Bayesian phylogenetic analysis of the influenza-A virus genomes isolated in Tunisia, and determination of potential recombination events.

The influenza-A virus (IAV) causes seasonal epidemics and presents a pandemic risk with the possibility of genetic re-assortment, allowing the emergence of new strains. The evolution of IAVes is done most often by relatively frequent re-assortment between gene segments, but the hypothesis of their evolution by recombination between RNA segments has not been justified to this date. Here, we examine this hypothesis by Bayesian phylogenetic analysis, to test if recombination events have occurred between genomic RNA segments. Different IAV subtypes are observed in co-circulation in Tunisia, which increases the probability of occurrence of double infections. Mixed infections are a prerequisite for recombination between co-infecting of viral strains. The aim of this work, and since understanding the evolutionary dynamics of IAV is essential for controlling human and avian influenza, phylogenetic analyzes (Bayesian approach) have been carried out for IAV strains isolated in Tunisia, to study their co-evolutionary history, trends, and possible recombination models. A set of IAV nucleic sequences, isolated in Tunisia from 2009 to 2013 (n = 102) were used in this study. These genomic segments encode various influenza A proteins. These viral strains studied were isolated following the 2009 H1N1 pandemic. The analyzes identified two large distinct groups of viral sequences and different subgroups. Assuming a relaxed molecular clock model (uncorrelated exponential (uced)) in a Bayesian coalescence approach and a constant effective time demographic history model (Coalescent: constant size), the substitution rate was estimated at 1.356 × 10-3 substitutions/site/year for segment 4 (haemagglutinin HA gene). Consistent estimates of the age of the most recent Common Ancestor (MRCA) were obtained for the different subgroups, the MRCA ages of the two viral populations corresponding to segment 4 and segment 6 (neuraminidase gene NA) of the genome are estimated at 443.737 years and 501.159 years respectively. A detailed phylogenetic study of the HA gene was performed. The incongruous phylogenetic models deduced for the three genomic subgroups studied corresponding to this gene were indicative of recombination events between the different subpopulations. The detection of these relative signals indicating the presence of recombination events can be considered as proof that recombination seems to play a role, even a small one, in the evolution of (IAV). Reliable recombination sites have been located with statistical significance between H3, H1 and H9 subtypes. MRCA age estimates of recombinants phylogenetic clades indicate directional gene transfers from the H1 and H9 populations to the H3 population, and from H1 and H3 to the H9 population, and their co-divergences during the study period.

Investigation of Extracts from Tunisian Ethnomedicinal Plants as Antioxidants, Cytotoxins, and Antimicrobials / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To determine the medicinal potential of various plants and their parts extracted with different solvents.</p><p><b>METHODS</b>The total phenolic content of acetonitrile/water (60%-40%) (ACN/W) and aqueous (W) extract fractions was determined by high-performance liquid chromatography (HPLC), and terpenic compounds were detected by gas chromatography/mass spectrometry (GC/MS). Antioxidant activity of the samples was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay and β-carotene bleaching method. Cell viability was investigated by thiazolyl blue tetrazolium bromide [3-(4,5-dimethylthiazol)-2-yl 2,5-diphenyltetrazolium bromide] (MTT) assay. The mechanisms involved in cytotoxic activity were investigated in a murine macrophage cell line (RAW 264.7) and cancer lines.</p><p><b>RESULTS</b>Our findings show that 11 plant species exhibited biological activity. In addition, moderate antibacterial activity was reported against one or more of the tested bacterial strains at two concentrations: 300 μg and 3 mg/disc. Furthermore, our data reveal that among all plants investigated, some extract and hydrophobic fractions were potent scavengers of the DPPH radical (6.78 μg/mL < EC50 < 8.55 μg/mL). Taken together, our results show that Nerium oleander (NOACN/W) and Pituranthos tortuosus (PTACN/W) were highly cytotoxic against RAW 264.7 cells with IC80 values of 0.36, and 1.55 μg/mL, respectively. In contrast, murine macrophage cell lines had low growth and were significantly sensitive to water extracts of Thymus hirtus sp. algeriensis (THW), Lavandula multifida (LMW), and ACN/W extract of Erica multiflora (EMACN/W) at doses > 400, 47.20, and 116.74 μg/mL, respectively. The current work demonstrates that RAW 264.7 cell proliferation was inhibited by samples in a dose-dependent manner.</p><p><b>CONCLUSION</b>Our findings, validated through free radical scavenging activity, agar diffusion assay, and cytotoxicity of essential oils towards cancer cells, show that ethnomedicinal plants used in this work have a novel application as a tumor suppressor.</p>

Assessing the effect of zooprophylaxis on zoonotic cutaneous leishmaniasis transmission: a system dynamics approach.

Capturing or diverting the disease carrying vector from humans can reduce the transmission of vector borne diseases such as leishmaniasis. The use of animals that act as dead-end hosts to relieve the vector (sandfly) bites on humans is called zooprophylaxis. However, as the number of blood meal providers especially domestic animals increases, the sandflies enhanced availability of blood meals will improve its number and survival, thereby countering the impact of diverting bites from humans. Thus, the transmission model exhibits the structure of a feedback loop characterizing complex dynamic systems. In order to rigorously assess the effect of zooprophylaxis, we propose a system dynamic model for zoonotic cutaneous leishmaniasis transmission with 3 blood-meal hosts: domestic animals, humans, and a reservoir (rodents). In this context, a simulation study of the proposed model with a follow-up period of 1000 days was performed. We explored how perturbations in the parameters characterizing the transmission, essentially the vector biting rates and the size of the domestic animal population, affect the zooprophylaxis outcome. The results show that the basic reproductive number R0 and the disease incidence in humans are decreasing function of the relative size of the domestic animal population. The speed of this decrease depends also on the vector biting rates of the different mammal species. The key factors influencing the magnitude of zooprophylaxis are: the sizes of the vector, rodent, and domestic animal populations, as well as, the biting rates which incorporate relative attraction and accessibility of the vectors to the mammalian populations.

Lack of protection of pre-immunization with saliva of long-term colonized Phlebotomus papatasi against experimental challenge with Leishmania major and saliva of wild-caught P. papatasi.

Immunity to saliva of Phlebotomus papatasi protects against Leishmania major infection as determined by co-inoculation of parasites with salivary gland homogenates (SGHs) of this vector. These results were obtained with long-term colonized female P. papatasi. We investigated the effect of pre-immunization with SGH of long-term colonized P. papatasi against L. major infection co-inoculated with SGH of wild-caught P. papatasi. Our results showed that pre-exposure to SGH of long-term, colonized P. papatasi do not confer protection against infection with L. major co-inoculated with SGH of wild-caught P. papatasi. These preliminary results strongly suggest that the effectiveness of a vector saliva-based vaccine derived from colonized sand fly populations may be affected by inconsistent immune response after natural exposure.

DNA based vaccination with a cocktail of plasmids encoding immunodominant Leishmania (Leishmania) major antigens confers full protection in BALB/c mice.

Despite the lack of effective vaccines against parasitic diseases, the prospects of developing a vaccine against leishmaniasis are still high. With this objective, we have tested four DNA based candidate vaccines encoding to immunodominant leishmania antigens (LACKp24, TSA, LmSTI1 and CPa). These candidates have been previously reported as capable of eliciting at least partial protections in the BALB/c mice model of experimental cutaneous leishmaniasis. When tested under similar experimental conditions, all of them were able to induce similar partial protective effects, but none could induce a full protection. In order to improve the level of protection we have explored the approach of DNA based vaccination with different cocktails of plasmids encoding to the different immunodominant Leishmania antigens. A substantial increase of protection was achieved when the cocktail is composed of all of the four antigens; however, no full protection was achieved when mice were challenged with a high dose of parasite in their hind footpad. The full protection was only achieved after a challenge with a low parasitic dose in the dermis of the ear. It was difficult to determine clear protection correlates, other than the mixture of immunogens induced specific Th1 immune responses against each component. Therefore, such an association of antigens increased the number of targeted epitopes by the immune system with the prospects that the responses are at least additive if not synergistic. Even though, any extrapolation of this approach when applied to other animal or human models is rather hazardous, it undoubtedly increases the hopes of developing an effective leishmania vaccine.

Zooprophylaxis: impact of breeding rabbits around houses on reducing the indoor abundance of Phlebotomus papatasi.

Zooprophylaxis is the use of animals to deviate vectors from humans. The indoor abundance of Phlebotomus papatasi in houses with rabbit holes in the peridomestic areas are significantly lower than the indoor abundance in houses without rabbit holes in their peridomestic areas. Introduction of rabbits in artificial underground holes in peridomestic areas reduced significantly the indoor abundance of P. papatasi. Cleaning rabbit holes in peridomestic area by removing all rabbit feces induced a significant increase in the abundance of P. papatasi inside bedrooms. The ecologic niche made around houses in endemic areas by creating active rabbit holes is a major source of attractiveness of P. papatasi, and therefore it may deviate the vector from humans to rabbits. Although rabbit holes are breeding sites for P. papatasi, rabbits are not competent reservoirs for Leishmania major. Our overall findings strongly suggest that zooprophylaxis could be effective in reducing the indoor abundance of P. papatasi and subsequently may be used to control the transmission L. major in rural areas.

Field trials of a very potent rabies DNA vaccine which induced long lasting virus neutralizing antibodies and protection in dogs in experimental conditions.

Kinetics of antibody responses and protection against rabies were investigated after injection of a single dose of rabies DNA vaccine and compared to those induced by one or two injections of cell culture-derived vaccine in dogs issued from the common local breed and reared in experimental conditions. Rabies DNA vaccine administered intradermally by a jet injector in the inner face of the ear was by far more efficient in inducing long lasting high titers of virus neutralizing antibodies compared to cell culture vaccine Rabisin administered either subcutaneously or intramuscularly. Four years after vaccine administration of either DNA or cell culture-derived rabies vaccines, full protection against a rabies peripheral challenge was achieved. Vaccine trials targeting dogs living in field conditions in Tunisia further established that rabies DNA-based vaccination induced a stronger induction of virus neutralizing antibodies compared to Rabisin. This report shows for the first time that DNA vaccination could be more efficient under experimental or field conditions in large size mammals than the best commercially available cell culture-derived vaccine. This improvement will hopefully allow a better rabies control in developing countries by using a more efficient vaccination with fewer doses and targeting all categories of dogs.

A comparative evaluation of different DNA vaccine candidates against experimental murine leishmaniasis due to L. major.

Over the past few years, several reports of DNA vaccines against murine cutaneous experimental leishmaniasis came out with promising but sometimes discordant results. The present studies were designed to compare, under similar conditions, the protective effects in the highly susceptible BALB/c mice of DNA vaccine candidates encoding to various Leishmania major antigens. The candidate DNA vaccines encode to the following antigens: LACK, PSA2, Gp63, LeIF and two newly identified p20 and Ribosomal like protein, in addition to different truncated portions of the LACK antigen. The most promising gene was LACK and it is more protective when it is used as a p24 truncated form. Furthermore, the presence of a tandem repeats of immunostimulating sequences (ISS) in the plasmid backbone played an important adjuvant effect in the observed protective effect induced by the DNA vaccine encoding to the LACKp24. Nevertheless, neither of the DNA vaccine candidates was able to mount a full protection in BALB/c mice challenged with a highly virulent L. major strain. Further improvements of the DNA vaccination approach are still needed to design a fully protective vaccine against leishmaniasis. Three directions of investigations are currently explored: DNA vaccines using a cocktail of antigens; Prime/Boost approach; and association of immune modulators with the candidate antigens.

Post-exposure therapy in mice against experimental rabies: a single injection of DNA vaccine is as effective as five injections of cell culture-derived vaccine.

Two rabies post-exposure therapies were comparatively evaluated: BALB/c mice were challenged at day 0 with rabies virus and then received either a single dose of rabies DNA vaccine administered at day 0, or five doses of cell culture-derived rabies vaccine administered at days 0, 3, 7, 15 and 28. Both regimens, rapidly triggered protective levels of neutralizing antibodies against rabies virus in vaccinated mice. In addition, one injection of DNA vaccine protected 53% of the challenged mice, compared to 40% of mice protected after five injections of cell culture-derived vaccine. We conclude that rabies post-exposure vaccination in BALB/c mice, based on a single administration of rabies DNA vaccine might be at least as effective as five injections of cell culture-derived vaccine.