A 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 Culex pipiens 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 C. pipiens 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.

Bluetongue is a major infectious disease of ruminants that is caused by bluetongue virus (BTV). In this study, we analyzed virulence and genetic differences of (i) three BTV field strains from Italy maintained at either a low (L strains) or high (H strains) passage number in cell culture and (ii) three South African "reference" wild-type strains and their corresponding live attenuated vaccine strains. The Italian BTV L strains, in general, were lethal for both newborn NIH-Swiss mice inoculated intracerebrally and adult type I interferon receptor-deficient (IFNAR ؊/؊ ) mice, while the virulence of the H strains was attenuated significantly in both experimental models. Similarly, the South African vaccine strains were not pathogenic for IFNAR ؊/؊ mice, while the corresponding wild-type strains were virulent. Thus, attenuation of the virulence of the BTV strains used in this study is not mediated by the presence of an intact interferon system. No clear distinction in virulence was observed for the South African BTV strains in newborn NIH-Swiss mice. Full genomic sequencing revealed relatively few amino acid substitutions, scattered in several different viral proteins, for the strains found to be attenuated in mice compared to the pathogenic related strains. However, only the genome segments encoding VP1, VP2, and NS2 consistently showed nonsynonymous changes between all virulent and attenuated strain pairs. This study established an experimental platform for investigating the determinants of BTV virulence. Future studies using reverse genetics will allow researchers to precisely map and "weight" the relative influences of the various genome segments and viral proteins on BTV virulence.

Bluetongue virus (BTV) is the causative agent of bluetongue, a major infectious disease of ruminants with serious consequences to both animal health and the economy. The clinical outcome of BTV infection is highly variable and dependent on a variety of factors related to both the virus and the host. In this study, we show that the BTV nonstructural protein NS4 favors viral replication in sheep, the animal species most affected by bluetongue. In addition, NS4 confers a replication advantage on the virus in interferon (IFN)-competent primary sheep endothelial cells and immortalized cell lines. We determined that in cells infected with an NS4 deletion mutant (BTV8ΔNS4), there is increased synthesis of type I IFN compared to cells infected with wild-type BTV-8. In addition, using RNA sequencing (RNA-seq), we show that NS4 modulates the host IFN response and downregulates mRNA levels of type I IFN and interferon-stimulated genes. Moreover, using reporter assays and protein synthesis assays, we show that NS4 downregulates the activities of a variety of promoters, such as the cytomegalovirus immediate-early promoter, the IFN-β promoter, and a promoter containing interferon-stimulated response elements (ISRE). We also show that the NS4 inhibitory activity on gene expression is related to its nucleolar localization. Furthermore, NS4 does not affect mRNA splicing or cellular translation. The data obtained in this study strongly suggest that BTV NS4 is an IFN antagonist and a key determinant of viral virulence. IMPORTANCE Bluetongue is one of the main infectious diseases of ruminants and is caused by bluetongue virus (BTV), an arthropod-borne virus transmitted from infected to susceptible animals by Culicoides biting midges. Bluetongue has a variable clinical outcome that can be related to both virus and host factors. It is therefore critical to understand the interplay between BTV and the host immune responses. In this study, we show that a nonstructural protein of BTV (NS4) is critical to counteract the innate immune response of the host. Infection of cells with a BTV mutant lacking NS4 results in increased synthesis of IFN-β and upregulation of interferon-stimulated genes. In addition, we show that NS4 is a virulence factor for BTV by favoring viral replication in sheep, the animal species most susceptible to bluetongue.

In the recent years, USUTU virus (USUV), a flavivirus of the Japanese encephalitis virus complex, has been reported in Central Europe. As part of a systematic surveillance programme to monitor possible entrance and/or circulation of vector-borne viruses, since 2001, sentinel-chicken flocks were placed throughout the Italian territory nearby areas considered at risk of virus introduction. They have been periodically checked for the presence of antibodies against flaviviruses by indirect ELISA, plaque reduction neutralization test for USUTU, West Nile and tick-borne encephalitis viruses. In July 2007, a sentinel chicken in a flock of 20 animals located within the Ravenna province seroconverted to USUV reaching neutralizing titres up to 1:5120. A second chicken seroconverted to the same virus 2 months later. Although no virus was rescued from these animals and from wild or farm birds sampled in the area, these results still provided evidence of the circulation of USUV in north-eastern Italy.

Bluetongue virus (BTV) causes bluetongue, a major hemorrhagic disease of ruminants. In order to investigate the molecular determinants of BTV virulence, we used a BTV8 strain minimally passaged in tissue culture (termed BTV8L in this study) and a derivative strain passaged extensively in tissue culture (BTV8H) in in vitro and in vivo studies. BTV8L was pathogenic in both IFNAR−/− mice and in sheep, while BTV8H was attenuated in both species. To identify genetic changes which led to BTV8H attenuation, we generated 34 reassortants between BTV8L and BTV8H. We found that partial attenuation of BTV8L in IFNAR−/− mice was achieved by simply replacing genomic segment 2 (Seg2, encoding VP2) or Seg10 (encoding NS3) with the BTV8H homologous segments. Fully attenuated viruses required at least two genome segments from BTV8H, including Seg2 with either Seg1 (encoding VP1), Seg6 (encoding VP6 and NS4), or Seg10 (encoding NS3). Conversely, full reversion of virulence of BTV8H required at least five genomic segments of BTV8L. We also demonstrated that BTV8H acquired an increased affinity for glycosaminoglycan receptors during passaging in cell culture due to mutations in its VP2 protein. Replication of BTV8H was relatively poor in interferon (IFN)-competent primary ovine endothelial cells compared to replication of BTV8L, and this phenotype was determined by several viral genomic segments, including Seg4 and Seg9. This study demonstrated that multiple viral proteins contribute to BTV8 virulence. VP2 and NS3 are primary determinants of BTV pathogenesis, but VP1, VP5, VP4, VP6, and VP7 also contribute to virulence.IMPORTANCE Bluetongue is one of the major infectious diseases of ruminants, and it is listed as a notifiable disease by the World Organization for Animal Health (OIE). The clinical outcome of BTV infection varies considerably and depends on environmental and host- and virus-specific factors. Over the years, BTV serotypes/strains with various degrees of virulence (including nonpathogenic strains) have been described in different geographical locations. However, no data are available to correlate the BTV genotype to virulence. This study shows that BTV virulence is determined by different viral genomic segments. The data obtained will help to characterize thoroughly the pathogenesis of bluetongue. The possibility to determine the pathogenicity of virus isolates on the basis of their genome sequences will help in the design of control strategies that fit the risk posed by new emerging BTV strains.