West Nile Virus lineage 1 in Italy: newly introduced or a re-occurrence of a previously circulating strain?

In Italy, West Nile virus (WNV) appeared for the first time in the Tuscany region in 1998. After 10 years of absence, it re-appeared in the areas surrounding the Po River delta, affecting eight provinces in three regions. Thereafter, WNV epidemics caused by genetically divergent isolates have been documented every year in the country. Since 2018, only WNV Lineage 2 has been reported in the Italian territory. In October 2020, WNV Lineage 1 (WNV-L1) re-emerged in Italy, in the Campania region. This is the first occurrence of WNV-L1 detection in the Italian territory since 2017. WNV was detected in the internal organs of a goshawk (Accipiter gentilis) and a kestrel (Falco tinnunculus). The RNA extracted in the goshawk tissue samples was sequenced, and a Bayesian phylogenetic analysis was performed by a maximum-likelihood tree. Genome analysis, conducted on the goshawk WNV complete genome sequence, indicates that the strain belongs to the WNV-L1 Western-Mediterranean (WMed) cluster. Moreover, a close phylogenetic similarity is observed between the goshawk strain, the 2008–2011 group of Italian sequences, and European strains belonging to the Wmed cluster. Our results evidence the possibility of both a new re-introduction or unnoticed silent circulation in Italy, and the strong importance of keeping the WNV surveillance system in the Italian territory activ

Evidence of rift valley fever seroprevalence in the Sahrawi semi-nomadic pastoralist system, Western Sahara

BACKGROUND: The increasing global importance of Rift Valley fever (RVF) is clearly demonstrated by its geographical expansion. The presence of a wide range of host and vector species, and the epidemiological characteristics of RVF, have led to concerns that epidemics will continue to occur in previously unaffected regions of Africa. The proximity of the Sahrawi territories of Western Sahara to endemic countries, such as Mauritania, Senegal, and Mali with periodic isolation of virus and serological evidence of RVF, and the intensive livestock trade in the region results in a serious risk of RVF spread in the Sahrawi territories, and potentially from there to the Maghreb and beyond. A sero-epidemiological survey was conducted in the Saharawi territories between March and April 2008 to investigate the possible presence of the RVF virus (RVFV) and associated risk factors. A two-stage cluster sampling design was used, incorporating 23 sampling sites. RESULTS: A total of 982 serum samples was collected from 461 sheep, 463 goats and 58 camels. Eleven samples (0.97%) tested positive for IgG against the RVFV. There were clusters of high seroprevalence located mostly in the Tifariti (7.69%) and Mehaires (7.14%) regions, with the Tifariti event having been found in one single flock (4/26 positive animals). Goats and older animals were at a significantly increased risk being seropositive (p = 0.007 and p = 0.007, respectively). CONCLUSION: The results suggest potential RVF activity in the study area, where intense livestock movement and trade with neighbouring countries might be considered as a primary determinant in the spread of the disease. The importance of a continuous field investigation is reinforced, in light of the risk of RVF expansion to historically unaffected regions of Africa

West Nile Virus lineage 2 overwintering in Italy

In January 2022, West Nile virus (WNV) lineage 2 (L2) was detected in an adult female goshawk rescued near Perugia in the region of Umbria (Italy). The animal showed neurological symptoms and died 15 days after its recovery in a wildlife rescue center. This was the second case of WNV infection recorded in birds in the Umbria region during the cold season, when mosquitoes, the main WNV vectors, are usually not active. According to the National Surveillance Plan, the Umbria region is included amongst the WNV low-risk areas. The necropsy evidenced generalized pallor of the mucous membranes, mild splenomegaly, and cerebral edema. WNV L2 was detected in the brain, heart, kidney, and spleen homogenate using specific RT-PCR. Subsequently, the extracted viral RNA was sequenced. A Bayesian phylogenetic analysis performed through a maximum-likelihood tree showed that the genome sequence clustered with the Italian strains within the European WNV strains among the central-southern European WNV L2 clade. These results, on the one hand, confirmed that the WNV L2 strains circulating in Italy are genetically stable and, on the other hand, evidenced a continuous WNV circulation in Italy throughout the year. In this report case, a bird-to-bird WNV transmission was suggested to support the virus overwintering. The potential transmission through the oral route in a predatory bird may explain the relatively rapid spread of WNV, as well as other flaviviruses characterized by similar transmission patterns. However, rodent-to-bird transmission or mosquito-to-bird transmission cannot be excluded, and further research is needed to better understand WNV transmission routes during the winter season in Ital

Lack of detection of West Nile virus in an islander population of chelonians during a West Nile virus outbreak.

In 2011, several outbreaks of West Nile disease occurred in Sardinia (Italy). The region hosts several chelonian species. Because of the increasing concern on the potential role that ectotherms may play in the ecology of West Nile virus (WNV), in October 2011 blood samples were collected from 41 endemic Sardinian chelonians and tested for the presence of active WNV infection or neutralizing antibodies by real time polymerase chain reaction (RT-PCR) and serumneutralisation, respectively. Neither WNV neutralising antibodies (0%; 95% CI: 0‑8.4%) nor WNV RNA (0%; 95% CI: 0‑6.8%) were found in the tested samples. According to the results of this screening survey, it is unlikely that chelonians are involved in the epidemiology of the 2011 WNV outbreaks in Sardinia

West Nile and Usutu Virus Introduction via Migratory Birds: A Retrospective Analysis in Italy

The actual contribution of migratory birds in spreading West Nile (WNV) and Usutu virus (USUV) across Europe and from Africa to old countries is still controversial. In this study, we reported the results of molecular and serological surveys on migrating birds sampled during peaks of spring and autumn migration at 11 Italian sites located along important flyways, from 2012 to 2014. A total of 1335 specimens made of individual or pooled sera, and organs from 275 dead birds were tested for WNV and USUV RNA by real time PCR (RT-PCR). Furthermore, sera were tested by serum neutralization assay for detecting WNV and USUV neutralizing antibodies. Molecular tests detected WNV lineage 2 RNA in a pool made of three Song Thrush (Turdus philomelos) sera sampled in autumn, and lineage 1 in kidneys of six trans-Saharan birds sampled in spring. Neutralizing antibodies against WNV and USUV were found in 5.80% (n = 72; 17 bird species) and 0.32% (n = 4; 4 bird species) of the tested sera, respectively. Our results do not exclude the role of migratory birds as potential spreaders of WNV and USUV from Africa and Central Europe to Mediterranean areas and highlight the importance of a more extensive active surveillance of zoonotic viruses

Transplacental transmission of the Italian Bluetongue virus serotype 2 in sheep

In order to study the capability of a Bluetongue virus serotype 2 (BTV‑2) field isolate to cross the placental barrier, 2 groups of 5 pregnant ewes were infected with a field BTV‑2 Italian strain (Group A) or with the same strain passaged once in Culicoides cells (Kc) (Group B). Following infection, EDTA‑blood and serum samples were collected weekly and tested for the presence of BTV RNA/infectious virus and anti‑BTV‑2 antibodies, respectively. At lambing, precolostral EDTA‑blood and serum samples were collected from lambs and tested as before. The lambs were then sampled as scheduled for the dams. All sheep seroconverted on day 12 post‑infection (pi) and remained seropositive throughout the sampling period (day 68 pi). BTV was isolated from day 7 pi to day 14 pi in animals of Group A and from day 5 pi to day 12 pi in animals of Group B. None of the 14 lambs born had pre‑colostral antibodies. Three lambs born from two ewes of Group B were viraemic at birth and in one lamb infectious virus was isolated from blood up to 11 days of age. This study proved for the first time that a single passage of BTV‑2 field strain in Kc cells is able to give to BTV the ability to cross the placenta barrier and infect foetal tissues

Antigenic relationship among zoonotic flaviviruses from Italy

Here we report studies of the antigenic relationship of West Nile virus (WNV) and Usutu virus (USUV), two zoonotic flaviviruses from Italy, together with a Japanese encephalitis virus (JEV) strain and compared them with their genetic relationship using the immunodominant viral E protein. Thirty-nine isolates and reference strains were inactivated and used to immunize rabbits to produce hyper immune sera. Serum samples were tested by neutralization against all isolates and results visualized by generating antigenic map. Strains of WNV, USUV, and JEV grouped in separate clusters on the antigenic map. JEV was closer antigenically to USUV (mean of 3.5 Antigenic Unit, AU, equivalent to a 2-fold change in antibody titer) than to WNV strains (mean of 6 AU). A linear regression model predicted, on average, one unit of antigenic change, equivalent to a 2-fold change in antibody titer, for every 22 amino acid substitutions in the E protein ectodomain. Overall, antigenic map was demonstrated to be robust and consistent with phylogeny of the E protein. Indeed, the map provided a reliable means of visualizing and quantifying the relationship between these flaviviruses. Further antigenic analyses employing representative strains of extant serocomplexes are currently underway. This will provide a more in deep knowledge of antigenic relationships between flaviviruses

USUTU VIRUS IN ITALY, AN EMERGENCE OR A SILENT INFECTION?

International audienceA two year study (2008-2009) was carried out to monitor the Usutu virus (USUV) circulation in Italy. Sentinel horses and chickens, wild birds and mosquitoes were sampled and tested for the presence of USUV and USUV antibodies within the WND National Surveillance plan. Seroconversion evidenced in sentinel animals proved that in these two years the virus has circulated in Tuscany, Emilia Romagna, Veneto and Friuli Venezia Giulia regions. In Veneto USUV caused a severe blackbird die-off disease involving at least a thousand birds. Eleven viral strains were detected in organs of 9 blackbirds (52.9%) and two magpies (0.5%) originating from Veneto and Emilia Romagna regions. USUV was also detected in a pool of caught in Tuscany. According to the alignment of the NS5 partial sequences, no differences between the Italian USUV strains isolated from Veneto, Friuli and Emilia Romagna regions were observed. The Italian North Eastern strain sequences were identical to those of the strain detected in the brain of a human patient and shared a high similarity with the isolates from Vienna and Budapest. Conversely, there were few differences between the Italian strains which circulated in the North Eastern regions and the USUV strain detected in a pool of caught in Tuscany. A high degree of similarity at both nucleotide and amino acid level was also found when the full genome sequence of the Italian North Eastern isolate was compared with that of the strains circulating in Europe. The North Eastern Italian strain sequence exhibited 97% identity to the South African reference strain SAAR-1776. The deduced amino acid sequences of the Italian strain differed by 10 and 11 amino-acids from the Budapest and Vienna strains, respectively, and by 28 from the SAAR-1776 strain. According to this study two strains of USUVs are likely to have circulated in Italy between 2008 and 2009. They have developed strategies of adaptation and evolution to spread into new areas and to become established

Descrizione dei focolai di west nile disease nel 2011 nella regione Sardegna, Italia

In 2011, strains of West Nile Virus (WNV) belonging to lineage 1 spread for the first time in Sardinia region (Italy). In contrast to previous WNV Italian incursion, the strains were found in Culex modestus and, more surprisingly, they were able to cause severe clinical signs in the affected birds. Based on the partial sequence of the NS3 encoding gene, the Sardinian WNV strains demonstrated a high similarity with the other WNV strains recently detected in the Mediterranean Basin. Nonetheless, the 2011 Sardinian sequences were grouped in a distinct sub-cluster. Both the NS3-249P and NS3-249T genotypes were detected in the Sardinian outbreaks confirming that the co-circulation of different genotypes in the affected population might be common for WNV as for many RNA viruses. No association, however, was observed between virulence and viral genotype

SARS-CoV-2 RNA Persistence in Naso-Pharyngeal Swabs

Since February 2020, Italy has been seriously affected by the SARS-CoV-2 pandemic. To support the National Health Care system, naso-pharyngeal/oropharyngeal swabs collected from suspected cases of Teramo province, Abruzzo region, are tested at Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise G. Caporale, for the presence of SARS-CoV-2 RNA. Out of 12,446 tested individuals, 605 returned positive results at least once, with prevalence significantly higher in men. A reduction of the level of viral RNA in the first swab per each positive patient collected over time was also observed. Moreover, 81 patients had at least one positive sample and two final negative tests: positivity in swabs lasted from 14 to 63 days, with a median value of 30 days. This shows the potential for the virus to coexist with patients for a long time, although we highlighted intermittent positivity in several cases. The evolution of the SARS-CoV-2 epidemiological situation and knowledge on viral shedding should be closely monitored, to interpret the findings correctly and adjust accordingly the surveillance activities