Results of the RT-PCR (two methods) performed on bodies and legs of virus-exposed females.

<p>Results of the RT-PCR (two methods) performed on bodies and legs of virus-exposed females.</p

Mean percent of adults and number of adults per gonotrophic cycle from virus-exposed females.

<p>HE-sA, human erythrocytes plus viral strain A. HE-sB, human erythrocytes plus viral strain B.</p><p>(*)Calculated on the total number of eggs laid by the females in the three gonotrophic cycles.</p

Percentage of blood fed females, fecundity and fertility after the three blood meals (°).

<p>R, whole rabbit erythrocytes based blood meal. HE, human erythrocytes based blood meal. HE-M, human erythrocytes plus virus culture medium. HE-sA, human erythrocytes plus viral strain A. HE-sB, human erythrocytes plus viral strain B. a, b, different letters indicate statistically significant differences at the probability level P = 0.05. NS, non significant.</p><p>(°) Statistics are referred to comparisons among females blood fed with different kinds of blood in the same gonotrophic cycle.</p

Number of adults obtained per gonotrophic cycle from non-infected control females.

<p>R, whole rabbit erythrocytes-based blood meal. HE, human erythrocytes -based blood meal. HE-M, human erythrocytes plus virus culture medium.</p><p>(*)Calculated on the total number of eggs laid by the females in the three gonotrophic cycles.</p><p>(°) The total number of adults is followed by the ratio number of females/number of males.</p

Number of viral copies detected by RT-PCR (method 2) at increasing time periods post infection.

<p>(□: A1; ○: B1).</p

Phlebotomine sand fly–borne pathogens in the Mediterranean Basin: Human leishmaniasis and phlebovirus infections

<div><p>Pathogens transmitted to humans by phlebotomine sand flies are neglected, as they cause infectious diseases that are not on the priority list of national and international public health systems. However, the infections caused by protozoa of the <i>Leishmania</i> genus and viruses belonging to the <i>Phlebovirus</i> genus (family Phenuiviridae)—the most significant group of viruses transmitted by sand flies—have a relevant role for human pathology. These infections are emerging in the Mediterranean region and will likely spread in forthcoming decades, posing a complex threat to human health. Four species and 2 hybrid strains of <i>Leishmania</i> are pathogenic for humans in the Mediterranean Basin, with an estimated annual incidence of 239,500–393,600 cases of cutaneous leishmaniasis and 1,200–2,000 cases of visceral leishmaniasis. Among the phleboviruses, Toscana virus can cause neuroinvasive infections, while other phleboviruses are responsible for a typical “3-day fever”; the actual incidence of <i>Phlebovirus</i> infections in the Mediterranean area is unknown, although at least 250 million people are exposed. Here, we reviewed the current literature on epidemiology of sand fly–borne infections in the Mediterranean Basin, with a focus on humans. Our analysis indicates the need for increased public health activities directed to determine the disease burden of these infections as well as to improve their surveillance. Among the emerging challenges concerning sand fly–borne pathogens, the relationships between sand fly–borne protozoa and viruses should be considered in future studies, including epidemiological links between <i>Leishmania</i> and phleboviruses as well as the conditional capacity for these pathogens to be involved in interactions that may evolve towards increased virulence.</p></div

Additional file 1: Figure S1. of Isolation of three novel reassortant phleboviruses, Ponticelli I, II, III, and of Toscana virus from field-collected sand flies in Italy

Pictures of two of the sampled sites: site 1 (left) and site 2 (right). (PDF 342 kb

Transmission cycle, geographical distribution, and proven/suspected reservoirs of <i>Leishmania</i> in the Mediterranean region.

<p>Transmission cycle, geographical distribution, and proven/suspected reservoirs of <i>Leishmania</i> in the Mediterranean region.</p

Phleboviruses phylogeny reconstruction.

<p>Phylogenetic relationships between selected Old World sand fly–borne phleboviruses based on partial large (L) RNA sequences; phylogenetic relationships of selected amino acid sequences were inferred by using the maximum likelihood method based on the Jones-Taylor-Thornton (JTT) model gamma distributed with invariant sites [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005660#pntd.0005660.ref035" target="_blank">35</a>]. A discrete gamma distribution was used to model evolutionary-rate differences among sites [5 categories]. The analysis involved 44 sequences. All positions with less than 95% site coverage were eliminated so that there was a total of 55 positions in the final dataset. Evolutionary analysis was conducted in MEGA6 using 500 bootstrap pseudoreplications [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005660#pntd.0005660.ref035" target="_blank">35</a>].</p

Leishmanial species circulating in the Mediterranean region and pathogenic for humans.

<p>Leishmanial species circulating in the Mediterranean region and pathogenic for humans.</p