Genomic monitoring to understand the emergence and spread of Usutu virus in the Netherlands, 2016-2018.

Usutu virus (USUV) is a mosquito-borne flavivirus circulating in Western Europe that causes die-offs of mainly common blackbirds (Turdus merula). In the Netherlands, USUV was first detected in 2016, when it was identified as the likely cause of an outbreak in birds. In this study, dead blackbirds were collected, screened for the presence of USUV and submitted to Nanopore-based sequencing. Genomic sequences of 112 USUV were obtained and phylogenetic analysis showed that most viruses identified belonged to the USUV Africa 3 lineage, and molecular clock analysis evaluated their most recent common ancestor to 10 to 4 years before first detection of USUV in the Netherlands. USUV Europe 3 lineage, commonly found in Germany, was less frequently detected. This analyses further suggest some extent of circulation of USUV between the Netherlands, Germany and Belgium, as well as likely overwintering of USUV in the Netherlands.

Towards high quality real-time whole genome sequencing during outbreaks using Usutu virus as example.

Recently, protocols for amplicon based whole genome sequencing using Nanopore technology have been described for Ebola virus, Zika virus, yellow fever virus and West Nile virus. However, there is some debate regarding reliability of sequencing using this technology, which is important for applications beyond diagnosis such as linking lineages to outbreaks, tracking transmission pathways and pockets of circulation, or mapping specific markers. To our knowledge, no in depth analyses of the required read coverage to compensate for the error profile in Nanopore sequencing have been described. Here, we describe the validation of a protocol for whole genome sequencing of USUV using Nanopore sequencing by direct comparison to Illumina sequencing. To that point we selected brain tissue samples with high viral loads, typical for birds which died from USUV infection. We conclude that the low-cost MinION Nanopore sequencing platform can be used for characterization and tracking of Usutu virus outbreaks.

Global status of Middle East respiratory syndrome coronavirus in dromedary camels: a systematic review.

Dromedary camels have been shown to be the main reservoir for human Middle East respiratory syndrome (MERS) infections. This systematic review aims to compile and analyse all published data on MERS-coronavirus (CoV) in the global camel population to provide an overview of current knowledge on the distribution, spread and risk factors of infections in dromedary camels. We included original research articles containing laboratory evidence of MERS-CoV infections in dromedary camels in the field from 2013 to April 2018. In general, camels only show minor clinical signs of disease after being infected with MERS-CoV. Serological evidence of MERS-CoV in camels has been found in 20 countries, with molecular evidence for virus circulation in 13 countries. The seroprevalence of MERS-CoV antibodies increases with age in camels, while the prevalence of viral shedding as determined by MERS-CoV RNA detection in nasal swabs decreases. In several studies, camels that were sampled at animal markets or quarantine facilities were seropositive more often than camels at farms as well as imported camels vs. locally bred camels. Some studies show a relatively higher seroprevalence and viral detection during the cooler winter months. Knowledge of the animal reservoir of MERS-CoV is essential to develop intervention and control measures to prevent human infections.

A Serological Protein Microarray for Detection of Multiple Cross-Reactive Flavivirus Infections in Horses for Veterinary and Public Health Surveillance.

The genus Flavivirus in the family Flaviviridae includes some of the most important examples of emerging zoonotic arboviruses that are rapidly spreading across the globe. Japanese encephalitis virus (JEV), West Nile virus (WNV), St. Louis encephalitis virus (SLEV) and Usutu virus (USUV) are mosquito-borne members of the JEV serological group. Although most infections in humans are asymptomatic or present with mild flu-like symptoms, clinical manifestations of JEV, WNV, SLEV, USUV and tick-borne encephalitis virus (TBEV) can include severe neurological disease and death. In horses, infection with WNV and JEV can lead to severe neurological disease and death, while USUV, SLEV and TBEV infections are mainly asymptomatic, however, and induce antibody responses. Horses often serve as sentinels to monitor active virus circulation in serological surveillance programmes specifically for WNV, USUV and JEV. Here, we developed and validated a NS1-antigen protein microarray for the serological differential diagnosis of flavivirus infections in horses using sera of experimentally and naturally infected symptomatic as well as asymptomatic horses. Using samples from experimentally infected horses, an IgG and IgM specificity of 100% and a sensitivity of 95% for WNV and 100% for JEV was achieved with a cut-off titre of 1 : 20 based on ROC calculation. In field settings, the microarray identified 93-100% of IgG-positive horses with recent WNV infections and 87% of TBEV IgG-positive horses. WNV IgM sensitivity was 80%. Differentiation between closely related flaviviruses by the NS1-antigen protein microarray is possible, even though we identified some instances of cross-reactivity among antibodies. However, the assay is not able to differentiate between naturally infected horses and animals vaccinated with an inactivated WNV whole-virus vaccine. We showed that the NS1-microarray can potentially be used for diagnosing and distinguishing flavivirus infections in horses and for public health purposes within a surveillance setting. This allows for fast, cheap, syndrome-based laboratory testing for multiple viruses simultaneously for veterinary and public health purposes.

Characterization of Puumala hantavirus in bank voles from two regions in the Netherlands where human cases occurred.

Puumala hantavirus (PUUV) is the most common and widespread hantavirus in Europe and is associated with a mild form of haemorrhagic fever with renal syndrome in humans, called nephropathia epidemica. This study presents the molecular characterization of PUUV circulating in bank voles in two regions of the Netherlands. Most human cases of hantavirus infection are from these two regions. Phylogenetic analysis of the (partial) S, M and L-segments indicated that the Dutch strains belong to the CE lineage, which includes PUUV strains from France, Germany and Belgium. We have identified two distinct groups of PUUV, corresponding with their geographic origin and with adjoining regions in neighbouring countries.

Zika virus and the current outbreak: an overview.

Zika virus (ZIKV), a mosquito-borne flavivirus closely related to yellow fever virus and dengue virus, is currently causing a large outbreak in the Americas. Historically, ZIKV infection was considered a sporadic, relatively mild disease characterised by fever, maculopapular rash, conjunctivitis and often arthralgia. However, current observational studies suggest that ZIKV may cause more severe neurological sequelae such as Guillain-Barre syndrome, and birth defects, mainly microcephaly, in babies of whom the mother was infected with ZIKV during pregnancy. This article provides a clinically focussed overview of ZIKV, with emphasis on the current outbreak, clinical manifestations, diagnostic tools and caveats.

The sample of choice for detecting Middle East respiratory syndrome coronavirus in asymptomatic dromedary camels using real-time reversetranscription polymerase chain reaction.

The newly identified Middle East respiratory syndrome coronavirus (MERS-CoV), which causes severe respiratory disease, particularly in people with comorbidities, requires further investigation. Studies in Qatar and elsewhere have provided evidence that dromedary camels are a reservoir for the virus, but the exact modes of transmission of MERS-CoV to humans remain unclear. In February 2014, an assessment was made of the suitability and sensitivity of different types of sample for the detection of MERSCoV by real-time reverse-transcription polymerase chain reaction (RT-PCR) for three gene targets: UpE (upstream of the E gene), the N (nucleocapsid) gene and open reading frame (ORF) 1a. Fifty-three animals presented for slaughter were sampled. A high percentage of the sampled camels (79% [95% confidence interval 66.9-91.5%, standard error 0.0625]; 42 out of 53) were shown to be shedding MERS-CoV at the time of slaughter, yet all the animals were apparently healthy. Among the virus-positive animals, nasal swabs were most often positive (97.6%). Oral swabs were the second most frequently positive (35.7%), followed by rectal swabs (28.5%). In addition, the highest viral load, expressed as a cycle threshold (Ct) value of 11.27, was obtained from a nasal swab. These findings lead to the conclusion that nasal swabs are the candidate sample of choice for detecting MERS-CoV using RT-PCR technology in apparently healthy camels.

Des travaux de recherche approfondis sont encore nécessaires concernant le coronavirus responsable du syndrome respiratoire du Moyen-Orient (MERSCoV), un virus identifié récemment et qui provoque des troubles respiratoires sévères en particulier chez les individus atteints de pathologies multiples. Les études effectuées au Qatar et ailleurs ont démontré que les dromadaires font office de réservoirs du virus ; toutefois, les modalités précises de la transmission du MERS-CoV à l'être humain demeurent obscures. En février 2014, une équipe de chercheurs a évalué l'adéquation et la sensibilité de plusieurs types d'échantillons pour détecter le MERS-CoV en utilisant l'amplification en chaîne par polymérase couplée à une transcription inverse en temps réel (RT-PCR) spécifique pour trois cibles génétiques, à savoir la séquence UpE (en amont du gène E), le gène N (nucléocapside) et le cadre de lecture ORF1a. Pour ce faire, divers prélèvements ont été effectués sur 53 dromadaires destinés à l'abattage. Un fort pourcentage de ces dromadaires (79 % [intervalle de confiance à 95 % compris entre 66,9 et 91,5 %, erreur standard : 0,0625], soit 42 sur 53) excrétaient le MERSCoV au moment de l'abattage, mais aucun ne présentait le moindre signe clinique. Les échantillons dans lesquels le plus de cas positifs ont été détectés étaient les écouvillons nasaux (97,6 %). Venaient ensuite les écouvillons oraux, qui ont détecté 35,7 % de cas positifs, puis les écouvillons rectaux (28,5 % de cas positifs détectés). Par ailleurs, ce sont les écouvillons nasaux qui ont permis d'obtenir l'intensité la plus élevée de la réponse de la RT-PCR, exprimée en une valeur du seuil de cycles de 11,27. Ces résultats permettent de conclure que les écouvillons nasaux sont les échantillons à privilégier pour la détection du MERS-CoV par RTPCR chez les dromadaires asymptomatiques.

Es preciso investigar más a fondo el coronavirus del síndrome respiratorio de Oriente Medio (MERS-CoV), recién identificado, que provoca una grave enfermedad respiratoria, sobre todo en personas con afecciones concomitantes. Estudios realizados en Qatar y otros lugares han deparado pruebas de que los dromedarios son un reservorio del virus, pero aún no están del todo claros los modelos exactos de transmisión del MERS-CoV al ser humano. Los autores describen un análisis realizado en febrero de 2014 de la idoneidad y sensibilidad de distintos tipos de muestra para detectar el MERS-CoV mediante una reacción en cadena de la polimerasa acoplada a transcripción inversa en tiempo real (RTPCR) dirigida contra tres genes: el gen UpE (upstream of the E gene: en dirección 5' desde el gen E); el gen N (nucleocápside) y el marco de lectura abierto (ORF) 1a. Para ello se tomaron muestras de 53 animales enviados al sacrificio. Se comprobó que un elevado porcentaje de los dromedarios analizados (un 79% [intervalo de confianza al 95%: 66,9­91,5%; error estándar: 0,0625], esto es, 42 de 53) excretaban virus en el momento del sacrificio, pese a que todos los animales parecían estar sanos. Entre los ejemplares positivos para el MERS-CoV, las muestras que con más frecuencia arrojaban resultado positivo eran los frotis nasales (97,6%). Las segundas, por orden de frecuencia, eran los frotis bucales (35,7%), seguidos de los frotis rectales (28,5%). Además, la carga viral más alta, expresada por un valor de ciclo umbral (Ct) (o punto de cruce) de 11,27, se obtuvo a partir de un frotis nasal. Estos resultados llevan a la conclusión de que los frotis nasales son el tipo de muestra más adaptado para detectar el MERS-CoV en dromedarios aparentemente sanos mediante la técnica de RT-PCR.

Seoul hantavirus in brown rats in the Netherlands: implications for physicians--Epidemiology, clinical aspects, treatment and diagnostics.

The recent discovery of Seoul hantavirus (SEOV) presence in wild rat populations in the Netherlands has direct implications for Dutch clinicians and hantavirus diagnostics. SEOV is amongst the Old World hantaviruses which cause haemorrhagic fever and renal syndrome (HFRS) in humans. HFRS is characterised by a classical triad of fever, acute kidney injury and haemorrhage, but can show different signs and symptoms in specific cases. SEOV is transmitted from infected rats to humans by inhalation of aerosolised excreta. When compared with the known circulating hantaviruses in the Netherlands, Puumala (PUUV) and Tula (TULV), SEOV causes a more severe form of HFRS. Data from cohort studies undertaken in China and Northern Europe show differences in signs and symptoms at onset of disease, (haemorrhagic) complications and mortality. Furthermore, routine diagnostics currently available for hantavirus diagnosis in the Netherlands are not optimised for SEOV detection. The clinical outcome of an SEOV and PUUV infection will greatly benefit from an early diagnosis which will reduce the costs of unnecessary tests and treatments as well. The discovery of SEOV circulation in the Netherlands follows recent findings of SEOV infections in both rodents and humans in England, Wales, France, Belgium and Sweden, indicating the emerging character of SEOV and a high importance of this hantavirus for Public Health in large areas of Europe. Here, we review the current knowledge on the clinical manifestation of SEOV versus PUUV infections in humans, the treatment of clinical cases and diagnostics.

Ebola virus disease: a review on epidemiology, symptoms, treatment and pathogenesis.

Currently, West Africa is facing the largest outbreak of Ebola virus disease (EVD) in history. The virus causing this outbreak, the Zaire Ebolavirus (EBOV), belongs to the genus Ebolavirus which together with the genus Marburgvirus forms the family of the Filoviridae. EBOV is one of the most virulent pathogens among the viral haemorrhagic fevers, and case fatality rates up to 90% have been reported. Mortality is the result of multi-organ failure and severe bleeding complications. By 18 September 2014, the WHO reported of 5335 cases (confirmed, suspected and probable) with 2622 deaths, resulting in a case fatality rate of around 50%. This review aims to provide an overview of EVD for clinicians, with the emphasis on pathogenesis, clinical manifestations, and treatment options.

Seroprevalence of hepatitis E virus in pigs from different farming systems in The Netherlands.

Sporadic nontravel-related hepatitis E virus (HEV) infections have been reported in industrialized countries. These infections are caused by zoonotic HEV genotypes 3 and 4 that circulate in swine, wild boar, and deer. In The Netherlands, HEV RNA has been detected in >50% of the pig farms, and HEV-specific antibodies were detected in ∼70% of the slaughter pigs. In the current study, HEV seroprevalences were investigated in pigs raised on conventional, free-range, and organic farms in The Netherlands. Differences in seroprevalence may indicate different exposure routes or transmission dynamics within pig herds for HEV. In 2004, serum samples of 846 fattening pigs were obtained from farms that applied conventional (265 pigs at 24 farms), organic (417 pigs at 42 farms), and free-range (164 pigs at 12 farms) farming. HEV-specific antibodies were detected in samples from all conventional and free-range pig farms and in 41 of 42 organic pig farms, indicating that the probability of introducing HEV on a farm appeared to be equal for the different farming types. The estimated average within-herd seroprevalence was significantly higher for pigs raised on organic farms (89%) than for pigs raised on conventional farms (72%, P = 0.04) and nearly significant for pigs raised on free-range farms (76%, P = 0.06). Six of ten organic farms were estimated to have a withinherd seroprevalence of >95%, compared with 1 of 10 and 4 of 10 of the free-range and conventional pig farms, respectively. This suggests a higher force of infection with HEV for pigs reared on organic farms compared with pigs reared on conventional or free-range farms. This may be due to repetitive exposure to HEV caused by farming system-specific housing conditions, such as a greater contact frequency between pigs and more exposure to pig manure, increasing the transmission rate.

Occurrence of methicillin-resistant Staphylococcus aureus in rats living on pig farms.

In The Netherlands, MRSA ST398 has emerged in hospitals and human carriers have been associated with exposure to pigs and cattle. High prevalences of MRSA ST398 in pigs and pig farmers have been determined and the transmission routes of MRSA on pig farms need to be elucidated. In the south of the Netherlands, in recent years, the black rat (Rattus rattus) has emerged as a prominent rodent on livestock farms. From March till May 2008, a survey on MRSA in rats living on livestock farms in the south of The Netherlands and the north of Belgium was conducted. In total, 40 black rats (R. rattus) and 3 brown rats (Rattus norvegicus) were collected on 12 farms including five pig farms, five poultry farms, one mixed pig and veal farm and one goat farm. MRSA ST398 was detected in black rats captured at two of the five pig farms as well as in a black rat living on the mixed pig and veal farm. From one black rat captured at another pig farm MRSA ST 97 was isolated. Considering the behaviour of rats on livestock farms, it is concluded that rats might play a role in the spread and persistence of MRSA on pig farms.

[Climate change influences the incidence of arthropod-borne diseases in the Netherlands]. / Klimaatverandering beïnvloedt het vóórkomen in Nederland van ziekten overgebracht door teken, muggen en zandvliegen.

Climate change is associated with changes in the occurrence of arthropod-borne diseases. It is difficult to foresee which arthropod-borne diseases will appear in the Netherlands due to climate change. Climate change influences the prevalence of ticks and may lead to a further increase in Lyme disease and an increased risk of the introduction of rickettsioses. With further warming of the climate there is a real possibility of settlement of the mosquito Aedes albopictus and introduction of the sandfly in the Netherlands. Whether this will lead to circulation of micro-organisms transmitted by these vectors (e.g. West Nile virus, Dengue virus, Leishmania) is not clear. Continued vigilance is necessary, even for vector-borne diseases that appear to be less relevant for the Netherlands.

Accidental importation of the mosquito Aedes albopictus into the Netherlands: a survey of mosquito distribution and the presence of dengue virus.

In the summer of 2005, the Asian tiger mosquito, Aedes albopictus (Skuse) (Diptera: Culicidae) was found for the first time in the Netherlands. It was collected on the premises of several horticultural companies that import the ornamental plant Dracaena sanderiana (Sparagalus: Dracaenaceae [Agavaceae]), known as Lucky bamboo, from southern China, an area endemic for this mosquito species and for arboviruses transmitted by this vector. Here we report the results of a 1-year survey of the distribution and vector status of Ae. albopictus in Lucky bamboo nurseries in the Netherlands (July 2006-June 2007). As it had been established previously that the presence of this species was linked to the import of Lucky bamboo, the survey was conducted only on sites owned by relevant import companies. In total, 569 adult Ae. albopictus were collected with mosquito traps from 15 of the 17 (88%) glasshouses used by Lucky bamboo importers, none of which were found to be infected with dengue virus. On two occasions there was evidence that Ae. albopictus had escaped from the glasshouses, but, overall, there was no evidence that a population had become established in the greenhouses or elsewhere.