Cloning of Bovine herpesvirus type 1 and type 5 as infectious bacterial artifical chromosomes

<p>Abstract</p> <p>Background</p> <p>Bovine herpesviruses type 1 (BoHV1) and type 5 (BoHV5) are two closely related pathogens of cattle. The identity of the two viruses on the amino acid level averages 82%. Despite their high antigenetic similarities the two pathogens induce distinctive clinical signs. BoHV1 causes respiratory and genital tract infections while BoHV5 leads to severe encephalitis in calves.</p> <p>Findings</p> <p>The viral genomes of BoHV1 and BoHV5 were cloned as infectious bacterial artificial chromosomes (BACs). First, recombinant viruses carrying the genetic elements for propagation in bacteria were generated. Second, DNA from these recombinant viruses were transferred into prokaryotic cells. Third, DNA from these bacteria were transferred into eukaryotic cells. Progeny viruses from BAC transfections showed similar kinetics as their corresponding wild types.</p> <p>Conclusion</p> <p>The two viral genomes of BoHV1 and BoHV5 cloned as BACs are accessible to the tools of bacterial genetics. The ability to easily manipulate the viral genomes on a molecular level in future experiments will lead to a better understanding of the difference in pathogenesis induced by these two closely related bovine herpesviruses.</p

Conserved rotavirus NSP5 and VP2 domains interact and affect viroplasm

One step of the life cycle common among all rotaviruses (RV) studied so far is the formation of viroplasms, membrane-less cytosolic inclusions providing a microenvironment for early morphogenesis and RNA replication. Viroplasm-like structures (VLS) are simplified viroplasm models consisting of complexes of non-structural protein 5 (NSP5) with either the RV core-shell VP2 or NSP2. We identified and characterized the domains required for NSP5-VP2 interaction and VLS formation. VP2 mutations L124A, V865A, or I878A impaired both NSP5 hyperphosphorylation and NSP5/VP2 VLS formation. Moreover, NSP5-VP2 interaction does not depend on NSP5 hyperphosphorylation. The NSP5 tail region is required for VP2 interaction. Notably, VP2 L124A expression acts as dominant-negative by disrupting the formation of either VLSs or viroplasms and blocking RNA synthesis. In silico analyses revealed that VP2 L124, V865, and I878 are conserved among RV A to H species. The detailed knowledge of the protein interaction interface required for viroplasm formation may facilitate the design of broad-spectrum antivirals to block RV replication. Importance Alternative treatments to combat rotavirus infection are a requirement for susceptible communities where vaccines cannot be applied. This demand is urgent for newborn infants, immunocompromised patients but also for adults traveling to high-risk regions and even for livestock industry. Aside from structural and physiological divergences among RV species studied until now, all replicate within cytosolic inclusions termed viroplasms. These inclusions are composed of viral and cellular proteins and viral RNA. Viroplasm-like structures (VLS), composed of RV proteins NSP5 with either NSP2 or VP2, are models for investigating viroplasms. In this study, we identified a conserved amino acid in the VP2 protein, L124, necessary for its interaction with NSP5 and the formation of both VLSs and viroplasms. As RV vaccines cover a narrow range of viral strains, the identification of VP2 L124 residue lays the foundations for the design of drugs that specifically block NSP5-VP2 interaction as a broad-spectrum RV antiviral

Novel snake papillomavirus does not cluster with other non-mammalian papillomaviruses

Papillomaviruses (PVs) are associated with the development of neoplasias and have been found in several different species, most of them in humans and other mammals. We identified, cloned and sequenced PV DNA from pigmented papilloma-like lesions of a diamond python (Morelia spilota spilota). This represents the first complete PV genome discovered in a Squamata host (MsPV1). It consists of 7048 nt and contains the characteristic open reading (ORF) frames E6, E7, E1, E2, L1 and L2. The L1 ORF sequence showed the highest percentage of sequence identities to human PV5 (57.9%) and Caribbean manatee (Trichechus manatus) PV1 (55.4%), thus, establishing a new clade. According to phylogenetic analysis, the MsPV1 genome clusters with PVs of mammalian rather than sauropsid hosts

Comparison of a continuous glucose monitoring system with a portable blood glucose meter to determine insulin dose in cats with diabetes mellitus.

Background:The continuous glucose monitoring system (CGMS) Guardian REAL‐Time® allows the generation of very detailed glucose profiles in cats. The performance of CGMS to generate short‐term glucose profiles to evaluate treatment response has not been yet evaluated in diabetic cats.Hypothesis:Analysis of glucose profiles generated using the CGMS produces insulin dose recommendations that differ from those of profiles generated using the portable blood glucose meter (PBGM) in diabetic cats.Animals:Thirteen client‐owned diabetic cats.Methods:Prospective, observational study. Simultaneous glucose profiles were generated over an 8‐10 hour period using the CGMS, blood glucose concentration was measured every 2 hours with the PBGM. Profiles were submitted to three internal medicine specialists who used them to determine the insulin dose. Differences between insulin doses deduced from paired profiles were compared. Percentages of nadirs recorded with the CGMS that were lower, higher, or equal to those derived with the PBGM were calculated.Results:Twenty‐one paired glucose profiles were obtained. There was no difference of insulin doses based on CGMS and PBGM profiles (median 0 U; range: −1 to +0.5). Treatment decisions did not differ among investigators. Compared with the observed PBGM nadir, the CGMS nadir was lower, higher, or equal in 17, 2, and 2 of 21 cases, respectively.Conclusions and Clinical Importance:Adjustments in insulin dose based on glucose profiles generated with the CGMS are similar to those based on the PBGM. The common occurrence of lower nadirs recorded with the CGMS suggests that this device detects hypoglycemic periods that are not identified with the PBGM

Identification of a small molecule that compromises the structural integrity of viroplasms and rotavirus double-layered particles

Despite the availability of two attenuated vaccines, rotavirus (RV) gastroenteritis remains an important cause of mortality among children in developing countries, causing about 215,000 infant deaths annually. Currently, there are no specific antiviral therapies available. RV is a nonenveloped virus with a segmented double-stranded RNA genome. Viral genome replication and assembly of transcriptionally active double-layered particles (DLPs) take place in cytoplasmic viral structures called viroplasms. In this study, we describe strong impairment of the early stages of RV replication induced by a small molecule known as an RNA polymerase III inhibitor, ML-60218 (ML). This compound was found to disrupt already assembled viroplasms and to hamper the formation of new ones without the need for de novo transcription of cellular RNAs. This phenotype was correlated with a reduction in accumulated viral proteins and newly made viral genome segments, disappearance of the hyperphosphorylated isoforms of the viroplasm-resident protein NSP5, and inhibition of infectious progeny virus production. In in vitro transcription assays with purified DLPs, ML showed dose-dependent inhibitory activity, indicating the viral nature of its target. ML was found to interfere with the formation of higher-order structures of VP6, the protein forming the DLP outer layer, without compromising its ability to trimerize. Electron microscopy of ML-treated DLPs showed dose-dependent structural damage. Our data suggest that interactions between VP6 trimers are essential, not only for DLP stability, but also for the structural integrity of viroplasms in infected cells

Multiparameter Telemetry as a Sensitive Screening Method to Detect Vaccine Reactogenicity in Mice

Refined vaccines and adjuvants are urgently needed to advance immunization against global infectious challenges such as HIV, hepatitis C, tuberculosis and malaria. Large-scale screening efforts are ongoing to identify adjuvants with improved efficacy profiles. Reactogenicity often represents a major hurdle to the clinical use of new substances. Yet, irrespective of its importance, this parameter has remained difficult to screen for, owing to a lack of sensitive small animal models with a capacity for high throughput testing. Here we report that continuous telemetric measurements of heart rate, heart rate variability, body core temperature and locomotor activity in laboratory mice readily unmasked systemic side-effects of vaccination, which went undetected by conventional observational assessment and clinical scoring. Even minor aberrations in homeostasis were readily detected, ranging from sympathetic activation over transient pyrogenic effects to reduced physical activity and apathy. Results in real-time combined with the potential of scalability and partial automation in the industrial context suggest multiparameter telemetry in laboratory mice as a first-line screen for vaccine reactogenicity. This may accelerate vaccine discovery in general and may further the success of vaccines in combating infectious disease and cancer