Unsupervised machine learning and geometric morphometrics as tools for the identification of inter and intraspecific variations in the Anopheles Maculipennis complex

Geometric morphometric analysis was combined with two different unsupervised machine learning algorithms, UMAP and HDBSCAN, to visualize morphological differences in wing shape among and within four Anopheles sibling species (An. atroparvus, An. melanoon, An. maculipennis s.s. and An. daciae sp. inq.) of the Maculipennis complex in Northern Italy. Specifically, we evaluated: (1) wing shape variation among and within species; (2) the consistencies between groups of An. maculipennis s.s. and An. daciae sp. inq. identified based on COI sequences and wing shape variability; and (3) the spatial and temporal distribution of different morphotypes. UMAP detected at least 13 main patterns of variation in wing shape among the four analyzed species and mapped intraspecific morphological variations. The relationship between the most abundant COI haplotypes of An. daciae sp. inq. and shape ordination/variation was not significant. However, morphological variation within haplotypes was reported. HDBSCAN also recognized different clusters of morphotypes within An. daciae sp. inq. (12) and An. maculipennis s.s. (4). All morphotypes shared a similar pattern of variation in the subcostal vein, in the anal vein and in the radio-medial cross-vein of the wing. On the contrary, the marginal part of the wings remained unchanged in all clusters of both species. Any spatial-temporal significant difference was observed in the frequency of the identified morphotypes. Our study demonstrated that machine learning algorithms are a useful tool combined with geometric morphometrics and suggest to deepen the analysis of inter and intra specific shape variability to evaluate evolutionary constrains related to wing functionality

Arbovirus Screening in Mosquitoes in Emilia-Romagna (Italy, 2021) and Isolation of Tahyna Virus

Several viruses can be transmitted by mosquitoes. We searched some of these viruses in 20,778 mosquitoes, collected in 95 traps on the plains of Emilia-Romagna (North of Italy) in 2021. We detected West Nile virus (WNV) and Usutu virus (USUV) in pools of Culex (Cx.) pipiens. In addition, we detected two insect-specific flaviviruses in three pools of Aedes (Ae.) caspius and in two of Ae. vexans. Tahyna virus (TAHV) was detected in six pools, three of Ae. caspius and three of Cx. pipiens, and one isolated strain was obtained from one of the Ae. caspius pools. Moreover, we detected TAHV in pools of several mosquito species (Ae. caspius, Ae. vexans, Ae. albopictus, Anopheles maculipennis s.l.) collected in the previous year of surveillance. Our data indicate Ae. caspius as the species most infected with TAHV in the surveyed area. Together with the likely plasticity of the cycle, we reported strong genome stability of the TAHV, probably linked to a successful adaptation of the virus to its ecological niche. Interestingly, in six pools of Cx. pipiens we detected two associated viruses among USUV, WNV, TAHV and all the three viruses in two pools. This result allows us to assume the presence of particular conditions that prompt the circulation of arboviruses, creating the conditions for viral hot spots. While no human diseases related to Tahyna virus were reported in Italy, its detection over the years suggests that it is worth investigating this virus as a potential cause of disease in humans in order to assess its health burden. IMPORTANCE We reported in this work the detection of three Arboviruses (Arthropod-borne viruses) in mosquitoes collected in Emilia-Romagna in 2021. In addition to West Nile and Usutu viruses, which were reported from more than 10 years in the study area, we detected and isolated Tahyna virus (TAHV). We also reported detections of TAHV obtained in previous years of surveillance in different species of mosquitoes. TAHV is the potential causative agent of summer influenza-like diseases and also of meningitis. Even if human cases of disease referable to this virus are not reported in Italy, its relevant presence in mosquitoes suggests investigating the possibility they could

Chikungunya Virus in Aedes albopictus, Italy

Chikungunya Virus in Aedes albopictus, Ital

Retrospective screening of serum and cerebrospinal fluid samples from patients with acute meningo-encephalitis does not reveal past Japanese encephalitis virus infection, Emilia Romagna, Italy, 2011.

P Gaibani1, A C Finarelli2, R Cagarelli2, A Pierro1, G Rossini1, M Calzolari3, M Dottori3, P Bonilauri3, M P Landini1, V Sambri ([email protected])1 1. Operative Unit of Clinical Microbiology, Regional Reference Centre for Microbiological Emergencies, S. Orsola-Malpighi University Hospital, Bologna, Italy 2. Public Health Authority, Emilia Romagna Region, Bologna, Italy 3. Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia-Romagna 'B. Ubertini' (IZSLER; Experimental veterinary institute of Lombardy and Emilia Romagna), Reggio Emilia, Ital

PCV2 infection in vaccinated conventional gilts inseminated with PCV2b-spiked semen

The present trial investigated the effect of PCV2 vaccination on viremia, virus shedding and viral load in maternal tissues and foetuses of conventional gilts inseminated with PCV2b-spiked semen. Twelve gilts were randomly divided into two groups of six animals each (vaccinated infected, VI; non-vaccinated infected, NVI). Estrus synchronization was followed by artificial insemination (AI) with a single PCV2 negative semen dose supplemented with 0.2 mL of a PCV2b suspension containing 104.4 TCID50/50 \u3bcL (total viral dose: 105 TCID50). Vaginal, nasal and faecal swabs, and blood samples were collected weekly from two days before artificial insemination till the end of the experimental period (55 days post AI; DPAI) and tested by real-time PCR (qPCR) for PCV2; sera were tested for anti- PCV2 antibodies. During necropsy foetal and maternal tissues were collected for qPCR and histopathology. In each of the VI and NVI groups three out of the six gilts were pregnant at 29 DPAI. The VI group showed a significantly lower proportion of PCR-positive swabs: 24.6% VI vs 71.3% NVI. PCV2 clearance was demonstrated by qPCR in lymphoid tissue during the trial in the VI group. Only one foetus was PCV2-positive (in the NVI group) and three amniotic fluids of the NVI group. PCV2 was found in a significantly lower proportion of the placenta of foetuses in the VI group (39%) than the NVI group (77%). The PCV2 vaccine seems to play an active role in reducing virus shedding, tissue viral load and foetoplacental infection

Mosquito, Bird and Human Surveillance of West Nile and Usutu Viruses in Emilia-Romagna Region (Italy) in 2010

<div><h3>Background</h3><p>In 2008, after the first West Nile virus (WNV) detection in the Emilia-Romagna region, a surveillance system, including mosquito- and bird-based surveillance, was established to evaluate the virus presence. Surveillance was improved in following years by extending the monitoring to larger areas and increasing the numbers of mosquitoes and birds tested.</p> <h3>Methodology/Principal Findings</h3><p>A network of mosquito traps, evenly distributed and regularly activated, was set up within the surveyed area. A total of 438,558 mosquitoes, grouped in 3,111 pools and 1,276 birds (1,130 actively sampled and 146 from passive surveillance), were tested by biomolecular analysis. The survey detected WNV in 3 <em>Culex pipiens</em> pools while Usutu virus (USUV) was found in 89 <em>Cx. pipiens</em> pools and in 2 <em>Aedes albopictus</em> pools. Two birds were WNV-positive and 12 were USUV-positive. Furthermore, 30 human cases of acute meningoencephalitis, possibly caused by WNV or USUV, were evaluated for both viruses and 1,053 blood bags were tested for WNV, without any positive result.</p> <h3>Conclusions/Significance</h3><p>Despite not finding symptomatic human WNV infections during 2010, the persistence of the virus, probably due to overwintering, was confirmed through viral circulation in mosquitoes and birds, as well as for USUV. In 2010, circulation of the two viruses was lower and more delayed than in 2009, but this decrease was not explained by the relative abundance of <em>Cx. pipiens</em> mosquito, which was greater in 2010. The USUV detection in mosquito species confirms the role of <em>Cx. pipiens</em> as the main vector and the possible involvement of <em>Ae. albopictus</em> in the virus cycle. The effects of meteorological conditions on the presence of USUV-positive mosquito pools were considered finding an association with drought conditions and a wide temperature range. The output produced by the surveillance system demonstrated its usefulness and reliability in terms of planning public health policies.</p> </div

West Nile virus transmission. results from the integrated surveillance system in Italy, 2008 to 2015

IIn Italy a national Plan for the surveillance of imported and autochthonous human vector-borne diseases (chikungunya, dengue, Zika virus disease and West Nile virus (WNV) disease) that integrates human and veterinary (animals and vectors) surveillance, is issued and revised annually according with the observed epidemiological changes. Here we describe results of the WNV integrated veterinary and human surveillance systems in Italy from 2008 to 2015. A real time data exchange protocol is in place between the surveillance systems to rapidly identify occurrence of human and animal cases and to define and update the map of affected areas i.e. provinces during the vector activity period from June to October. WNV continues to cause severe illnesses in Italy during every transmission season, albeit cases are sporadic and the epidemiology varies by virus lineage and geographic area. The integration of surveillance activities and a multidisciplinary approach made it possible and have been fundamental in supporting implementation of and/or strengthening preventive measures aimed at reducing the risk of transmission of WNV trough blood, tissues and organ donation and to implementing further measures for vector control

Inventory of available data and data sources and proposal for data collection on vector‐borne zoonoses in animals

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Evidence of Simultaneous Circulation of West Nile and Usutu Viruses in Mosquitoes Sampled in Emilia-Romagna Region (Italy) in 2009

BACKGROUND: In recent years human diseases due to mosquito-borne viruses were increasingly reported in Emilia-Romagna region (Italy), from the chikungunya virus in 2007 to the West Nile virus (WNV) in 2008. An extensive entomological survey was performed in 2009 to establish the presence and distribution of mosquito arboviruses in this region, with particular reference to flaviviruses. METHODOLOGY/PRINCIPAL FINDINGS: From May 6 to October 31, a total of 190,516 mosquitoes were sampled in georeferenced stations, grouped in 1,789 pools according date of collection, location, and species, and analyzed by reverse transcription polymerase chain reaction (RT-PCR) to detect the presence of RNA belong to Flavivirus genus. WNV was detected in 27 mosquito pools, producing sequences similar to those of birds and human strains obtained in 2008 outbreak, pointed out the probable virus overwintering. Isolation of WNV was achieved from one of these pools. Moreover 56 pools of mosquitoes tested positive for Usutu virus (USUV). Most PCR positive pools consisted of Culex pipiens, which also was the most analyzed mosquito species (81.4% of specimens); interestingly, USUV RNA was also found in two Aedes albopictus mosquito pools. Simultaneous circulation of WNV and USUV in the survey area was highlighted by occurrence of 8 mosquito WNV- and USUV-positive pools and by the overlaying of the viruses "hot spots", obtained by kernel density estimation (KDE) analysis. Land use of sampled stations pointed out a higher proportion of WNV-positive Cx. pipiens pool in rural environments respect the provenience of total sampled pool, while the USUV-positive pools were uniformly captured in the different environments. CONCLUSIONS/SIGNIFICANCE: Obtained data highlighting the possible role of Cx. pipiens mosquito as the main vector for WNV and USUV in Northern Italy, and the possible involvement of Ae. albopictus mosquito in USUV cycle. The described mosquito-based surveillance could constitute the foundation for a public health alert system targeting mosquito borne arboviruses