High genetic variability of Schmallenberg virus M-segment leads to efficient immune escape from neutralizing antibodies.

Schmallenberg virus (SBV) is the cause of severe fetal malformations when immunologically naïve pregnant ruminants are infected. In those malformed fetuses, a "hot-spot"-region of high genetic variability within the N-terminal region of the viral envelope protein Gc has been observed previously, and this region co-localizes with a known key immunogenic domain. We studied a series of M-segments of those SBV variants from malformed fetuses with point mutations, insertions or large in-frame deletions of up to 612 nucleotides. Furthermore, a unique cell-culture isolate from a malformed fetus with large in-frame deletions within the M-segment was analyzed. Each Gc-protein with amino acid deletions within the "hot spot" of mutations failed to react with any neutralizing anti-SBV monoclonal antibodies or a domain specific antiserum. In addition, in vitro virus replication of the natural deletion variant could not be markedly reduced by neutralizing monoclonal antibodies or antisera from the field. The large-deletion variant of SBV that could be isolated in cell culture was highly attenuated with an impaired in vivo replication following the inoculation of sheep. In conclusion, the observed amino acid sequence mutations within the N-terminal main immunogenic domain of glycoprotein Gc result in an efficient immune evasion from neutralizing antibodies in the special environment of a developing fetus. These SBV-variants were never detected as circulating viruses, and therefore should be considered to be dead-end virus variants, which are not able to spread further. The observations described here may be transferred to other orthobunyaviruses, particularly those of the Simbu serogroup that have been shown to infect fetuses. Importantly, such mutant strains should not be included in attempts to trace the spatial-temporal evolution of orthobunyaviruses in molecular-epidemiolocal approaches during outbreak investigations.

A simplified digital workflow for the prosthetic finishing of implant rehabilitations: a case report.

The aim of this article is to describe how, during the provisional and definitive prosthetic phases, using new digital technologies, it is possible to improve the ergonomics of the prosthetist's work and reduce the discomfort of patients, subjecting them to the fewest possible appointments at the dentist. The proposal of a full digital protocol, described by the following case report, for the realization of a definitive prosthetic rehabilitation supported by a reduced number of implants, in fact, allows to considerably reduce the number of appointments and reduce any bias. A 67-year-old male patient presents for the first visit to the Department of Dentistry of the San Raffaele Hospital, wearing a removable upper prosthesis and with the request to heal the aesthetic and functional situation through prosthetics fixed. An initial panoramic radiograph was performed, intra and extra oral photos were taken and also intraoral impressions. A stereo-lithographic models are obtained from intraoral scans, and two total prostheses, upper and lower, were packaged for the provisional post-surgical phase was performed. In accordance with the All-on-4 method 8 implant fixtures were placed. For the final prosthetic phase, the patient underwent only two operative sessions. In the first session, scans were taken with the provisionals in situ, of the patient's mucous membranes and with the Scan-abutments in place. In the second session using specific CADSoftware the matching of the STL files of the three scans were created, the opposing arches of the patient were related on a digital articulator, and the milled titanium bars were immediately constructed and finished with the resin. Finally, the definitive prostheses were delivered to the patient without any other test. Digital technology has allowed a clear reduction in working times and costs and has allowed the reduction of stress for patients who undergo invasive and extensive treatments to recover aesthetics and function, and for clinicians who must manage complex cases with fewer appointments possible.

Examining bull semen for residues of Schmallenberg virus RNA.

Schmallenberg orthobunyavirus (SBV) was initially detected in 2011 in Germany from dairy cattle with fever and decreased milk yield. The virus infection is now established in many parts of the world with recurrent epidemics. SBV is transmitted through midges and transplacental. No direct virus transmission including via breeding has ever been demonstrated. In some bulls, however, the virus is detectable transiently, in low to minute quantities, in semen post-infection. While the infection is considered of low impact for the dairy industry, some SBV-free countries have adopted a zero-risk approach requiring bull semen batches to be tested for SBV RNA residues prior to import. This, in turn, obligates a protocol to enable sensitive detection of SBV RNA in semen samples for export purposes. Here, we describe how we established a now ISO/IEC 17025 accredited protocol that can effectively detect minute quantities of SBV RNA in semen and also its application to monitor bull semen during two outbreaks in the United Kingdom in 2012 and 2016. The data demonstrate that only a small number of bulls temporarily shed low amounts of SBV.

Investigation into an outbreak of Border disease virus in pigs in England.

Border disease (BD) was first reported in 1959 in lambs from the border region of England and Wales. The causative virus (BD virus; BDV) has since been identified in several other ruminant species and pigs. The virus is prevalent in sheep flocks of UK, Europe and USA and has potential to inflict substantial economic losses. Natural BDV infection of pigs was first reported in the UK in 1992 from pigs with haemorrhagic lesions and more recently from healthy pigs in Spain and Japan. Here, a persistent problem of poor growth and anaemia in a small proportion of growing pigs on a mixed pig and sheep holding was investigated and tissues were tested in a pan viral microarray. The microarray detected BDV RNA in several tissues which was further confirmed by sequencing, specific BDV PCR and immunohistochemistry. Phylogenetically, the virus clustered with other BDVs in the sub-genotype 1b. This investigation highlights likely interspecies transmission of pestiviruses and their impact on pestivirus detection and eradication programs.

Mitf cooperates with Rb1 and activates p21Cip1 expression to regulate cell cycle progression.

The controls that enable melanoblasts and melanoma cells to proliferate are likely to be related, but so far no key regulator of cell cycle progression specific to the melanocyte lineage has been identified. The microphthalmia-associated transcription factor Mitf has a crucial but poorly defined role in melanoblast and melanocyte survival and in differentiation. Here we show that Mitf can act as a novel anti-proliferative transcription factor able to induce a G1 cell-cycle arrest that is dependent on Mitf-mediated activation of the p21(Cip1) (CDKN1A) cyclin-dependent kinase inhibitor gene. Moreover, cooperation between Mitf and the retinoblastoma protein Rb1 potentiates the ability of Mitf to activate transcription. The results indicate that Mitf-mediated activation of p21Cip1 expression and consequent hypophosphorylation of Rb1 will contribute to cell cycle exit and activation of the differentiation programme. The mutation of genes associated with melanoma, such as INK4a or BRAF that would affect either Mitf cooperation with Rb1 or Mitf stability respectively, would impair Mitf-mediated cell cycle control.

Classical swine fever virus infection protects aortic endothelial cells from pIpC-mediated apoptosis.

Classical swine fever virus (CSFV) causes severe disease in pigs associated with leukopenia, haemorrhage and fever. We show that CSFV infection protects endothelial cells from apoptosis induced by the dsRNA mimic, pIpC, but not from other apoptotic stimuli, FasL or staurosporine. CSFV infection inhibits pIpC-induced caspase activation, mitochondrial membrane potential loss and cytochrome c release as well as the pro-apoptotic effects of truncated Bid (tBid) overexpression. The CSFV proteins N(pro) and E(rns) both contribute to CSFV inhibition of apoptosis. We conclude that CSFV infection can inhibit apoptotic signalling at multiple levels, including at the caspase-8 and the mitochondrial checkpoints. By supporting viral replication, endothelial cells may promote CSFV pathogenesis.

Timing of oral feeding changes in premature infants who underwent osteopathic manipulative treatment.

BACKGROUND: The delayed transition from gavage-to-nipple feeding is one of the most significant factors that may prolong hospital length of stay (LOS). Osteopathic manipulative treatment (OMT) has been demonstrated to be effective regarding LOS reduction, but no investigations have documented its clinical validity for attaining oral feeding. OBJECTIVES: To assess OMT utility regarding the timing of oral feeding in healthy preterm infants. DESIGN: Preliminary propensity score-matched retrospective cohort study. SETTING: Data were extrapolated from the neonatal intensive care unit (NICU) of Del Ponte Hospital in Varese, Italy, during the period between March 2012 and December 2013. INTERVENTIONS: Two propensity score-matched groups of healthy preterm infants aged 28+0 to 33+6 were compared, observing those supported with OMT until hospital discharge and control subjects. MAIN OUTCOME MEASURES: Days from birth to the attainment of oral feeding was the primary endpoint. Body weight, body length, head circumference and LOS were considered as secondary endpoints. RESULTS: Seventy premature infants were included in the study as the control group (n = 35; body weight (BW) = 1457.9 ± 316.2 g; gestational age (GA) = 31.5 ± 1.73 wk) and the osteopathic group (n = 35; BW = 1509.6 ± 250.8 g; GA = 31.8 ± 1.64 wk). The two groups had analogous characteristics at study entry. In this cohort, we observed a significant reduction in TOF (-5.00 days; p = 0.042) in the osteopathic group with a greater effect in very low birth weight infants. CONCLUSIONS: These data demonstrate the utility and potential efficacy of OMT for the attainment of oral feeding. Further adequately powered clinical trials are recommended.

Characterization of catalytic and non-catalytic activities of EgGST2-3, a heterodimeric glutathione transferase from Echinococcus granulosus.

Glutathione transferases (GSTs) perform several catalytic and non-catalytic roles in the defense against toxicities of electrophile compounds and oxidative stress, and therefore are involved in stress-response and cell detoxification. Previously, we have provided evidence indicating that EgGST2 and EgGST3, two phylogenetically distant Echinococcus granulosus GSTs, can naturally form a heterodimeric structure (EgGST2-3). In the present work, the recombinant heterodimer GST (rEgGST2-3) is characterized. Hence, rEgGST2-3 was able to conjugate GSH to three substrates: 1-chloro-2,4-dinitrobenzene (CDNB, general substrate for GSTs), 1,2-dichloro-4-nitrobenzene (specific substrate for mammalian Mu class) and trans,trans-deca-2,4-dienal (reactive carbonyl). The canonical activity was considerably reduced by all the conventional inhibitors (cybacron blue, triphenylthin chloride and bromosulfophthalein) and by other inhibitors (ellagic acid, alizarin and chenodeoxycholic acid). Besides this, rEgGST2-3 activity was inhibited by a number of anthelmintic drugs, where the halogenated phenolic drugs (mainly bithionol and hexachlorophene) acted as stronger inhibitors, suggesting they may bind to the EgGST2-3. Moreover, rEgGST2-3 exhibited glutathione-peroxidase activity, and its specific constant (kcat/KM) was calculated. Finally, rEgGST2-3 displayed the ability to bind non-substrate molecules, particularly anthelmintic drugs, suggesting that ligandin activity may have potential to act as a passive protection parasite mechanism. Overall, the rEgGST2-3 behavior was shown to be both complementary and redundant to that reported for rEgGST1, another characterized GST from E. granulosus. It would be appropriate that different enzymes in the same organism do not have exactly the same functional properties to develop a better adaptation to life in the host.

Incursion of Schmallenberg virus into Great Britain in 2011 and emergence of variant sequences in 2016.

Schmallenberg virus (SBV) is a vector-borne orthobunyavirus in the family Bunyaviridae, first identified in Germany before rapidly spreading throughout Europe. To investigate the events surrounding the incursion of this virus into Great Britain (GB) and its subsequent spread, archived sheep serum samples from an unrelated field survey in 2011 were analysed for the presence of SBV specific antibodies, to determine the earliest date of seroconversion. This serological study, along with analysis of the spatial spread of the sources of samples submitted for SBV analysis after January 2012, suggests that SBV entered GB on more than one occasion and in more than one location. Phylogenetic analysis of SBV sequences from 2012 ovine samples, from a variety of counties and dates, demonstrated a non-linear evolution of the virus, i.e. there was no distinct clustering between host species, geographical locations or during the outbreak. This also supports the notion of multiple viruses entering GB, rather than a single virus incursion. Premature termination signals were present in several non-structural putative protein sequences. One SBV sequence exhibited large deletions in the M segment of the genome. After the first outbreak in 2011-2012, interest in SBV in GB waned and continuous surveillance was not upheld. The re-emergence of SBV in 2016 has raised renewed concern and ended speculation that SBV might have been eradicated permanently from GB. When SBV sequences from 2012 were compared with those from the re-emergence in 2016-2017, a second distinct clade of SBV was identified that separates recent strains from those observed during the first outbreak.

Establishment of a Bovine Viral Diarrhea Virus Type 2 Intranasal Challenge Model for Assessing Vaccine Efficacy.

The objective of this study was to develop a bovine viral diarrhea virus type 2 (BVDV-2) challenge model suitable for evaluation of efficacy of BVDV vaccines; a model that mimics natural infection and induces clear leukopenia and viremia. Clinical, hematological and virological parameters were evaluated after infection of two age groups of calves (3 and 9 months) with two BVDV-2 strains (1362727 and 502643). Calves became pyrexic between 8 and 9 days post inoculation and exhibited symptoms, such as nasal discharge, mild depression, cough, and inappetence. Leukopenia with associated lymphopenia and neutropenia was evident in all groups with lowest leukocyte and lymphocyte counts reached 8 dpi and granulocyte counts between 11 and 16 dpi, dependent on the strain and age of the calves. A more severe thrombocytopenia was seen in those animals inoculated with strain 1362727. Leukocyte and nasal swab samples were positive by virus isolation, as early as 3 dpi and 2 dpi respectively, independent of the inocula used. All calves seroconverted with high levels of BVDV-2 neutralizing antibodies. BVDV RNA was evident as late as 90 dpi and provides the first evidence of the presence of replicating virus long after recovery from BVDV-2 experimental infection. In summary, moderate disease can be induced after experimental infection of calves with a low titer of virulent BVDV-2, with leukopenia, thrombocytopenia, viremia, and virus shedding. These strains represent an attractive model to assess the protective efficacy of existing and new vaccines against BVDV-2.

Comparative analysis of adaptive immune responses following experimental infections of cattle with bovine viral diarrhoea virus-1 and an Asiatic atypical ruminant pestivirus.

Atypical ruminant pestiviruses are closely related to the two bovine viral diarrhoea virus (BVDV) species, BVDV-1 and BVDV-2. While there is evidence of cross-protective immune responses between BVDV-1 and BVDV-2, despite antigenic differences, there is little information on the antigenic cross-reactivity with atypical ruminant pestiviruses. The aim of this study was therefore to assess the specificity of antibody and T cell responses induced by experimental infection of calves with BVDV-1 strain Ho916, Th/04_KhonKaen (TKK), an Asiatic atypical ruminant pestivirus, or co-infection with both viruses. Homologous virus neutralization was observed in sera from both single virus infected and co-infected groups, while cross-neutralization was only observed in the TKK infected group. T cell IFN-γ responses to both viruses were observed in the TKK infected animals, whereas Ho916 infected calves responded better to homologous virus. Specifically, IFN-γ responses to viral non-structural protein, NS3, were observed in all infected groups while responses to viral glycoprotein, E2, were virus-specific. Broader antigen-specific cytokine responses were observed with similar trends between inoculation groups and virus species. The limited T cell and antibody immune reactivity of Ho916 inoculated animals to TKK suggests that animals vaccinated with current BVDV-1-based vaccines may not be protected against atypical ruminant pestiviruses.

Viral mutations enhance the Max binding properties of the vMyc b-HLH-LZ domain.

Interaction with Max via the helix-loop-helix/leucine zipper (HLH-LZ) domain is essential for Myc to function as a transcription factor. Myc is commonly upregulated in tumours, however, its activity can also be potentiated by virally derived mutations. vMyc, derived from the virus, MC29 gag-Myc, differs from its cellular counterpart by five amino acids. The N-terminal mutation stabilizes the protein, however, the significance of the other mutations is not known. We now show that vMyc can sustain longer deletions in the LZ domain than cMyc before complete loss in transforming activity, implicating the viral mutations in contributing to Myc:Max complex formation. We confirmed this both in vitro and in vivo, with loss of Max binding correlating with a loss in the biological activity of Myc. A specific viral mutation, isoleucine383>leucine (I383>L) in helix 2 of the HLH domain, extends the LZ domain from four to five heptad repeats. Significantly, introduction of I383>L into a Myc mutant that is defective for Max binding substantially restored its ability to complex with Max in vitro and in vivo. We therefore propose that this virally derived mutation is functional by significantly contributing to establishing a more hydrophobic interface between the LZs of Myc and Max.

A retrospective study detects a novel variant of porcine epidemic diarrhea virus in England in archived material from the year 2000.

Outbreaks of porcine epidemic diarrhea (PED) were first recorded in England in the 1970s and continued to be confirmed until 2002. Retrospective analysis of archived material from one of the last confirmed cases in England in the year 2000 demonstrates the previous existence of a very diverse PED virus strain. Following the outbreaks of PED in North America in 2013, there has been renewed interest in phylogenetic analysis of sequences from PEDV strains worldwide. There is a gap in the available sequence data between the mid 1980s and the mid 2000s. This work is an example of how this gap can be at least partially filled by the examination of archived material.

Differential influence of adjacent normal cells on the proliferation of mammalian cells transformed by the viral oncogenes myc, ras and src.

We investigated the role of adjacent normal cells in the modulation of focal outgrowth of mammalian fibroblasts transformed by different viral oncogenes (myc, src and ras). NIH3T3 cells transformed by these three oncogenes were derived by transfection or infection and showed comparable cloning efficiencies in semi-solid medium. However, upon replating in liquid medium a small number of transformed cells together with a vast excess of normal mouse embryo fibroblasts C3H10T1/2, ras- and src-transformed cells were able to overgrow the monolayer and formed distinct foci, whereas myc-transformed cells lacked this ability. Conditioned medium from normal cells did not affect the proliferation of myc-transformed cells at clonal density. Addition of phorbol ester tumour promoters, either at the time of plating or as late as after one week, efficiently rescued focus formation by myc-transformed cells. In contrast, when myc-transformed cells were cultivated alone, their clone size and cloning efficiency were slightly reduced by the addition of tumour promoters. These results indicate that cell-cell contacts between transformed cells and adjacent normal cells specifically inhibit the growth of myc- but not of ras- or src-transformed cells. The ability of tumour promoters and phospholipase-C to rescue the focus forming ability of myc-transformed cells is consistent with the possibility that activation of protein-kinase C is involved in the clonal expansion of 'suppressed' myc-bearing cells.

c-Myc inhibits myogenic differentiation and myoD expression by a mechanism which can be dissociated from cell transformation.

The c-Myc oncoprotein is a basic-helix-loop-helix-leucine zipper (b-HLH-LZ) transcription factor involved in regulating cell proliferation and differentiation. We have used retrovirus-mediated gene transfer to investigate the effect of ectopic c-Myc expression on the spontaneous differentiation of primary quail myoblasts in vitro. Unlike normal myoblasts, c-Myc-expressing myoblasts are unable to form myotubes or express muscle-specific genes, such as myosin, and show severely reduced expression of the myogenic regulatory factors myoD, myogenin, and myf5. The c-Myc leucine zipper (LZ) motif is essential for the differentiation block since myoblasts expressing a mutant with a partial deletion of this region, c-Myc delta 7, differentiate and express myoD family regulators and muscle-specific genes normally. Remarkably, c-Myc delta 7, like wild-type c-Myc, retains the capacity to transform the growth phenotype of myoblasts, and associates with the b-HLH-LZ Myc partner protein Max in transformed cells. We conclude that the block to myogenic differentiation induced by c-Myc can be dissociated from cell transformation per se, and that this attribute correlates more closely with down-regulation of myoD family gene expression. These findings are discussed in the light of current models of Myc function.

Role of Gas1 down-regulation in mitogenic stimulation of quiescent NIH3T3 cells by v-Src.

Quiescent mammalian fibroblasts can be induced to reenter the cell cycle by growth factors and oncoproteins. We studied the pathway(s) through which v-Src, the oncogenic tyrosine kinase encoded by the v-src oncogene of Rous sarcoma virus, forces serum-starved NIH3T3 cells to enter S-phase. To this purpose, we isolated and characterized a polyclonal population of NIH3T3 cells transformed by the MR31 retroviral vector, encoding G418 resistance and the v-src temperature-sensitive allele from the mutant ts LA31 PR-A. NIH(MR31) cells displayed a temperature-conditional transformed phenotype and could be made quiescent by serum deprivation at the restrictive temperature. Serum stimulation or thermolabile v-Src reactivation induced entry into S-phase to a comparable extent, although with different kinetics. The data suggest that v-Src mitogenic activity involves early activation of the Erk1/Erk2 MAP kinases with very little tyrosine phosphorylation of the Shc adaptor proteins at least during the early stages of v-Src reactivation and that v-Src-induced S-phase entry was strongly inhibited by drugs affecting MEK or PI 3-kinase. Our results also suggest that down-regulation of gas1 gene expression plays an important role in regulating the efficiency of entry into S-phase triggered by reactivated v-Src and that Gas1 down-regulation does not require PI 3-kinase dependent signals.

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.

Soybean protein diets in the management of type II hyperlipoproteinaemia.

The efficacy of soybean protein treatment of stable type II hyperlipoproteinaemia was evaluated in 57 patients assigned to the following protocols: (i) substitution of animal protein with soybean protein (19 subjects) and (ii) addition of soybean protein to a standard low-lipid diet (38 subjects). After 16 weeks of treatment, plasma cholesterol was reduced by 29.5% in the first group, and by 29.9% in the second; the difference was not significant. Similarly the reduction in LDL cholesterol was not significantly different between the 2 groups (39% in the first group and 36% in the second). Plasma triglycerides fell by 11.8% and 18.2%, respectively. HDL cholesterol was not modified to any significant extent by soybean protein regimens. These results provide that the addition of soybean protein to a standard low-lipid diet is effective in inducing a significant cholesterol decrease in patients with type II hyperlipoproteinaemia.

Insulin and glucagon degradation in liver are not affected by hepatic cirrhosis.

Hyperinsulinemia and impaired glucose tolerance are associated with liver cirrhosis. To investigate whether insulin-degrading activity in liver tissue plays a role in hyperinsulinemia, we assayed this activity in biopsy tissue from healthy and cirrhotic subjects. There was no difference in insulin degradation between these two groups. Also glucagon-degrading activity in liver tissue, which is catalyzed by the same enzyme as insulin-degrading activity, did not differ between the two groups studied. Therefore, insulin-degrading activity does not appear to be involved in the hyperinsulinemia that occurs in liver cirrhosis. The study provides indirect evidence that hyperinsulinemia and impaired glucose metabolism in liver cirrhosis are due to different mechanisms (receptorial and post-receptorial defects, and altered feedback inhibition of insulin secretion).

The case for pedicle fixation of the lumbar spine.

STUDY DESIGN: This was a retrospective review of one surgeon's results using three different lumbosacral arthrodesis techniques: Group 1, no instrumentation; Group 2, Luque Rod and sublaminar wire technique; and Group 3, AO intrapedicular screw and plate technique. OBJECTIVE: To determine whether the use of metal implants results in a higher fusion rate. Once a solid arthrodesis is achieved, is this correlated with a good clinical result? SUMMARY OF BACKGROUND DATA: Controversy persists regarding the value of the use of intrapedicular fixation to augment arthrodesis of the lumbosacral junction. Controversy also exists regarding the correlation of solid arthrodesis with relief of preoperative symptoms. METHODS: Three serial sequential populations (50 subjects each) undergoing varied primary multiple-level lumbosacral arthrodesis procedures were studied retrospectively. The ultimate clinical results of these three different surgical populations were studied after prolonged follow-up. RESULTS: Group one had a 14% fusion rate and a 4% complication rate. Group two had a 36% fusion rate and an 8% complication rate. Group three had a 64% fusion rate and an 18% complication rate. Complications were intraoperative dural tears and nerve root injuries. Patient satisfaction with each operative procedure to relieve preoperative low back pain was statistically correlated with whether a solid arthrodesis was obtained. CONCLUSION: Intrapedicular fixation technique is the most reliable method for obtaining a solid multiple-level lumbosacral arthrodesis. Solid arthrodesis is correlated with a successful clinical result. Complications associated with the use of intrapedicular fixation were frequent but their occurrence demonstrated a "learning curve pattern."