Neutralising antibodies to West Nile virus detected in horses in Windhoek, Namibia.

ABSTRACT: West Nile virus (WNV) is a vector-borne virus maintained in nature by a bird-mosquito cycle. However, it can occasionally and accidentally infect horses and human beings, leading to sometimes severe or even fatal outcomes in these species. Therefore, the monitoring of its circulation and disease occurrence is of relevance. Unfortunately, it is underdiagnosed or not diagnosed in several African counties, including Namibia, where no data is currently available for horses. In this study, 98 horses in three different stables in the Windhoek city area were investigated. They were found to have a seroprevalence of approximately 7%. Positive reactions were seen at all three stables, suggesting a greater than expected prevalence of the virus. This is the first report of serological evidence for the presence of the virus in horses in Nambia. Even though clinical signs were not reported in any of the stables from which the sera were derived, the seroprevalence to the virus suggests that horses with high genetic and/or economic value could benefit from vaccination against WNV. Because of the zoonotic potential of the virus, these findings are also of significance to human health authorities.

Artificial intelligence-based radiomics on computed tomography of lumbar spine in subjects with fragility vertebral fractures.

PURPOSE: There is emerging evidence that radiomics analyses can improve detection of skeletal fragility. In this cross-sectional study, we evaluated radiomics features (RFs) on computed tomography (CT) images of the lumbar spine in subjects with or without fragility vertebral fractures (VFs). METHODS: Two-hundred-forty consecutive individuals (mean age 60.4 ± 15.4, 130 males) were evaluated by radiomics analyses on opportunistic lumbar spine CT. VFs were diagnosed in 58 subjects by morphometric approach on CT or XR-ray spine (D4-L4) images. DXA measurement of bone mineral density (BMD) was performed on 17 subjects with VFs. RESULTS: Twenty RFs were used to develop the machine learning model reaching 0.839 and 0.789 of AUROC in the train and test datasets, respectively. After correction for age, VFs were significantly associated with RFs obtained from non-fractured vertebrae indicating altered trabecular microarchitecture, such as low-gray level zone emphasis (LGLZE) [odds ratio (OR) 1.675, 95% confidence interval (CI) 1.215-2.310], gray level non-uniformity (GLN) (OR 1.403, 95% CI 1.023-1.924) and neighboring gray-tone difference matrix (NGTDM) contrast (OR 0.692, 95% CI 0.493-0.971). Noteworthy, no significant differences in LGLZE (p = 0.94), GLN (p = 0.40) and NGDTM contrast (p = 0.54) were found between fractured subjects with BMD T score < - 2.5 SD and those in whom VFs developed in absence of densitometric diagnosis of osteoporosis. CONCLUSIONS: Artificial intelligence-based analyses on spine CT images identified RFs associated with fragility VFs. Future studies are needed to test the predictive value of RFs on opportunistic CT scans in identifying subjects with primary and secondary osteoporosis at high risk of fracture.

Sero-surveillance of emerging viral diseases in camels and cattle in Nouakchott, Mauritania: an abattoir study.

This study reports the monitoring of several emerging viral pathogens in Mauritania, which was carried out by the analysis of bovine and camel samples taken at the slaughterhouse of Nouakchott. Blood and serum were collected by random sampling from 159 camels and 118 cattle in March 2013 at the large animals abattoir in Nouakchott. Serological tests for Rift Valley Fever (RVF), Peste des Petits Ruminants (PPR), West Nile disease (WND), epizootic haemorrhagic disease (EHD) and African horse sickness (AHS) were carried out using commercial ELISA kits. The samples, which resulted positives for PPR, WND and AHS, were tested with the confirmatory virus neutralization test (VNT). According to ELISA results, serological prevalence of RVF was 45% (95% CI 52.3-37.7) in camels and 16% (95% CI 22.6-9.4) in cattle. The difference between the observed prevalences in camels and in cattle was significant (p value ≤ 0.01). PPR was absent in camels and had 12% prevalence (95% CI, 17.86-6.14) in cattle. Furthermore, camels showed 92% (95% CI, 96.1-87.9) prevalence of WNV, 73% (95% CI, 82.3-63.64) of EHD and 3% (95% CI, 5.6-0.4) of AHS. This data are of relevance since provided useful feedbacks on the circulation of the pathogens in field. Moreover, this survey provided new information on the susceptibility of camels to several emerging pathogens and on the possible use of this species as sentinel animal.

Western Bluetongue virus serotype 3 in Sardinia, diagnosis and characterization.

Over the last 20 years, Italy has experienced multiple incursions of different serotypes of Bluetongue virus (BTV), a Culicoides-borne arbovirus, the causative agent of bluetongue (BT), a major disease of ruminants. The majority of these incursions originated from Northern Africa, likely because of wind-blown dissemination of infected midges. Here, we report the first identification of BTV-3 in Sardinia, Italy. BTV-3 circulation was evidenced in sentinel animals located in the province of Sud Sardegna on September 19, 2018. Prototype strain BTV-3 SAR2018 was isolated on cell culture. BTV-3 SAR2018 sequence and partial sequences obtained by next-generation sequencing from nucleic acids purified from the isolate and blood samples, respectively, were demonstrated to be almost identical (99-100% of nucleotide identity) to BTV-3 TUN2016 identified in Tunisia in 2016 and 2017, a scenario already observed in past incursions of other BTV serotypes originating from Northern Africa.

A novel Bluetongue virus serotype 3 strain in Tunisia, November 2016.

Since 1998, southern Europe has experienced multiple incursions of different serotypes and topotypes of Bluetongue virus, a vector-borne transmitted virus, the causative agent of Bluetongue (BT), a major disease of ruminants. Some of these incursions originated from northern Africa, likely because of wind-blown dissemination of infected midges. In this report, we describe the detection and whole genome characterization of a novel BTV-3 strain identified in a symptomatic sheep in Tunisia. Sequences were immediately deposited with the GenBank Database under Accession Nos KY432369-KY432378. Alert and preparedness are requested to face the next vector seasons in northern Africa and the potential incursion of this novel strain in southern Europe.

Usutu virus infections in humans: a retrospective analysis in the municipality of Modena, Italy.

OBJECTIVE: To monitor the spread and to evaluate the role for public health of Usutu virus (USUV) in an endemic area of Italy. METHODS: The survey was retrospectively conducted by detecting USUV RNA and USUV antibodies in cerebrospinal fluid and serum samples collected between 2008 and 2011 from 915 patients with or without neurologic impairments in the area of the municipality of Modena, Italy. Organs of birds and pools of mosquitoes were also tested for USUV RNA. Positive samples were partially sequenced and used for phylogenetic analysis. RESULTS: The presence of USUV RNA (1.1%; 95% confidence interval (CI) 0.6-2.0) was significantly (p <0.05) higher than that of West Nile virus (0%; 95% CI 0-0.33). USUV antibody level was 6.57% (95% CI 4.87-8.82), and it was significantly higher (p <0.05) compared to that of West Nile virus (p 2.96, 95% CI 1.89-4.62). Partial genome sequencing of USUV strains detected in humans, birds and mosquitoes revealed high nucleotide sequence identity within them and with the USUV strains isolated in Central Europe. CONCLUSIONS: USUV infection in humans is not a sporadic event in the studied area, and USUV neuroinvasiveness has been confirmed.

Factors Affecting Seroconversion Rates in Cattle Vaccinated with Two Commercial Inactivated BTV-8 Vaccines Under Field Conditions.

The immunogenicity of two inactivated bluetongue virus serotype 8 (BTV-8) vaccines was evaluated in 880 cattle under field conditions. The effect of selected factors on vaccine performance was also analysed at the herd and animal levels (vaccine, herd size and production, age, sex, time interval between vaccination and blood sampling and veterinary training). The immunogenicity elicited by vaccination with the two vaccines was monitored with the aid of a competitive enzyme-linked immunosorbent assay (c-ELISA) and serum neutralization test (SNT). To investigate whether the selected factors influenced seroconversion at the herd and animal levels, a multilevel logistic regression model developed in a mixed model was applied. Of the 880 cattle vaccinated, 76.0% yielded BTV c-ELISA antibodies, whereas only 25.0% seroconverted based on SNT. Type of vaccine (odds ratio [OR] 4.5; 95% confidence interval [CI], 2.2-9.0 for SNT and OR 3.5; 95% CI, 2.1-5.9 for c-ELISA), veterinary training in vaccine administration (OR 8.1; 95% CI, 4.7-14.1 for SNT and OR 2.4; 95% CI, 1.3-4.2 for c-ELISA), animal age (OR 1.4; 95% CI, 1.1-1.8 for SNT and OR 1.7; 95% CI, 1.4-2.1 for c-ELISA) and days between first vaccine administration and blood collection (OR 1.9; 95% CI, 1.1-3.1 for SNT and OR 2.6; 95% CI, 1.7-3.8 for c-ELISA) were the major factors affecting vaccine performance under field conditions. This is the first study to use multilevel logistic regression in the evaluation of selected risk factors affecting BTV-8 vaccine performance in cattle.

Experimental infection of rock pigeons (Columba livia) with three West Nile virus lineage 1 strains isolated in Italy between 2009 and 2012.

West Nile virus (WNV) circulation dynamics in the context of the urban environment is not yet elucidated. In this perspective, three groups of eight rock pigeons (Columbia livia) were inoculated with three WNV lineage 1 strains isolated in Italy between 2009 and 2012. The pigeons did not develop any clinical signs consistent with WNV acute infection. All animals seroconverted and shed virus up to 15 days post-infection by the oral or cloacal routes. In all infected groups viraemia lasted for 4 days post-infection. No WNV-specific gross or histological lesions were found in infected birds compared to control birds and immunohistochemistry remained constantly negative from all tissues. The reservoir competence index was also assessed and it ranged between 0·11 and 0·14. This study demonstrates that pigeons are competent reservoir hosts for Italian WNV lineage 1 circulating strains thus potentially posing a risk to the public health system.

Bluetongue.

Summary Bluetongue (BT) is an arthropod-transmitted viral disease of non-African ungulates, principally sheep. The disease results from vascular injury analogous to that of human haemorrhagic viral fevers, with characteristic tissue infarction, haemorrhage, vascular leakage, oedema, and hypovolaemic shock. Importantly, BT is not zoonotic. Bluetongue virus (BTV) infection of ruminants and vector Culicoides midges is endemic throughout many tropical and temperate regions of the world; however, within this global range the virus exists within relatively discrete ecosystems (syn. episystems) where specific constellations of BTV serotypes are spread by different species of biting Culicoides midges. Recently discovered goat-associated BTVs, notably BTV serotype 25 (BTV-25) in central Europe, appear to have distinctive biological properties and an epidemiology that is not reliant on Culicoides midges as vectors for virus transmission. Bluetongue virus infection of ruminants is often subclinical, but outbreaks of severe disease occur regularly at the upper and lower limits of the virus's global range, where infection is distinctly seasonal. There have been recent regional alterations in the global distribution of BTV infection, particularly in Europe. It is proposed that climate change is responsible for these events through its impact on vector midges. However, the role of anthropogenic factors in mediating emergence of BTV into new areas remains poorly defined; for example, it is not clear to what extent anthropogenic factors were responsible for the recent translocation to northern and eastern Europe of live attenuated vaccine viruses and an especially virulent strain of BTV-8 with distinctive properties. Without thorough characterisation of all environmental and anthropogenic drivers of the recent emergence of BT in northern Europe and elsewhere, it is difficult to predict what the future holds in terms of global emergence of BTV infection. Accurate and convenient laboratory tests are available for the sensitive and specific serological and virological diagnosis of BTV infection and confirmation of BT in animals. Prevention and control strategies for BT are largely reactive in nature, and typically are reliant on vaccination of susceptible livestock and restrictions on animal trade and movement.

Epizootic haemorrhagic disease.

Summary Epizootic haemorrhagic disease (EHD) is an arthropod-transmitted viral disease of certain wild ungulates, notably North American white-tailed deer and, more rarely, cattle. The disease in white-tailed deer results from vascular injury analogous to that caused by bluetongue virus (BTV), to which EHD virus (EHDV) is closely related. There are seven serotypes of EHDV recognised, and Ibaraki virus, which is the cause of sporadic disease outbreaks in cattle in Asia, is included in EHDV serotype 2. The global distribution and epidemiology of BTV and EHDV infections are also similar, as both viruses occur throughout temperate and tropical regions of the world where they are transmitted by biting Culicoides midges and infect a wide variety of domestic and wild ungulates. However, the global distribution and epidemiology of EHDV infection are less well characterised than they are for BTV. Whereas most natural and experimental EHDV infections (other than Ibaraki virus infection) of livestock are subclinical or asymptomatic, outbreaks of EHD have recently been reported among cattle in the Mediterranean Basin, Reunion Island, South Africa, and the United States. Accurate and convenient laboratory tests are increasingly available for the sensitive and specific serological and virological diagnosis of EHDV infection and confirmation of EHD in animals, but commercial vaccines are available only for prevention of Ibaraki disease and not for protection against other strains and serotypes of EHDV.

First detection of co-circulation of West Nile and Usutu viruses in equids in the south-west of Tunisia.

In the last fifteen years, West Nile Virus (WNV) has dramatically expanded its geographic range and is now considered the most widespread arbovirus in the world. In Tunisia, West Nile Fever (WNF) outbreaks were reported in humans in 1997, 2003 and 2012. Usutu Virus (USUV), which is a 'new' emerging Flavivirus antigenically close to WNV, has never been reported in Tunisia. A serological investigation in 284 equids was conducted in 2012 in the southern west region of the country to assess the presence and prevalence of the WNV and USUV infection. Of the 284 samples tested by competitive enzyme-linked immunoassay, 129 were positive. Of these, 120 (42.3%) had WNV-specific neutralizing antibodies. The prevalence was significantly higher in areas closer to the oasis compared with that of the surrounding arid areas. Antibody titres against USUV were also reported in 10 equids. This was the first evidence of USUV circulation in Tunisia. Data recorded by this study indicate that WNV and USUV have circulated/are circulating in the region and that there is an urgent need to adapt the current surveillance programmes to this new scenario.

First evidence of simultaneous occurrence of West Nile virus and Usutu virus neuroinvasive disease in humans in Croatia during the 2013 outbreak.

PURPOSE: We report on first evidence of simultaneous occurrence of West Nile virus (WNV) and Usutu virus (USUV) neuroinvasive infection in humans in Croatia during the transmission season 2013. METHODS: From June to December 2013, a total of 95 patients with clinically suspected WNV infection (WNV fever and neuroinvasive disease) were tested for WNV IgM/IgG antibodies using enzyme-linked immunosorbent assay. Twenty-six reactive samples were further tested by virus neutralization test for confirmation. RESULTS: WNV neuroinvasive infection was confirmed in 20 patients, while in three patients USUV neutralizing antibodies were detected. Cases occurred during the 11-week interval (from 24 July to 07 October 2013). Both WNV and USUV cases were distributed in three north-western Croatian counties. In addition to human cases, recent asymptomatic WNV infection (detection of IgM antibodies) was recorded in 9/3,460 (0.3 %) tested sentinel horses. Infected animals were recorded in two eastern and one north-western county. CONCLUSIONS: Our results indicate co-circulation of WNV and USUV in Croatia. WNV infection could be misdiagnosed with other emerging infectious diseases presenting with neurological symptoms such as USUV infection.

Bluetongue serotype 2 and 9 modified live vaccine viruses as causative agents of abortion in livestock: a retrospective analysis in Italy.

The recent outbreak caused by Schmallenberg virus, which affected sheep, goats and cattle in Europe, highlighted the importance of having a robust surveillance plan capable of monitoring abortions and malformations in the livestock offspring. In this context, bluetongue viruses (BTVs) represented and represent one of the major threats to the European livestock industry. Aiming to improve the understanding on BTV cross placental transmission and serotype involvement, in this retrospective study foetal spleens and/or brains of 663 ovines, 429 bovines, 155 goats and 17 buffaloes were tested for the presence of BTV by virus isolation. BTV vaccine strains were isolated from 31 foetuses (2.4%; 95% CI: 1.7-3.4%): 24 (3.6%; 95% CI: 2.4-5.3%) from ovine foetal tissues; 6 (1.4%; 95% CI: 0.6-3.0%) from bovine foetal tissues and 1 (0.6%; 95% CI: 0.2-3.5%) from the spleen of a caprine foetus. All foetuses were from animals vaccinated with either BTV-2 or BTV-2, and BTV-9 modified live vaccines (MLVs) produced by Onderstepoort Biological Products (OBP), South Africa. Among the 31 isolated vaccine strains, serotype 9 (n = 28) was more frequently isolated (P < 0.05) than serotype 2 (n = 3). In two cases infectious vaccine strains were found in the foetal tissues 2 months after the vaccine administration. Other pathogens known to be causative agents of abortion in ruminants were not detected nor isolated. This study demonstrates, for the first time, that BTV-2 and BTV-9 vaccine strains are able to cross the placental barrier of sheep, cattle and goats. BTV-2 and BTV-9 vaccine strains are able to infect foetuses and cause abortions or malformations depending on the period of pregnancy at the time of vaccination.

Bluetongue virus in Lebanon.

Since 2000, several incursions of bluetongue virus (BTV) occurred in the Mediterranean Basin involving European and surrounding Countries. The Middle East represents one of the most important gateways for the access of BTV in Europe. Limited data on the BTV situation in this area are available. In this perspective, an epidemiological survey on the presence of BTV in Lebanon was conducted. Of the 181 serum samples tested, 97 (mean = 53.6%; 95% CI: 46.3-60.7) resulted positive when tested for the presence of BTV antibodies by c-ELISA, of these 42 (mean = 42%; 95% CI: 32.8-51.8) serum samples were from sheep and 55 (mean = 67.9%; 95% CI: 57.1-77.1) serum samples were from goats. Fourteen blood samples (14/110; mean = 12.7%; 95% CI: 7.8-20.3), 6 (6/66; mean = 9.1%; 95% CI: 4.4-18.5) from sheep and 8 (8/44; mean = 18.2%; 95% CI: 9.6-32.0) from goats, were positive by qRT-PCR. The results with serum-neutralization assay and typing performed by RT-PCR confirmed that six BTV serotypes are currently circulating in Lebanon, and these serotypes are as follows: 1, 4, 6, 8, 16 and 24. This study is the first report that confirms the presence and circulation of BTV in Lebanon.

Schmallenberg virus in Italy: a retrospective survey in Culicoides stored during the bluetongue Italian surveillance program.

Following the first case of Schmallenberg (SBV) in northern Italy in February 2012, virus detection was conducted on midges collected during the national entomological surveillance program for bluetongue (BT). Six cattle farms, within a radius of 50 km from the SBV case, were selected for a 12 month study, aiming to determine when the virus entered the area, if it was capable of overwintering, and the possible role played by each species of the Obsoletus complex in disseminating the infection. A total of 33,724 Culicoides were collected at the six sites between June 2011 and June 2012. Species belonging to the Obsoletus Complex were the most abundant (94.44%) and, within the complex, Culicoides obsoletus was the most prevalent species in the studying area (65.4%). Nearly 7000 Culicoides midges were screened, either in pools or individually, for SBV by real-time RT-PCR. Viral genome was detected in six pools of the Obsoletus complex, collected at three sites between September and November 2011, and in a single parous female of C. obsoletus, collected in May 2012. As a result of the BT surveillance program in Italy it was possible to demonstrate, retrospectively, that SBV has circulated in at least three Italian provinces since early September 2011, nearly 5 months prior and as far as 40 km away from the first detected case. Similarly, the survey confirmed the presence of SBV in the vector population 3 months after the outbreak, following a cold winter during which the blacklight traps failed to catch active adult midges.

West Nile virus lineage 2 in Sardinian wild birds in 2012: a further threat to public health.

West Nile virus (WNV) strains belonging to lineage 2 were detected and isolated from the tissues of a goshawk and two carrion crows in Sardinia in August 2012. According to NS3 sequence analysis, the Sardinian isolates shared a high level of similarity with those of Italian lineage 2 strains which circulated in 2011 and with the homologous sequence of the 2004 Hungarian isolate. Following the human fatality reported in 2011 in Olbia, this study is the first to report the spread and enzootic circulation of WNV lineage 2 in Sardinia.

Loss of temporal retinal nerve fibers in Parkinson disease: a mitochondrial pattern?

BACKGROUND AND PURPOSE: Optic nerve involvement is frequent in mitochondrial disease, and retinal abnormalities are described in Parkinson's disease (PD). METHODS: We evaluated retinal nerve fiber layer (RNFL) thickness by optical coherence tomography in 43 patients with PD and in 86 age-matched controls. We considered separately the eyes ipsilateral and contralateral to the most affected body side in patients with PD. ancova analysis, Pearson test, and multiple regression analysis were used (P < 0.05). RESULTS: Patients with PD showed significantly thinner temporal RNFL thickness compared to controls (P = 0.004), more evident in the eye contralateral to the most affected body side. Average RNFL thickness significantly correlated with age in both controls and patients with PD (P-values ranging from 0.001 to 0.019), whereas in patients with PD RNFL thickness did not correlate with clinical variables. CONCLUSIONS: Our study reveals a loss of retinal nerve fibers in the temporal quadrant in PD, which is typically susceptible in mitochondrial optic neuropathies.

Evidence of West Nile virus lineage 2 circulation in Northern Italy.

A West Nile virus (WNV) strain belonging to lineage 2 was for the first time detected in two pools of Culex pipiens collected in the province of Udine and in tissues of a wild collared dove (Streptopelia decaocto) found dead in the province of Treviso, in North East of Italy. It was molecularly identified by group and WNV lineage specific RT-PCRs and characterized by partial sequencing of the NS3 and NS5 genes. When compared with the sequences of same fragments of NS3 and NS5 of the WNV lineage 2 strain isolated from birds of prey in Hungary (2004), the phylogenetic analysis of these sequences revealed 100% and 99% similarity, respectively. As the Hungarian strain, the NS3 selected sequence differed from the 2010 Greek isolate by one amino-acid located at 249 site which is the site involved in genetic modulation of WNV pathogenicity. The Italian and Hungarian strains have histidine rather than proline at this site. The presence of a lineage 2 strain in regions where the lineage 1 strain is still circulating, creates a new scenario with unpredictable consequences. In this situation comprehensive investigations on the occurrence, ecology, and epidemiology of these different WNV strains circulating in Italy become the highest priority.

The shear mode of multilayer graphene.

The quest for materials capable of realizing the next generation of electronic and photonic devices continues to fuel research on the electronic, optical and vibrational properties of graphene. Few-layer graphene (FLG) flakes with less than ten layers each show a distinctive band structure. Thus, there is an increasing interest in the physics and applications of FLGs. Raman spectroscopy is one of the most useful and versatile tools to probe graphene samples. Here, we uncover the interlayer shear mode of FLGs, ranging from bilayer graphene (BLG) to bulk graphite, and suggest that the corresponding Raman peak measures the interlayer coupling. This peak scales from ~43 cm(-1) in bulk graphite to ~31 cm(-1) in BLG. Its low energy makes it sensitive to near-Dirac point quasiparticles. Similar shear modes are expected in all layered materials, providing a direct probe of interlayer interactions.