Crystal structure of Schmallenberg orthobunyavirus nucleoprotein–RNA complex reveals a novel RNA sequestration mechanism

Schmallenberg virus (SBV) is a newly emerged orthobunyavirus (family Bunyaviridae) that has caused severe disease in the offspring of farm animals across Europe. Like all orthobunyaviruses, SBV contains a tripartite negative-sense RNA genome that is encapsidated by the viral nucleocapsid (N) protein in the form of a ribonucleoprotein complex (RNP). We recently reported the three-dimensional structure of SBV N that revealed a novel fold. Here we report the crystal structure of the SBV N protein in complex with a 42-nt-long RNA to 2.16 Å resolution. The complex comprises a tetramer of N that encapsidates the RNA as a cross-shape inside the protein ring structure, with each protomer bound to 11 ribonucleotides. Eight bases are bound in the positively charged cleft between the N- and C-terminal domains of N, and three bases are shielded by the extended N-terminal arm. SBV N appears to sequester RNA using a different mechanism compared with the nucleoproteins of other negative-sense RNA viruses. Furthermore, the structure suggests that RNA binding results in conformational changes of some residues in the RNA-binding cleft and the N- and C-terminal arms. Our results provide new insights into the novel mechanism of RNA encapsidation by orthobunyaviruses.     

Schmallenberg Virus Circulation in Culicoides in Belgium in 2012: Field Validation of a Real Time RT-PCR Approach to Assess Virus Replication and Dissemination in Midges

Indigenous Culicoides biting midges are suggested to be putative vectors for the recently emerged Schmallenberg virus (SBV) based on SBV RNA detection in field-caught midges. Furthermore, SBV replication and dissemination has been evidenced in C. sonorensis under laboratory conditions. After SBV had been detected in Culicoides biting midges from Belgium in August 2011, it spread all over the country by the end of 2011, as evidenced by very high between-herd seroprevalence rates in sheep and cattle. This study investigated if a renewed SBV circulation in midges occurred in 2012 in the context of high seroprevalence in the animal host population and evaluated if a recently proposed realtime RT-PCR approach that is meant to allow assessing the vector competence of Culicoides for SBV and bluetongue virus under laboratory conditions was applicable to field-caught midges. Therefore midges caught with 12 OVI traps in four different regions in Belgium between May and November 2012, were morphologically identified, age graded, pooled and tested for the presence of SBV RNA by realtime RT-PCR. The results demonstrate that although no SBV could be detected in nulliparous midges caught in May 2012, a renewed but short lived circulation of SBV in parous midges belonging to the subgenus Avaritia occured in August 2012 at all four regions. The infection prevalence reached up to 2.86% in the south of Belgium, the region where a lower seroprevalence was found at the end of 2011 than in the rest of the country. Furthermore, a frequency analysis of the Ct values obtained for 31 SBV-S segment positive pools of Avaritia midges showed a clear bimodal distribution with peaks of Ct values between 21–24 and 33–36. This closely resembles the laboratory results obtained for SBV infection of C. sonorensis and implicates indigenous midges belonging to the subgenus Avaritia as competent vectors for SBV.     

Genetic and phylogenetic analysis of the honey bee sacbrood virus from jiangxi isolates

The high prevalence of honeybee viral diseases poses a severe threat to the health of honeybees and causes substantial economic losses worldwide. Sacbrood virus (SBV) is a single-strand RNA virus that infects honeybees at all life stages. The infection can shorten the lifespan of adult bees and is lethal to larvae. SBV is the major cause of honeybee losses in Asia. Genetic and phylogenetic analyses of SBV isolates from different areas have been previously conducted. However, the impact of Apis mellifera Linnaeus and Apis cerana Fabricius coexistence on the infection and phylogeny of SBV remains unknown. In this study, we collected A. cerana and A. mellifera samples from commercial apiaries, only A. cerana in mountainous region. SBV prevalence was evaluated in three commercial apiaries of Jinxi, Tonggu and Nanchang and two mountainous regions of Zixi and Yifeng. In our sampling location, we found a higher SBV prevalence in the mountainous regions than in commercial apiaries. Partial structural polyprotein coding sequences were sequenced and compared with other GenBank SBV isolates. Phylogenetic tree topologies showed that SBV isolates form two major groups based on their host specificity, and isolates from same country tend to cluster together in subclades, indicating that the host and geographic region has significant effects on SBV strain specificity.     

Exposure of Asian Elephants and Other Exotic Ungulates to Schmallenberg Virus

Schmallenberg virus (SBV) is an emerging Orthobunyavirus, first described in 2011 in cattle in Germany and subsequently spread throughout Europe, affecting mainly ruminant livestock through the induction of foetal malformations. To gain a better understanding of the spectrum of susceptible species and to assess the value of current SBV serological assays, screening of serum samples from exotic artiodactyls and perissodactyls collected at the Living Collections from the Zoological Society of London (Whipsnade and London Zoos) and Chester Zoo was carried out. There was compelling evidence of SBV infection in both zoological collections. The competitive ELISA has proved to be applicable for the detection of SBV in exotic Bovidae, Cervidae, Suidae, Giraffidae and most notably in endangered Asian elephants (Elephas maximus), but unreliable for the screening of Camelidae, for which the plaque reduction neutralisation test was considered the assay of choice.     

Generation and characterization of a potentially applicable Vero cell line constitutively expressing the Schmallenberg virus nucleocapsid protein

Schmallenberg virus (SBV) is a Culicoides-transmitted orthobunyavirus that poses a threat to susceptible livestock species such as cattle, sheep and goats. The nucleocapsid (N) protein of SBV is an ideal diagnostic antigen for the detection of viral infection. In this study, a stable Vero cell line, Vero-EGFP-SBV-N, constitutively expressing the SBV-N protein was established using a lentivirus system combined with puromycin selection. This cell line spontaneously emitted green fluorescent signals distributed throughout the cytoplasm, in which the expression of SBV-N fusion protein was confirmed by western blot analysis. The expression of SBV-N protein in Vero-EGFP-SBV-N cells was stable for more than fifty passages without puromycin pressure. The SBV-N fusion protein contained both an N-terminal enhanced green fluorescent protein (EGFP) tag and a C-terminal hexa-histidine (6 × His) tag, by which the N protein was successfully purified using Ni–NTA affinity chromatography. The cell line was further demonstrated to be reactive with SBV antisera and an anti-SBV monoclonal antibody in indirect immunofluorescence assays. Taken together, our results demonstrate that the Vero-EGFP-SBV-N cell line has potential for application in the serological diagnosis of SBV infection.     

Testing of UK Populations of Culex pipiens L. for Schmallenberg Virus Vector Competence and Their Colonization

Background

Schmallenberg virus (SBV), an arboviral pathogen of ruminants, emerged in northern Europe during 2011 and has subsequently spread across a vast geographic area. While Culicoides biting midges (Diptera: Ceratopogonidae) have been identified as a biological transmission agent of SBV, the role of mosquitoes (Diptera: Culicidae) as potential vectors has not been defined beyond small-scale field collections in affected areas. Culex pipiens L. are one of the most widespread mosquitoes in northern Europe; they are present on farms across the region and have previously been implicated as vectors of several other arboviruses. We assessed the ability of three colony lines of Cx. pipiens, originating from geographically diverse field populations, to become fully infected by SBV using semi-quantitative real-time RT-PCR (sqPCR).

Findings

Two colony lines of Cx. pipiens were created in the UK (‘Brookwood’ and ‘Caldbeck’) from field collections of larvae and pupae and characterised using genetic markers. A third strain of Cx. pipiens from CVI Wageningen, The Netherlands, was also screened during experiments. Intrathoracic inoculation of the Brookwood line resulted in infections after 14 days that were characterised by high levels of RNA throughout individuals, but which demonstrated indirect evidence of salivary gland barriers. Feeding of 322 individuals across the three colony lines on a membrane based infection system resulted in no evidence of full dissemination of SBV, although infections did occur in a small proportion of Cx. pipiens from each line.

Conclusions/Significance

This study established two novel lines of Cx. pipiens mosquitoes of UK origin in the laboratory and subsequently tested their competence for SBV. Schmallenberg virus replication and dissemination was restricted, demonstrating that Cx. pipiens is unlikely to be an epidemiologically important vector of the virus in northern Europe.     

Culicoides Species Communities Associated with Wild Ruminant Ecosystems in Spain: Tracking the Way to Determine Potential Bridge Vectors for Arboviruses

The genus Culicoides Latreille 1809 is a well-known vector for protozoa, filarial worms and, above all, numerous viruses. The Bluetongue virus (BTV) and the recently emerged Schmallenberg virus (SBV) are responsible for important infectious, non-contagious, insect-borne viral diseases found in domestic ruminants and transmitted by Culicoides spp. Both of these diseases have been detected in wild ruminants, but their role as reservoirs during the vector-free season still remains relatively unknown. In fact, we tend to ignore the possibility of wild ruminants acting as a source of disease (BTV, SBV) and permitting its reintroduction to domestic ruminants during the following vector season. In this context, a knowledge of the composition of the Culicoides species communities that inhabit areas where there are wild ruminants is of major importance as the presence of a vector species is a prerequisite for disease transmission. In this study, samplings were conducted in areas inhabited by different wild ruminant species; samples were taken in both 2009 and 2010, on a monthly basis, during the peak season for midge activity (in summer and autumn). A total of 102,693 specimens of 40 different species of the genus Culicoides were trapped; these included major BTV and SBV vector species. The most abundant vector species were C. imicola and species of the Obsoletus group, which represented 15% and 11% of total numbers of specimens, respectively. At the local scale, the presence of major BTV and SBV vector species in areas with wild ruminants coincided with that of the nearest sentinel farms included in the Spanish Bluetongue Entomological Surveillance Programme, although their relative abundance varied. The data suggest that such species do not exhibit strong host specificity towards either domestic or wild ruminants and that they could consequently play a prominent role as bridge vectors for different pathogens between both types of ruminants. This finding would support the hypothesis that wild ruminants could act as reservoirs for such pathogens, and subsequently be involved in the reintroduction of disease to livestock on neighbouring farms.     

Experimental Infection of Sheep at 45 and 60 Days of Gestation with Schmallenberg Virus Readily Led to Placental Colonization without Causing Congenital Malformations

Background

Main impact of Schmallenberg virus (SBV) on livestock consists in reproductive disorders, with teratogenic effects, abortions and stillbirths. SBV pathogenesis and viral placental crossing remain currently poorly understood. Therefore, we implemented an experimental infection of ewes, inoculated with SBV at 45 or 60 days of gestation (dg).

Methodology

“Mourerous” breed ewes were randomly separated in three groups: eight and nine ewes were subcutaneously inoculated with 1 ml of SBV infectious serum at 45 and 60 dg, respectively (G45 and G60). Six other ewes were inoculated subcutaneously with sterile phosphate buffer saline as control group. All SBV inoculated ewes showed RNAemia consistent with previously published studies, they seroconverted and no clinical sign was reported. Lambs were born at term via caesarian-section, and right after birth they were blood sampled and clinically examined. Then both lambs and ewes were euthanatized and necropsied.

Principal Findings/Significance

No lambs showed any malformation suggestive of SBV infection and none of them had RNAemia or anti-SBV antibodies prior to colostrum uptake. Positive SBV RNA detection in organs was rare in both G45 and G60 lambs (2/11 and 1/10, respectively). Nevertheless most of the lambs in G45 (9/11) and G60 (9/10) had at least one extraembryonic structure SBV positive by RTqPCR. The number of positive extraembryonic structures was significantly higher in G60 lambs. Time of inoculation (45 or 60 dg) had no impact on the placental colonization success rate but affected the frequency of detecting the virus in the offspring extraembryonic structures by the time of lambing. SBV readily colonized the placenta when ewes were infected at 45 or 60 dg but infection of the fetuses was limited and did not lead to congenital malformations.     

Development and evaluation of an indirect enzyme-linked immunosorbent assay for serological detection of Schmallenberg virus antibodies in ruminants using whole virus antigen

Background

In late 2011, a new Orthobunyavirus of the Simbu serogroup named Schmallenberg virus (SBV) emerged in continental Europe. The virus is transmitted by hematophagous arthropods, with the Culicoides species as, so far known, main vectors. Infection with the virus can cause clinical signs in adult ruminants including diarrhea, fever and reduced milk production. Transplacental infection of the developing fetus can lead to malformations of varying severity. To assess seroprevalence of SBV in Sweden an indirect enzyme-linked immunosorbent assay (ELISA) was established in connection with the surveys. Here, we describe the development and evaluation of the indirect ELISA, based on whole virus as the coating antigen and a monoclonal antibody for the detection of antibodies to SBV in ruminant sera. The evaluation includes comparison between the in-house ELISA, virus neutralization test and an indirect commercial ELISA.

Results

The optimal working dilutions of antigens and conjugate were estimated with checkerboard titrations. Comparative studies, including ROC analyses, were used for the selection of an optimal cut-off (S/P value?=?sample value as percentage of positive control value). With an estimated S/P value of 15% the whole virus ELISA showed a specificity of 100% and a sensitivity of 99.19% compared to virus neutralization test (VNT) and with a good consistency as shown in reproducibility and variability experiments. Furthermore, the comparison of our whole virus indirect ELISA to an indirect ELISA with a SBV nucleoprotein antigen, demonstrated a higher sensitivity of our test.

Conclusion

The indirect whole virus ELISA described in this paper is a readily available test for serological analysis of SBV antibodies. Since this in-house ELISA demonstrates a specificity and sensitivity comparable to virus neutralization test and also shows a higher sensitivity compared to commercially available indirect ELISA, it is a useful alternative for surveillance and screening purposes of SBV.

     

No evidence for the persistence of Schmallenberg virus in overwintering mosquitoes

In 2011, Schmallenberg virus (SBV), a novel member of the Simbu serogroup, genus Orthobunyavirus, was identified as the causative agent of a disease in ruminants in Europe. Based on the current knowledge on arthropods involved in the transmission of Simbu group viruses, a role of both midges and mosquitoes in the SBV transmission cycle cannot be excluded beforehand. The persistence of SBV in mosquitoes overwintering at SBV‐affected farms in the Netherlands was investigated. No evidence for the presence of SBV in 868 hibernating mosquitoes (Culex, Anopheles, and Culiseta spp., collected from January to March 2012) was found. This suggests that mosquitoes do not play an important role, if any, in the persistence of SBV during the winter months in northwestern Europe.     

Reversible inhibition of cell multiplication in vitro by inhibitors of arginine esteroproteases

Two classes of active-site specific inhibitors of trypsin-like proteases have been shown to inhibit reversibly the multiplication of eukaryotic cells in vitro. The competitive inhibitors p-aminobenzamidine and benzamidine were found to arrest the growth of normal and transformed mouse fibroblasts and human KB cells. The inhibition of cell multiplication occurred within 24 h and was accompanied by substantial decreases in the rates of DNA and protein synthesis. The rate of RNA synthesis was relatively unaffected by the protease inhibitors. In agreement with the known inhibition constants (K_i) for their action against trypsin, p-aminobenzamidine was a much more effective inhibitor of cell multiplication than benzamidine. In addition, tosyllysine chloromethyl ketone (Tos-LysCH₂Cl), an active-site titrant and irreversible inhibitor of trypsin, was found to cause a reversible inhibition of growth. These results suggest that an essential protease activity is necessary for cell multiplication. However, in the case of mouse L-cells, all of the inhibitors and particulary p-aminobenzamidine caused excessive accumulation of lactate in the extracellular medium. This observation, which suggests the possibility of additional sites of action of these compounds in cells, was found to depend upon the cell type and appears to be unrelated to the inhibition of growth.     

Transpuparial transmission of Kashmir bee virus and sacbrood virus in the honey bee (Apis mellifera)

When particles of Kashmir bee virus (KBV) and sacbrood virus (SBV) were fed to larvae of the honey bee, Apis mellifera, in Australian colonies, the resulting pupae became in apparently infected. There was no statistically significant difference in the susceptibility of 1, 2, 3 or 4-day-old larvae for either virus, but 5-day-old larvae were significantly less susceptible to SBV than younger larvae. There was no significant difference in the proportions of pupae that became in apparently infected when, as larvae, they were fed various concentrations of each virus, but significantly more larvae were removed from their cells when fed concentrated preparations of each virus than when fed diluted preparations. Susceptible larvae that became in apparently infected with KBV and SBV developed normally into in apparently infected pupae and later, emerged as in apparently infected worker bees.     

Differential hygienic behavior of Apis cerana F. and Apis mellifera L. to Sacbrood virus infection

Beekeeping with Apis cerana of Korean apiculture is facing with serious colony collapse caused by invasive Sacbrood virus (SBV) disease. This fatal brood disease was the main reason of more than 90% colony lost in Korea leading almost the extinct crisis. Sacbrood virus can infect either larvae or adult honeybees, with a higher sensibility of larvae to the infection. Since SBV has spread to all over the country, efforts have been made to treat and prevent this devastating disease although no effective results have so far been obtained. Several studies have demonstrated that Apis mellifera bee colonies that express an efficient hygienic behavior exhibit a higher resistance to the brood disease. In this study we demonstrated that the differences of hygienic behavior between A. cerana and A. mellifera. A. cerana more efficiently removed the pin-killed brood than A. mellifera. On the other hand, A. mellifera more efficiently removed SBV-infected larvae and SBV-dead brood than A. cerana. However, it remains unclear whether the advantage of hygienic bee could have efficacy against Sacbrood disease on A. cerana colonies.     

Schmallenberg virus infection of adult type I interferon receptor knock-out mice

Schmallenberg virus (SBV), a novel orthobunyavirus, was discovered in Europe in late 2011. It causes mild and transient disease in adult ruminants, but fetal infection can lead to abortion or severe malformations. There is considerable demand for SBV research, but in vivo studies in large animals are complicated by their long gestation periods and the cost of high containment housing. The goal of this study was to investigate whether type I interferon receptor knock-out (IFNAR(-/-)) mice are a suitable small animal model for SBV. Twenty IFNAR(-/-) mice were inoculated with SBV, four were kept as controls. After inoculation, all were observed and weighed daily; two mice per day were sacrificed and blood, brain, lungs, liver, spleen, and intestine were harvested. All but one inoculated mouse lost weight, and two mice died spontaneously at the end of the first week, while another two had to be euthanized. Real-time RT-PCR detected large amounts of SBV RNA in all dead or sick mice; the controls were healthy and PCR-negative. IFNAR(-/-) mice are susceptible to SBV infection and can develop fatal disease, making them a handy and versatile tool for SBV vaccine research.     

Life cycle of Helicosporidium parasiticum in the navel orangeworm, Paramyelois transitella

Germination of Helicosporidium parasiticum spores in the gut of the naval orangeworm, Paramyelois transitella, was characterized by a rapid expulsion of filaments and sporoplasms. Two stages of multiplication were observed: The first consisted of elongate cells that developed directly from sporoplasms and divided to form 4–8 daughter cells enclosed in a thin-walled elongate pellicle, which subsequently ruptured to release the enclosed cells, and the second consisted of spherical cells that divided to form 4–8 daughter cells enclosed in an oval pellicle. This second cycle of multiplication was repeated several times and ended in the formation of sporonts. Sporonts then developed a spore wall before they formed a filament and sporoplasms. The pathogen apparently does not belong to the Protozoa; however, its possible relationship to the primitive ascomycetes still remains to be clarified.     

TaqMan MGB Probe Fluorescence Real-Time Quantitative PCR for Rapid Detection of Chinese Sacbrood Virus

Sacbrood virus (SBV) is a picorna-like virus that affects honey bees (Apis mellifera) and results in the death of the larvae. Several procedures are available to detect Chinese SBV (CSBV) in clinical samples, but not to estimate the level of CSBV infection. The aim of this study was develop an assay for rapid detection and quantification of this virus. Primers and probes were designed that were specific for CSBV structural protein genes. A TaqMan minor groove binder (MGB) probe-based, fluorescence real-time quantitative PCR was established. The specificity, sensitivity and stability of the assay were assessed; specificity was high and there were no cross-reactivity with healthy larvae or other bee viruses. The assay was applied to detect CSBV in 37 clinical samples and its efficiency was compared with clinical diagnosis, electron microscopy observation, and conventional RT-PCR. The TaqMan MGB-based probe fluorescence real-time quantitative PCR for CSBV was more sensitive than other methods tested. This assay was a reliable, fast, and sensitive method that was used successfully to detect CSBV in clinical samples. The technology can provide a useful tool for rapid detection of CSBV. This study has established a useful protocol for CSBV testing, epidemiological investigation, and development of animal models.     

Prevalence and Seasonal Variations of Six Bee Viruses in Apis mellifera L. and Varroa destructor Mite Populations in France

A survey of six bee viruses on a large geographic scale was undertaken by using seemingly healthy bee colonies and the PCR technique. Samples of adult bees and pupae were collected from 36 apiaries in the spring, summer, and autumn during 2002. Varroa destructor samples were collected at the end of summer following acaricide treatment. In adult bees, during the year deformed wing virus (DWV) was found at least once in 97% of the apiaries, sacbrood virus (SBV) was found in 86% of the apiaries, chronic bee paralysis virus (CBPV) was found in 28% of the apiaries, acute bee paralysis virus (ABPV) was found in 58% of the apiaries, black queen cell virus (BQCV) was found in 86% of the apiaries, and Kashmir bee virus (KBV) was found in 17% of the apiaries. For pupae, the following frequencies were obtained: DWV, 94% of the apiaries; SBV, 80% of the apiaries; CBPV, none of the apiaries; ABPV, 23% of the apiaries; BQCV, 23% of the apiaries; and KBV, 6% of the apiaries. In Varroa samples, the following four viruses were identified: DWV (100% of the apiaries), SBV (45% of the apiaries), ABPV (36% of the apiaries), and KBV (5% of the apiaries). The latter findings support the putative role of mites in transmitting these viruses. Taken together, these data indicate that bee virus infections occur persistently in bee populations despite the lack of clinical signs, suggesting that colony disease outbreaks might result from environmental factors that lead to activation of viral replication in bees.     

Three-dimensional structure of the Chinese Sacbrood bee virus

The RNA of Chinese Sacbrood Bee Virus (CSBV) was purified and used as template to obtain a 1096 bp cDNA fragment by RT-PCR amplification. This DNA fragment was cloned into pGEM-T Easy Vector for sequencing. Analyses of the sequenced CSBV RNA fragment revealed a nucleotide sequence homology of 87.6% and a deduced amino-acid sequence homology of 94.6% with that of the Sacbrood Virus (SBV), indicating that CSBV is a different but highly homologous virus of SBV. The three-dimensional (3D) structure of CSBV was determined at 2.5 nm resolution by using electron cryo-microscopy (cryoEM) and computer reconstruction methods. The 3-D structure showed that the capsid has aT = 1 (orP = 3) icosahedral capsid shell with a smooth surface. There were 12 pentons at its icosahedral vertices (5-fold axes) and 132 holes penetrating the shell. The 3-D structure also revealed densities corresponding to the CSBV genome, suggesting icosahedrally-ordered RNA organization, a novel feature not previously reported for any picornaviruses.     

Short-Term and Long-Term Survival and Virulence of Legionella pneumophila in the Defined Freshwater Medium Fraquil

Legionella pneumophila (Lp) is the etiological agent responsible for Legionnaires’ disease, a potentially fatal pulmonary infection. Lp lives and multiplies inside protozoa in a variety of natural and man-made water systems prior to human infection. Fraquil, a defined freshwater medium, was used as a highly reproducible medium to study the behaviour of Lp in water. Adopting a reductionist approach, Fraquil was used to study the impact of temperature, pH and trace metal levels on the survival and subsequent intracellular multiplication of Lp in Acanthamoeba castellanii, a freshwater protozoan and a natural host of Legionella. We show that temperature has a significant impact on the short- and long-term survival of Lp, but that the bacterium retains intracellular multiplication potential for over six months in Fraquil. Moreover, incubation in Fraquil at pH 4.0 resulted in a rapid decline in colony forming units, but was not detrimental to intracellular multiplication. In contrast, variations in trace metal concentrations had no impact on either survival or intracellular multiplication in amoeba. Our data show that Lp is a resilient bacterium in the water environment, remaining infectious to host cells after six months under the nutrient-deprived conditions of Fraquil.