A longstanding enigmatic feature of the group 1 coronaviruses is the uncleaved phenotype of their spike protein, an exceptional property among class I fusion proteins. Here, however, we show that some group 1 coronavirus spike proteins carry a furin enzyme recognition motif and can actually be cleaved, as demonstrated for a feline coronavirus. Interestingly, this feature can be lost during cell culture adaptation by a single mutation in the cleavage motif; this, however, preserves a heparan sulfate binding motif and renders infection by the virus heparan sulfate dependent. We identified a similar cell culture adaptation for the human coronavirus OC43.Enveloped viruses use different types of fusion proteins to realize the membrane fusion by which they initiate their infection. For coronaviruses, it is the spike (S) protein that is responsible for cell entry, and this S protein has been shown to belong to the class I fusion proteins (4). These proteins typically occur in virions as homotrimeric complexes primed for fusion through cleavage by furin-like enzymes. Membrane fusion by these activated proteins can then be triggered upon receptor binding (e.g., human immunodeficiency virus type 1) or by conditions such as low pH after endosomal uptake (e.g., influenza A virus) (for a recent review, see reference 50).One of the puzzling questions about coronavirus S proteinmediated membrane fusion regards the cleavage requirement of the S protein. Coronaviruses have been assigned to different groups based on antigenic and genetic criteria (41). Interestingly, while the group 1 coronaviruses carry uncleaved S proteins, the S proteins of almost all viruses from groups 2 and 3 are furin activated (10) by processing at a characteristic multibasic motif (often RRXRR) present in these proteins. The importance of cleavage for infectivity was underscored recently by the revelation that the two prominent group 2 viruses lacking such a furin recognition site, and hence carrying uncleaved spikes, appeared to depend on a different, new processing mechanism. Thus, the severe acute respiratory syndrome coronavirus (SARS-CoV) and the murine hepatitis virus strain 2 (MHV-2) were both shown to require proteolytic cleavage in their target cell, which is mediated by cathepsin enzymes (23,36,42). The cathepsin cleavage site of the SARS-CoV spike protein was mapped to the same region as that in which, in other viruses, the S protein is activated by furin (B. J. Bosch and P. J. M. Rottier, unpublished observations), hence similarly generating an amino-terminal, receptor binding domain (S1) and a membrane-anchored carboxy-terminal domain (S2) responsible for membrane fusion (for reviews, see references 3 and 9).When looking closer into the enigmatic lack of cleavage of the group 1 coronavirus spike proteins, we established that the infection of cells by two of those viruses, human coronavirus (HCoV) NL63 (23) and feline infectious peritonitis virus strain 79-1146 (our unpublished observations), is insensitive to cathepsin inhibitors. However, w...

Background. Streptococcus suis has emerged as an important cause of bacterial meningitis in adults. The ingestion of undercooked pork is a risk factor for human S. suis serotype 2 (SS2) infection. Here we provide experimental evidence indicating that the gastrointestinal tract is an entry site of SS2 infection.Methods. We developed a noninvasive in vivo model to study oral SS2 infection in piglets. We compared in vitro interaction of S. suis with human and porcine intestinal epithelial cells (IEC).Results. Two out of 15 piglets showed clinical symptoms compatible with S. suis infection 24–48 hours after ingestion of SS2. SS2 was detected in mesenteric lymph nodes of 40% of challenged piglets. SS2 strains isolated from patients showed significantly higher adhesion to human IEC compared to invasive strains isolated from pigs. In contrast, invasive SS9 strains showed significantly higher adhesion to porcine IEC. Translocation across human IEC, which occurred predominately via a paracellular route, was significantly associated with clonal complex 1, the predominant zoonotic genotype. Adhesion and translocation were dependent on capsular polysaccharide production.Conclusions. SS2 should be considered a food-borne pathogen. S. suis interaction with human and pig IEC correlates with S. suis serotype and genotype, which can explain the zoonotic potential of SS2.

Background Rift Valley fever virus (RVFV) is an arbovirus of the order Bunyavirales that causes severe disease in ruminants and humans. Outbreaks in sheep herds are characterised by newborn fatalities and abortion storms. The association of RVFV infections with abortions of ovines and other ruminants is well recognized, whereas the pathology resulting in abortion has remained undescribed. Accumulating evidence suggests that RVFV is abortogenic in humans as well, warranting more research on the interaction of RVFV with the ruminant and human placenta. Methodology/Principal findings Pregnant ewes were inoculated with a highly virulent strain of RVFV and necropsied at different days post infection. Tissues were collected and analysed by PCR, virus isolation, and immunohistochemistry. The results show that RVFV replicates efficiently in maternal placental epithelial cells before the virus infects foetal trophoblasts. Moreover, the virus was shown to bypass the maternal epithelial cell layer by directly targeting foetal trophoblasts in the haemophagous zone, a region of the ovine placenta where maternal blood is in direct contact with foetal cells. Abortion was associated with widespread necrosis of placental tissues accompanied with severe haemorrhages. Experiments with human placental explants revealed that the same virus strain replicates efficiently in both cyto-and syncytiotrophoblasts. Conclusions/Significance This study demonstrates that RVFV targets the foetal-maternal interface in both ovine and human placentas. The virus was shown to cross the ovine placental barrier via two distinct routes, ultimately resulting in placental and foetal demise followed by abortion. Our finding that RVFV replicates efficiently in human trophoblasts underscores the risk of RVFV infection for human pregnancy.

The bunyavirus genome comprises a small (S), medium (M), and large (L) RNA segment of negative polarity. Although genome segmentation confers evolutionary advantages by enabling genome reassortment events with related viruses, genome segmentation also complicates genome replication and packaging. Accumulating evidence suggests that genomes of viruses with eight or more genome segments are incorporated into virions by highly selective processes. Remarkably, little is known about the genome packaging process of the tri-segmented bunyaviruses. Here, we evaluated, by single-molecule RNA fluorescence in situ hybridization (FISH), the intracellular spatio-temporal distribution and replication kinetics of the Rift Valley fever virus (RVFV) genome and determined the segment composition of mature virions. The results reveal that the RVFV genome segments start to replicate near the site of infection before spreading and replicating throughout the cytoplasm followed by translocation to the virion assembly site at the Golgi network. Despite the average intracellular S, M and L genome segments approached a 1:1:1 ratio, major differences in genome segment ratios were observed among cells. We also observed a significant amount of cells lacking evidence of M-segment replication. Analysis of two-segmented replicons and four-segmented viruses subsequently confirmed the previous notion that Golgi recruitment is mediated by the Gn glycoprotein. The absence of colocalization of the different segments in the cytoplasm and the successful rescue of a tri-segmented variant with a codon shuffled M-segment suggested that inter-segment interactions are unlikely to drive the copackaging of the different segments into a single virion. The latter was confirmed by direct visualization of RNPs inside mature virions which showed that the majority of virions lack one or more genome segments. Altogether, this study suggests that RVFV genome packaging is a non-selective process.

Streptococcus suis causes infections in pigs and occasionally in humans, resulting in manifestations as meningitis, sepsis, arthritis, and septic shock. For survival within the host, S. suis requires numerous nutrients including trace metals. Little is known about the specific proteins involved in metal scavenging in S. suis. In this study we evaluated the role of the putative high-affinity metal binding lipoprotein TroA in metal acquisition and virulence. A mutant strain deficient in the expression of TroA (⌬troA mutant) was constructed. Growth of the ⌬troA mutant in Todd-Hewitt broth was similar to wild-type growth; however, growth of the ⌬troA mutant in cation-deprived Todd-Hewitt broth and in porcine serum was strongly reduced compared to growth of wild-type bacteria. Supplementing the medium with extra manganese but not with magnesium, zinc, copper, nickel, or iron restored growth to wild-type levels, indicating that TroA is specifically required for growth in environments low in manganese. The ⌬troA mutant also showed increased susceptibility to H 2 O 2 , suggesting that TroA is involved in counteracting oxidative stress. Furthermore, the expression of the troA gene was subject to environmental regulation at the transcript level. In a murine S. suis infection model, the ⌬troA mutant displayed a nonvirulent phenotype. These data indicate that S. suis TroA is involved in manganese acquisition and is required for full virulence in mice.Streptococcus suis is an important pathogen of pigs and may cause meningitis, sepsis, arthritis, and septic shock. Occasionally, S. suis is able to infect humans. Infected humans may show symptoms similar to those in pigs (1,5,11,32). Although human infections are exceptional, a large outbreak in humans was reported in 2005 in China, with 215 cases and 39 deaths (52). Of the 33 known S. suis serotypes, serotype 2 is most frequently isolated from diseased pigs and humans. However, serotype 9 infections are emerging in pigs, especially in Europe (7,35,48). Current control measures are insufficient and mainly rely on antibiotic treatment and vaccination with homologous bacterins. Increased antibiotic resistance has been reported for S. suis (17, 49), and bacterin-based vaccines do not provide protection against multiple serotypes (6).For growth and function, bacteria have to acquire numerous nutrients from their surrounding environment. For pathogenic bacteria, an important group of essential nutrients are the trace metals. Metals such as iron, zinc, and manganese have been shown to be essential structural and catalytic cofactors for several bacterial proteins (2). However, the concentration of free available trace metals within an infected host is relatively low compared to the metal concentrations in medium usually applied for in vitro growth. Within the host, several trace metals are sequestered; for instance, iron binds to hemoglobin, and zinc and manganese bind to the S100 family of proteins produced by neutrophils (14,15). This recruitment of trace metals by host proteins ha...

BackgroundRift Valley fever virus (RVFV) is a mosquito-borne bunyavirus of the genus Phlebovirus that is highly pathogenic to ruminants and humans. The disease is currently confined to Africa and the Arabian Peninsula, but globalization and climate change may facilitate introductions of the virus into currently unaffected areas via infected animals or mosquitoes. The consequences of such an introduction will depend on environmental factors, the availability of susceptible ruminants and the capacity of local mosquitoes to transmit the virus. We have previously demonstrated that lambs native to the Netherlands are highly susceptible to RVFV and we here report the vector competence of Culex (Cx.) pipiens, the most abundant and widespread mosquito species in the country. Vector competence was first determined after artificial blood feeding of laboratory-reared mosquitoes using the attenuated Clone 13 strain. Subsequently, experiments with wild-type RVFV and mosquitoes hatched from field-collected eggs were performed. Finally, the transmission of RVFV from viremic lambs to mosquitoes was studied.Principal findingsArtificial feeding experiments using Clone 13 demonstrated that indigenous, laboratory-reared Cx. pipiens mosquitoes are susceptible to RVFV and that the virus can be transmitted via their saliva. Experiments with wild-type RVFV and mosquitoes hatched from field-collected eggs confirmed the vector competence of Cx. pipiens mosquitoes from the Netherlands. To subsequently investigate transmission of the virus under more natural conditions, mosquitoes were allowed to feed on RVFV-infected lambs during the viremic period. We found that RVFV is efficiently transmitted from lambs to mosquitoes, although transmission was restricted to peak viremia. Interestingly, in the mosquito-exposed skin samples, replication of RVFV was detected in previously unrecognized target cells.SignificanceWe here report the vector competence of Cx. pipiens mosquitoes from the Netherlands for RVFV. Both laboratory-reared mosquitoes and well as those hatched from field-collected eggs were found to be competent vectors. Moreover, RVFV was transmitted efficiently from indigenous lambs to mosquitoes, although the duration of host infectivity was found to be shorter than previously assumed. Interestingly, analysis of mosquito-exposed skin samples revealed previously unidentified target cells of the virus. Our findings underscore the value of including natural target species in vector competence experiments.

Rift Valley fever virus (RVFV) is a mosquito-borne pathogen that affects domesticated ruminants and occasionally humans. Classical RVF vaccines are based on formalin-inactivated virus or the live-attenuated Smithburn strain. The inactivated vaccine is highly safe but requires multiple administrations and yearly re-vaccinations. Although the Smithburn vaccine provides solid protection after a single vaccination, this vaccine is not safe for pregnant animals. An alternative live-attenuated vaccine, named Clone 13, carries a large natural deletion in the NSs gene which encodes the major virulence factor of the virus. The Clone 13 vaccine was previously shown to be safe for young lambs and calves. Moreover, a study in pregnant ewes suggested that the vaccine could also be applied safely during gestation. To anticipate on a possible future incursion of RVFV in Europe, we have evaluated the safety of Clone 13 for young lambs and pregnant ewes. In line with the guidelines from the World Organisation for Animal health (Office International des Epizooties, OIE) and regulations of the European Pharmacopeia (EP), these studies were performed with an overdose. Our studies with lambs showed that Clone 13 dissemination within vaccinated animals is very limited. Moreover, the Clone 13 vaccine virus was not shed nor spread to in-contact sentinels and did not revert to virulence upon animal-to-animal passage. Importantly, a large experiment with pregnant ewes demonstrated that the Clone 13 virus is able to spread to the fetus, resulting in malformations and stillbirths. Altogether, our results suggest that Clone 13 can be applied safely in lambs, but that caution should be taken when Clone 13 is used in pregnant animals, particularly during the first trimester of gestation.

Bunyaviruses have a genome that is divided over multiple segments. Genome segmentation complicates the generation of progeny virus, since each newly formed virus particle should preferably contain a full set of genome segments in order to disseminate efficiently within and between hosts. Here, we combine immunofluorescence and fluorescence in situ hybridization techniques to simultaneously visualize bunyavirus progeny virions and their genomic content at single-molecule resolution in the context of singly infected cells. Using Rift Valley fever virus and Schmallenberg virus as prototype tri-segmented bunyaviruses, we show that bunyavirus genome packaging is influenced by the intracellular viral genome content of individual cells, which results in greatly variable packaging efficiencies within a cell population. We further show that bunyavirus genome packaging is more efficient in insect cells compared to mammalian cells and provide new insights on the possibility that incomplete particles may contribute to bunyavirus spread as well.