Giovanni Faggioni

Genotyping of Bacillus anthracis strains based on automated capillary 25-loci Multiple Locus Variable-Number Tandem Repeats Analysis

BackgroundThe genome of Bacillus anthracis, the etiological agent of anthrax, is highly monomorphic which makes differentiation between strains difficult. A Multiple Locus Variable-number tandem repeats (VNTR) Analysis (MLVA) assay based on 20 markers was previously described. It has considerable discrimination power, reproducibility, and low cost, especially since the markers proposed can be typed by agarose-gel electrophoresis. However in an emergency situation, faster genotyping and access to representative databases is necessary.ResultsGenotyping of B. anthracis reference strains and isolates from France and Italy was done using a 25 loci MLVA assay combining 21 previously described loci and 4 new ones. DNA was amplified in 4 multiplex PCR reactions and the length of the resulting 25 amplicons was estimated by automated capillary electrophoresis. The results were reproducible and the data were consistent with other gel based methods once differences in mobility patterns were taken into account. Some alleles previously unresolved by agarose gel electrophoresis could be resolved by capillary electrophoresis, thus further increasing the assay resolution. One particular locus, Bams30, is the result of a recombination between a 27 bp tandem repeat and a 9 bp tandem repeat. The analysis of the array illustrates the evolution process of tandem repeats.ConclusionIn a crisis situation of suspected bioterrorism, standardization, speed and accuracy, together with the availability of reference typing data are important issues, as illustrated by the 2001 anthrax letters event. In this report we describe an upgrade of the previously published MLVA method for genotyping of B. anthracis and apply the method to the typing of French and Italian B. anthracis strain collections. The increased number of markers studied compared to reports using only 8 loci greatly improves the discrimination power of the technique. An Italian strain belonging to the B branch was described, and two new branches, D and E, are proposed. Owing to the upgrading achieved here, precise genotyping can now be produced either by automated capillary electrophoresis, or by the more accessible but slower and for some markers slightly less accurate agarose gel methodology.

Fieldable genotyping of Bacillus anthracis and Yersinia pestis based on 25-loci Multi Locus VNTR Analysis

BackgroundAnthrax and plague are diseases caused by Bacillus anthracis and Yersinia pestis respectively. These bacteria are etiological agents for worldwide zoonotic diseases and are considered among the most feared potential bioterror agents. Strain differentiation is difficult for these microorganisms because of their high intraspecies genome homogeneity. Moreover, fast strain identification and comparison with known genotypes may be crucial for naturally occurring outbreaks versus bioterrorist events discrimination.ResultsThirty-nine B. anthracis and ten Y. pestis strains, representative of the species genetic diversity, were genotyped by Agilent 2100 Bioanalyzer using previously described Multiple Locus VNTR Analysis assays (MLVA). Results were compared to previous data obtained by standard genotyping system (capillary electrophoresis on automatic sequencer) and, when necessary, direct amplicon sequencing. A reference comparison table containing actual fragment sizes, sequencer sizes and Agilent sizes was produced.ConclusionIn this report an automated DNA electrophoresis apparatus which provides a cheaper alternative compared to capillary electrophoresis approaches was applied for genotyping of B. anthracis and Y. pesti s. This equipment, uses pre-cast gels and provides easy transportation, low maintenance and overall general logistic requirements and costs, is easy to set up and provides rapid analysis. This platform is a candidate for on-site MLVA genotyping of biothreat agents as well as other bacterial pathogens. It is an alternative to the more expensive and demanding capillary electrophoresis methods, and to the less expensive but more time-consuming classical gel electrophoresis approach.

Co-circulation of Dengue and Chikungunya Viruses, Al Hudaydah, Yemen, 2012

We investigated 400 cases of dengue-like illness in persons hospitalized during an outbreak in Al Hudaydah, Yemen, in 2012. Overall, 116 dengue and 49 chikungunya cases were diagnosed. Dengue virus type 2 was the predominant serotype. The co-circulation of these viruses indicates that mosquitoborne infections represent a public health threat in Yemen.

High throughput MLVA-16 typing for Brucella based on the microfluidics technology.

BackgroundBrucellosis, a zoonosis caused by the genus Brucella, has been eradicated in Northern Europe, Australia, the USA and Canada, but remains endemic in most areas of the world. The strain and biovar typing of Brucella field samples isolated in outbreaks is useful for tracing back source of infection and may be crucial for discriminating naturally occurring outbreaks versus bioterrorist events, being Brucella a potential biological warfare agent. In the last years MLVA-16 has been described for Brucella spp. genotyping. The MLVA band profiles may be resolved by different techniques i.e. the manual agarose gels, the capillary electrophoresis sequencing systems or the microfluidic Lab-on-Chip electrophoresis. In this paper we described a high throughput system of MLVA-16 typing for Brucella spp. by using of the microfluidics technology.ResultsThe Caliper LabChip 90 equipment was evaluated for MLVA-16 typing of sixty-three Brucella samples. Furthermore, in order to validate the system, DNA samples previously resolved by sequencing system and Agilent technology, were de novo genotyped. The comparison of the MLVA typing data obtained by the Caliper equipment and those previously obtained by the other analysis methods showed a good correlation. However the outputs were not accurate as the Caliper DNA fragment sizes showed discrepancies compared with real data and a conversion table from observed to expected data was created.ConclusionIn this paper we described the MLVA-16 using a rapid, sophisticated microfluidics technology for detection of amplification product sizes. The comparison of the MLVA typing data produced by Caliper LabChip 90 system with the data obtained by different techniques showed a general concordance of the results. Furthermore this platform represents a significant improvement in terms of handling, data acquiring, computational efficiency and rapidity, allowing to perform the strain genotyping in a time equal to one sixth respect to other microfluidics systems as e.g. the Agilent 2100 bioanalyzer.Finally, this platform can be considered a valid alternative to standard genotyping techniques, particularly useful dealing with a large number of samples in short time. These data confirmed that this technology represents a significative advancement in high-throughput accurate Brucella genotyping.

Lab on a chip genotyping for Brucella spp. based on 15-loci multi locus VNTR analysis

BackgroundBrucellosis is an important zoonosis caused by the genus Brucella. In addition Brucella represents potential biological warfare agents due to the high contagious rates for humans and animals. Therefore, the strain typing epidemiological tool may be crucial for tracing back source of infection in outbreaks and discriminating naturally occurring outbreaks versus bioterroristic event. A Multiple Locus Variable-number tandem repeats (VNTR) Analysis (MLVA) assay based on 15 polymorphic markers was previously described. The obtained MLVA band profiles may be resolved by techniques ranging from low cost manual agarose gels to the more expensive capillary electrophoresis sequencing. In this paper a rapid, accurate and reproducible system, based on the Lab on a chip technology was set up for Brucella spp. genotyping.ResultsSeventeen DNA samples of Brucella strains isolated in Sicily, previously genotyped, and twelve DNA samples, provided by MLVA Brucella VNTR ring trial, were analyzed by MLVA-15 on Agilent 2100. The DNA fragment sizes produced by Agilent, compared with those expected, showed discrepancies; therefore, in order to assign the correct alleles to the Agilent DNA fragment sizes, a conversion table was produced. In order to validate the system twelve unknown DNA samples were analyzed by this method obtaining a full concordance with the VNTR ring trial results.ConclusionIn this paper we described a rapid and specific detection method for the characterization of Brucella isolates. The comparison of the MLVA typing data produced by Agilent system with the data obtained by standard sequencing or ethidium bromide slab gel electrophoresis showed a general concordance of the results. Therefore this platform represents a fair compromise among costs, speed and specificity compared to any conventional molecular typing technique.

West Nile alternative open reading frame (N-NS4B/WARF4) is produced in infected West Nile Virus (WNV) cells and induces humoral response in WNV infected individuals

BackgroundWest Nile Virus (WNV) is a flavivirus that requires an efficient humoral and cellular host response for the control of neuroinvasive infection. We previously reported the existence of six alternative open reading frame proteins in WNV genome, one of which entitled WARF4 is exclusively restricted to the lineage I of the virus. WARF4 is able to elicit antibodies in WNV infected horses; however, there was no direct experimental proof of the existence of this novel protein. The purpose of this study was to demonstrate the in vitro production of WARF4 protein following WNV infection of cultured VERO cells and its immunity in WNV infected individuals.ResultsWe produced a monoclonal antibody against WARF4 protein (MAb 3A12) which detected the novel protein in WNV lineage I-infected, cultured VERO cells while it did not react with WNV lineage II infected cells. MAb 3A12 specificity to WARF4 protein was confirmed by its reactivity to only one peptide among four analyzed that cover the full WARF4 amino acids sequence. In addition, WARF4 protein was expressed in the late phase of WNV lineage I infection. Western blotting and bioinformatics analyses strongly suggest that the protein could be translated by programmed −1 ribosomal frameshifting process. Since WARF4 is embedded in the NS4B gene, we rename this novel protein N-NS4B/WARF4. Furthermore, serological analysis shows that N-NS4B/WARF4 is able to elicit antibodies in WNV infected individuals.ConclusionsN-NS4B/WARF4 is the second Alternative Reading Frame (ARF) protein that has been demonstrated to be produced following WNV infection and might represent a novel tool for a better characterization of immune response in WNV infected individuals. Further serological as well as functional studies are required to characterize the function of the N-NS4B/WARF4 protein. Since the virus might actually make an extensive use of ARFs, it appears important to investigate the novel six ARF putative proteins of WNV.

A rapid allele variant discrimination method for Yersinia pestis strains based on high-resolution melting curve analysis.

Yersinia pestis isolates were genotyped analyzing the polymorphic DNA regions named variable number tandem repeats (VNTR). Allele variants were studied by high-resolution melting analysis (HRMA) of polymerase chain reaction fragments obtained for 25 VNTR loci. After comparison with previous results, 14 loci gave distinguishable normalized melting curves and allowed to correctly assign alleles. This HRMA typing technique permits to differentiate Y. pestis isolates and turned out to be robust, reproducible, and cheap.

Phylogeny of Dengue and Chikungunya viruses in Al Hudayda governorate, Yemen.

Yemen, which is located in the southwestern end of the Arabian Peninsula, is one of countries most affected by recurrent epidemics caused by emerging vector-borne viruses. Dengue virus (DENV) outbreaks have been reported with increasing frequency in several governorates since the year 2000, and the Chikungunya virus (CHIKV) has been also responsible of large outbreaks and it is now a major public health problem in Yemen. We report the results of the phylogenetic analysis of DENV-2 and CHIKV isolates (NS1 and E1 genes, respectively) detected in an outbreak occurred in Al-Hudayda in 2012. Estimates of the introduction date of CHIKV and DENV-2, and the phylogeographic analysis of DENV-2 are also presented. Phylogenetic analysis showed that the Yemen isolates of DENV belonged to the lineage 2 Cosmopolitan subtype, whereas CHIKV isolates from Yemen belonged to the ECSA genotype. All the CHIKV isolates from Yemen were statistically supported and dated back to the year 2010 (95% HPD: 2009-2011); these sequences showed an alanine in the aminoacid position 226 of the E1 protein. Phylogeographic analysis of DENV-2 virus showed that cluster 1, which included Yemen isolates, dated back to 2003 Burkina Faso strains (95% HPD 1999-2007). The Yemen, cluster dated back to 2011 (95% HPD 2009-2012). Our study sheds light on the global spatiotemporal dynamics of DENV-2 and CHIKV in Yemen. This study reinforces both the need to monitor the spread of CHIKV and DENV, and to apply significant measures for vector control.

Natural humoral immune response to ribosomal P0 protein in colorectal cancer patients

BackgroundTumor associated antigens are useful in colorectal cancer (CRC) management. The ribosomal P proteins (P0, P1, P2) play an important role in protein synthesis and tumor formation. The immunogenicity of the ribosomal P0 protein in head and neck, in breast and prostate cancer patients and the overexpression of the carboxyl-terminal P0 epitope (C-22 P0) in some tumors were reported.MethodsSera from 72 colorectal tumor patients (67 malignant and 5 benign tumors) were compared with 73 healthy donor sera for the presence of antibodies to CEA, EGFR, ErbB2 and ribosomal P proteins by western blotting or ELISA. Expression of the C-22 P0 epitope on tissues and colon cancer cells was determined by immunoperoxidase staining and indirect immunofluorescence/western blotting, respectively, employing MAb 2B2. Biological effects of MAb 2B2 on colon cancer cells were assessed by the Sulforhodamine B cell proliferation assay, trypan blue exclusion test and cleaved caspase-3 detection. Fisher’s exact test was used to compare the number of auto-antibodies positive patients with healthy donors. Variation in the C-22 P0 expression, and in the number of apoptotic cells was evaluated by Student’s t-test. Variation in cell survival and cell death was evaluated by Newman-Keuls test.ResultsNo significant humoral response was observed to CEA, EGFR and ErbB2 in CRC patients. Conversely, 7 out of 67 CRC patient sera reacted to ribosomal P proteins. The prevalence of P proteins auto-antibodies in CRC patients was significant. Five patients showed restricted P0 immunoreactivity, while two patients reacted simultaneously to all P proteins. The C-22 P0 epitope was homogenously expressed both in malignant tumors and the adjacent mucosa, but the intensity of expression was higher in the tumor. Starved colon cancer cells showed a higher C-22 P0 epitope plasma membrane expression compared to control cells. MAb 2B2 inhibited colon cancer cell growth and induced cell death in a dose dependent manner.ConclusionsOur study shows a spontaneous humoral immune response to ribosomal P0 protein in CRC patients and the inhibition of in vitro cancer cell growth after C-22 P0 epitope targeting. The ribosomal P0 protein might be a useful immunological target in CRC patients.

A FRET based melting curve analysis to detect nucleotide variations in HA receptor-binding site of H5N1 virus.

Outbreaks of highly pathogenic H5N1 influenza A virus represent a major public health problem because of the possibility of direct transmission of these viruses from avian species to humans. For influenza H5N1 hemagglutinin, a switch from SA-a-2, 3-Gal to SA-a-2, 6-Gal receptor specificity is a critical step that could lead to inter-human transmission. The monitoring of the receptor-binding preference of H5N1 viruses represents an instrument to detect a potential pandemic virus. The aim of this study was to develop a method based on the fluorescence resonance energy transfer (FRET) technology and melting peaks analysis for rapid screening of pandemic H5N1 influenza A virus. Three selected probes corresponding to a 23bp nucleotide sequence of the avian receptor-binding site were used in a real-time RT-PCR to detect nucleotide variations. Five strains of avian influenza A viruses isolated from avian species and two synthesized HA gene were tested. The results showed that the melting peaks analysis is a reliable screening method for detecting the variability of the H5N1 receptor-binding site.