2019.

Changed epidemiology of anthrax and molecular characteristics of Bacillus anthracis in Inner Mongolia Autonomous Region, China.

Anthrax is a natural foci disease in Inner Mongolia, which poses a severe threat to public health. In this study, the incidence number, rate and constituent ratio were used to describe the epidemiological characteristics of anthrax in the region from 1956-2018. The molecular correlation and genetic characteristics of the strains were investigated using canonical single nucleotide polymorphisms (CanSNP), multiple-locus variable-number tandem repeat analysis (MLVA-15) and whole genome sequencing (WGS). The epidemiological characteristics of anthrax in Inner Mongolia have altered significantly. The incidence of anthrax has decreased annually without vaccination, and the regional distribution of anthrax gradually transferred from central and western regions to the eastern. Moreover, the occupation distribution evolved from multiple early occupations to predominated by farmers and herdsmen. This change is closely related to policy factors and to changes in the means of production and the living habits of the local population. This indicates that reformulating the control and prevention strategies is essential. Both A. Br. Ames and A. Br. 001/002 subgroups were the predominant CanSNP genotypes of Bacillus anthracis in Inner Mongolia. A total of 36 strains constituted six shared MLVA-15 genotypes, suggesting an epidemiological link between the strains of each shared genotype. The six shared genotypes ([GT1, 9, 11 and 15] and [GT8 and 12]) consisting of 2-7 strains confirmed the occurrence of multiple point outbreaks and cross-regional transmission caused by multiple common sources of infection. Phylogenetic analysis based on the WGS core genome showed that strains from this study formed an independent clade (C.V.), and they were positioned close to each other, suggesting a common origin. Further comparison analysis should be performed to ascertain the geographic origin of these strains.

Complete genome sequences of Yersinia pestis from natural foci in China.

Yersinia pestis, the causative agent of plague, is a deadly bacterium that affects humans. Strain D106004 was isolated from a new plague focus in Yulong County, China, in 2006. To gain insights into the epidemic origin, we have sequenced the genomes of D106004 and strains Z176003 and D182038, isolated from neighboring regions.

Genome rearrangements of completely sequenced strains of Yersinia pestis.

Yersinia pestis has caused three worldwide plagues in human history that have led to innumerable deaths. We have completely sequenced the genomes of two strains (D106004 and D182038) of Y. pestis isolated from Yunnan Province of China. The most striking finding of our study is that large amounts of genome rearrangement events exist between the genomes of two Yunnan strains despite being isolated from two foci only 50 kilometers apart. When we compared the genome sequences of the Yunnan strains with six strains (CO92, KIM, 91001, Antiqua, Nepal516, and Pestoides F) of Y. pestis sequenced previously, we found that the genomes of Y. pestis were divided into 61 relatively independent segments. Pairwise comparisons of all 61 segments among eight strains showed that the Yunnan strains were most closely related to strain CO92. We concluded that Y. pestis genomes consist of segments that can change their positions and directions within the genomes caused by genome rearrangements, and our study confirmed the inference that the third plague pandemic originated in Yunnan since the genome sequences of Yunnan strains were closest to the strain CO92 isolated from the United States.

The location of endogenous wild-type p53 protein in 293T and HEK293 cells expressing low-risk HPV-6E6 fusion protein with GFP.

The mechanism underlining human papillomaviruses (HPVs) causing cancer has been studied extensively, and it was concluded that the high-risk HPVs' E6 targeted and degraded tumor suppressor protein p53, leading to infected cells malignant transformation. In contrast, the low-risk HPVs only cause proliferative but non-invasive lesions of infected epithelia. Therefore, we hypothesized that low-risk HPVs' E6 might interact with p53 in a different pattern. We used a mammalian green fluorescent protein (GFP) expression system to express HPV-18E6 and HPV-6E6 fusion proteins in wild-type (wt) p53 cell lines, 293T and HEK293 cells, to investigate the traffic and location of E6s and p53. The results indicated GFP-18E6 was mainly expressed in nucleus, whereas GFP-6E6 was expressed exclusively in cytoplasm. Endogenous wt p53 was shown to be localized in the nuclei of cells transfected with GFP- 18E6. Interestingly, for the first time, we observed that p53 was trapped in the cytoplasm and never translocated into the cell nuclei transfected with GFP-6E6. In conclusion, HPV-6E6 was responsible for the cytoplasmic localization of p53. Therefore, our experiments provide a new insight into the pathogenesis of HPV.

MLVA distribution characteristics of Yersinia pestis in China and the correlation analysis.

BACKGROUND: Yersinia pestis, the aetiological agent of plague, has been well defined genotypically on local and worldwide scales. In November 2005, five cases of severe pneumonia of unknown causes, resulting in two deaths, were reported in Yulong, Yunnan province. In this study, we compared Y. pestis isolated from the Yulong focus to strains from other areas. RESULTS: Two hundred and thirteen Y. pestis strains collected from different plague foci in China and a live attenuated vaccine strain of Y. pestis (EV76) were genotyped using multiple-locus variable-number tandem repeat analysis (MLVA) on 14 loci. A total of 214 Y. pestis strains were divided into 85 MLVA types, and Nei's genetic diversity indices of the various loci ranged between 0.02 - 0.76. Minimum spanning tree analysis showed that Y. pestis in China could be divided into six complexes. It was observed that Microtus strains were different from the other three biovar strains. Each plague focus had its own unique MLVA types. CONCLUSION: The strains isolated from Yulong, Yunnan province had a unique MLVA type, indicating a new clone group. Our results suggest that Yulong strains may have a close relationship with strains from the Qinghai-Tibet Plateau plague focus.

Genetic characteristics of Bacillus anthracis isolated from northwestern China from 1990 to 2016.

Anthrax is a global re-emerging zoonotic disease and is an endemic disease in China, especially in rural regions. In this study, the general characteristics of human anthrax outbreaks that occurred in areas of northwestern China over the past decade have been described. Meanwhile, the genetic characteristics of Bacillus anthracis isolated from these areas from 1990 to 2016 were analyzed by means of canonical single-nucleotide polymorphism (canSNP) analysis and multilocus variable-number tandem repeat analysis (MLVA) with 15 markers. Five sublineages/subgroups, namely, A.Br.001/002, A.Br.Vollum, A.Br.Aust94, A.Br.Ames and A.Br.008/009, were detected by using 13 canSNP sites. All of the sublineages were found in Xinjiang province, while one sublineage was found in Shaanxi, two in Gansu, three in Qinghai and four in Inner Mongolia. However, the geographical distribution of the B. anthracis populations exhibited different canSNP characteristics from those of the strains isolated before 1990 in China. In contrast to previous data, the A.Br.Ames subgroup was also observed to be scattered from Inner Mongolia to other provinces. All 106 strains were assigned to 36 MLVA15 genotypes, and 21 of these types were first observed in this study. The strains collected from anthrax outbreaks in recent decade were classified as subgroups A.Br.001/002 and A.Br.Ames and identified as genotypes MLVA15-28, MLVA15-30, MLVA15-31, MLVA15-38, MLVA15-CHN3, and MLVA15-CHN18. By canSNP analysis and MLVA, we found that the diversification of MLVA genotypes and the geographical distribution of B. anthracis populations is gradually becoming balanced across northwestern China. This study also provides preliminary survey results regarding the population diversity of B. anthracis in China, which will help promote the prevention and control of this important disease.

Investigation and source-tracing of an anthrax outbreak in Gansu Province, China.

Anthrax is an endemic disease in China. Cases are reported every year, especially in the northwestern areas. In August 2016, an outbreak of 21 cutaneous anthrax cases was reported in Min County, Gansu Province, China. In this study, the general characteristics of the anthrax outbreak are described. Two molecular typing methods, canonical single-nucleotide polymorphism (canSNP) and multiple-locus variable-number tandem repeat analysis with 15 markers (MLVA15), were used to investigate the possible source of transmission and to identify the genetic relationship among the strains/samples isolated in this outbreak as well as previous isolates. In this outbreak, all patients were infected through contact with diseased livestock or contaminated animal products. Livestock had been introduced into the local area shortly before the outbreak from Gannan Prefecture (in Gansu Province), Sichuan and Qinghai Provinces. In the molecular typing analysis, there were two canSNP subgroups found in Gansu, A.Br.001/002 and A.Br.Ames, and five MLVA15 genotypes were observed. The strains collected from the anthrax outbreak in Min County in 2016 belonged to the A.Br.001/002 canSNP subgroup and the MLVA15-28 and MLVA15-30 genotype. Strains previously isolated from Sichuan, Inner Mongolia and Maqu County (in Gannan Prefecture, Gansu Province) were clustered with these outbreak-related strains/samples according to the MLVA15-30 genotype. The MLVA15-28 genotype was found in strains isolated from Gansu and Xinjiang in previous studies. Combining the epidemiological investigation and molecular typing results, we conclude that the patients in this outbreak were infected by a local pathogen present in the adjoining area of Gansu, Sichuan and Qinghai Provinces.

A Single Nucleotide Polymorphism Is Involved in Regulation of Growth and Spore Formation of Bacillus anthracis Pasteur II Strain.

Anthrax toxins and capsules, which are encoded by genes located on pXO1 and pXO2, respectively, are major virulence factors of Bacillus anthracis. Our previous studies demonstrated that exposure to high-temperatures is unable to abolish the pXO1 plasmid of the Pasteur II strain, but the growth of the strain was obviously slower than that of the Sterne strain and wild-type virulent strain. To elucidate a potential regulatory mechanism of slowing growth, we employed comparative genome and bioinformatic analysis and revealed a unique SNP (G to T) at the 143135 bp position in pXO1 that is possibly involved in the mediation of growth of Pasteur II. However, the T to G mutation in groR did not result in any change of the amino acid sequence. A predominant nucleotide G existed at the 143135 bp in pXO1 of 100 wild-type B. anthracis isolates and 9 isolates documented in GenBank, whereas T replaced G in pXO1 of the Pasteur II strain. Further analysis indicate that the SNP is located in a gene between 143042 and 143173 bp, and that it encodes a small protein of 43 amino acids and is termed as a growth regulator (GroR). Site-directed mutagenesis and gene deletion demonstrates that groR regulates the growth and spore formation of B. anthracis. Our results indicate that the pXO1 plasmid is involved in the regulation of growth and spore formation in B. anthracis.

The pag Gene of pXO1 Is Involved in Capsule Biosynthesis of Bacillus anthracis Pasteur II Strain.

The poly-γ-D-glutamic acid capsule and anthrax toxins are major virulence factors of Bacillus anthracis. Genes responsible for capsule biosynthesis are located on pXO2, whereas genes encoding the toxins, which are composed of edema factors, lethal factors, and protective antigens (PA), are located on pXO1. In this study, we found that the pag null mutation not only eliminated the production of the protective antigen, it also eliminated the ability of the B. anthracis Pasteur II strain to form capsules. qPCR analysis revealed that the deletion of pag decreased the transcription levels of the capABCD operon and its regulatory genes acpA and acpB. The introduction of the acpA or acpB plasmid complemented the effect of the pag null mutation on capsule formation. Taken together, the above results suggest that PA probably affects capsule biosynthesis by altering the expression of acpA and acpB. In addition, we found that the deletion mutation of pag remarkably attenuated bacterial pathogenicity in a mouse model of infection. Our results indicate that besides encoding the protective antigen, the pag gene of pXO1 is also involved in the modulation of capsule biosynthesis. Our findings provide new insight into the regulation mechanisms of capsule formation in B. anthracis Pasteur II strain.

Genetic source tracking of an anthrax outbreak in Shaanxi province, China.

BACKGROUND: Anthrax is an acute zoonotic infectious disease caused by the bacterium known as Bacillus anthracis. From 26 July to 8 August 2015, an outbreak with 20 suspected cutaneous anthrax cases was reported in Ganquan County, Shaanxi province in China. The genetic source tracking analysis of the anthrax outbreak was performed by molecular epidemiological methods in this study. METHODS: Three molecular typing methods, namely canonical single nucleotide polymorphisms (canSNP), multiple-locus variable-number tandem repeat analysis (MLVA), and single nucleotide repeat (SNR) analysis, were used to investigate the possible source of transmission and identify the genetic relationship among the strains isolated from human cases and diseased animals during the outbreak. RESULTS: Five strains isolated from diseased mules were clustered together with patients' isolates using canSNP typing and MLVA. The causative B. anthracis lineages in this outbreak belonged to the A.Br.001/002 canSNP subgroup and the MLVA15-31 genotype (the 31 genotype in MLVA15 scheme). Because nine isolates from another four provinces in China were clustered together with outbreak-related strains by the canSNP (A.Br.001/002 subgroup) and MLVA15 method (MLVA15-31 genotype), still another SNR analysis (CL10, CL12, CL33, and CL35) was used to source track the outbreak, and the results suggesting that these patients in the anthrax outbreak were probably infected by the same pathogen clone. CONCLUSIONS: It was deduced that the anthrax outbreak occurred in Shaanxi province, China in 2015 was a local occurrence.

Involvement of the pagR gene of pXO2 in anthrax pathogenesis.

Anthrax is a disease caused by Bacillus anthracis. Specifically, the anthrax toxins and capsules encoded by the pXO1 and pXO2 plasmids, respectively, are the major virulence factors. We previously reported that the pXO1 plasmid was retained in the attenuated strain of B. anthracis vaccine strains even after subculturing at high temperatures. In the present study, we reinvestigate the attenuation mechanism of Pasteur II. Sequencing of pXO1 and pXO2 from Pasteur II strain revealed mutations in these plasmids as compared to the reference sequences. Two deletions on these plasmids, one each on pXO1 and pXO2, were confirmed to be unique to the Pasteur II strain as compared to the wild-type strains. Gene replacement with homologous recombination revealed that the mutation in the promoter region of the pagR gene on pXO2, but not the mutation on pXO1, contributes to lethal levels of toxin production. This result was further confirmed by RT-PCR, western blot, and animal toxicity assays. Taken together, our results signify that the attenuation of the Pasteur II vaccine strain is caused by a mutation in the pagR gene on its pXO2 plasmid. Moreover, these data suggest that pXO2 plasmid encoded proteins are involved in the virulence of B. anthracis.

Identification of the pXO1 plasmid in attenuated Bacillus anthracis vaccine strains.

Anthrax toxins and capsule are the major virulence factors of Bacillus anthracis. They are encoded by genes located on the plasmids pXO1 and pXO2, respectively. The vaccine strain Pasteur II was produced from high temperature subcultures of B. anthracis, which resulted in virulence attenuation through the loss of the plasmid pXO1. However, it is unclear whether the high temperature culture completely abolishes the plasmid DNA or affects the replication of the plasmid pXO1. In this study, we tested 3 B. anthracis vaccine strains, including Pasteur II from France, Qiankefusiji II from Russia, and Rentian II from Japan, which were all generated from subcultures at high temperatures. Surprisingly, we detected the presence of pXO1 plasmid DNA using overlap PCR in all these vaccine strains. DNA sequencing analysis of overlap PCR products further confirmed the presence of pXO1. Moreover, the expression of the protective antigen (PA) encoded on pXO1 was determined by using SDS-PAGE and western blotting. In addition, we mimicked Pasteur's method and exposed the A16R vaccine strain, which lacks the pXO2 plasmid, to high temperature, and identified the pXO1 plasmid in the subcultures at high temperatures. This indicated that the high temperature treatment at 42.5°C was unable to eliminate pXO1 plasmid DNA from B. anthracis. Our results suggest that the attenuation of the Pasteur II vaccine strain is likely due to the impact of high temperature stress on plasmid replication, which in turn limits the copy number of pXO1. Our data provide new insights into the mechanisms of the remaining immunogenicity and toxicity of the vaccine strains.

Electroporation enhances protective immune response of a DNA vaccine against Japanese encephalitis in mice and pigs.

Japanese encephalitis virus (JEV) is a pathogenic cause of Japanese Encephalitis (JE), which is a zoonotic disease transmitted by mosquitoes and amplified by pigs. Infection of JEV may lead to severe neurological sequelae, even death in humans and reproductive disorders in pigs. Vaccination is the only way to control JEV infection in humans. For pigs play important role in the JEV transmission cycle, developing a new veterinary vaccine is considered as a useful strategy for cutting off the transmission route of JEV. We have previously reported that DNA vaccine pCAG-JME, expressing prM-E proteins of JEV, is effective in mice through intramuscular injection (IM). However, the poor immunogenicity, due to low expression of immunogen, is the major obstacle for the development of DNA vaccine in large animals. In the present study, therefore, we immunized mice and pigs with pCAG-JME intramuscularly accompanied with electroporation (EP) stimulation, the attractive gene delivery approach. As compared with IM, EP-mediated vaccination markedly increased the expression of immunogen in the injection site and induced a dose- and time-dependent immune response. 100% survival rate was observed in groups vaccinated with doses ranged from 10 to 100µg, indicating that 10µg of DNA with EP for individual was enough for inducing effective protection in mice. Surprisingly, survival rate and end-point titers of anti-JEV antibodies were higher in mice even at lower dose of DNA (5µg) than that in mice inoculated 100µg through IM. Notably, the prM-E antigens also induced high antibody response in pig, while the neutralizing antibody titer achieved 1:320. Our results suggested that EP-mediated DNA immunization might act as an effective strategy against JEV, at least in pig, and that EP has a potential application prospect in DNA vaccination.