Glycoprotein D of bovine herpesvirus 5 (BoHV-5) confers an extended host range to BoHV-1 but does not contribute to invasion of the brain

Bovine herpesvirus 1 (BoHV-1) and BoHV-5 are closely related pathogens of cattle, but only BoHV-5 is considered a neuropathogen. We engineered intertypic gD exchange mutants with BoHV-1 and BoHV-5 backbones in order to address their in vitro and in vivo host ranges, with particular interest in invasion of the brain. The new viruses replicated in cell culture with similar dynamics and to titers comparable to those of their wild-type parents. However, gD of BoHV-5 (gD5) was able to interact with a surprisingly broad range of nectins. In vivo, gD5 provided a virulent phenotype to BoHV-1 in AR129 mice, featuring a high incidence of neurological symptoms and early onset of disease. However, only virus with the BoHV-5 backbone, independent of the gD type, was detected in the brain by immunohistology. Thus, gD of BoHV-5 confers an extended cellular host range to BoHV-1 and may be considered a virulence factor but does not contribute to the invasion of the brain.

Canine distemper virus persistence in demyelinating encephalitis by swift intracellular cell-to-cell spread in astrocytes is controlled by the viral attachment protein

The mechanism of viral persistence, the driving force behind the chronic progression of inflammatory demyelination in canine distemper virus (CDV) infection, is associated with non-cytolytic viral cell-to-cell spread. Here, we studied the molecular mechanisms of viral spread of a recombinant fluorescent protein-expressing virulent CDV in primary canine astrocyte cultures. Time-lapse video microscopy documented that CDV spread was very efficient using cell processes contacting remote target cells. Strikingly, CDV transmission to remote cells could occur in less than 6 h, suggesting that a complete viral cycle with production of extracellular free particles was not essential in enabling CDV to spread in glial cells. Titration experiments and electron microscopy confirmed a very low CDV particle production despite higher titers of membrane-associated viruses. Interestingly, confocal laser microscopy and lentivirus transduction indicated expression and functionality of the viral fusion machinery, consisting of the viral fusion (F) and attachment (H) glycoproteins, at the cell surface. Importantly, using a single-cycle infectious recombinant H-knockout, H-complemented virus, we demonstrated that H, and thus potentially the viral fusion complex, was necessary to enable CDV spread. Furthermore, since we could not detect CD150/SLAM expression in brain cells, the presence of a yet non-identified glial receptor for CDV was suggested. Altogether, our findings indicate that persistence in CDV infection results from intracellular cell-to-cell transmission requiring the CDV-H protein. Viral transfer, happening selectively at the tip of astrocytic processes, may help the virus to cover long distances in the astroglial network, "outrunning" the host's immune response in demyelinating plaques, thus continuously eliciting new lesions.

Intralesional bovine papillomavirus DNA loads reflect severity of equine sarcoid disease

REASONS FOR PERFORMING STUDY: Sarcoids are nonmetastasising, yet locally aggressive skin tumours that constitute the most frequent neoplasm in equids. Infection by bovine papillomaviruses types 1 and 2 (BPV-1, BPV-2) has been recognised as major causative factor in sarcoid pathogenesis, but a possible correlation of intralesional virus load with disease severity has not been established thus far. HYPOTHESIS: Given the pathogenic role of BPV-1 and BPV-2 in sarcoid disease, we suggest that intralesional viral DNA concentration may reflect the degree of affection. METHODS: Severity of disease was addressed by recording the tumour growth kinetics, lesion number and tumour type for 37 sarcoid-bearing horses and one donkey. Viral load was estimated via quantitative real-time PCR (qPCR) of the E2, E5, L1 and L2 genes from the BPV-1/-2 genome for one randomly selected lesion per horse and correlated with disease severity. RESULTS: Quantitative PCR against E2 identified viral DNA concentrations ranging from 0-556 copies/tumour cell. Of 16 horses affected by quiescent, slowly growing single tumours or multiple mild-type lesions, 15 showed a viral load up to 1.4 copies per cell. In stark contrast, all equids (22/22) bearing rapidly growing and/or multiple aggressive sarcoids had a viral load between 3 and 569 copies per cell. Consistent results were obtained with qPCR against E5, L1 and L2. CONCLUSIONS: While tumours of the same clinical type carried variable virus load, confirming that viral titre does not determine clinical appearance, we identified a highly significant correlation between intralesional viral load and disease severity. POTENTIAL RELEVANCE: The rapid determination of BPV viral load will give a reliable marker for disease severity and may also be considered when establishing a therapeutic strategy.

Clinical Characteristics of Children with Viral Single- and Co-Infections and a Petechial Rash

BACKGROUND:: Children with petechial rash are more likely to undergo invasive diagnostics, to be treated with antibiotics for potential bacterial infection and to be hospitalized. However, viruses have also been associated with petechial rash. Nonetheless, a systematic analysis of viral infections with modern available techniques as quantitative real time polymerase chain reaction (q-PCR) in the context of petechial rash is lacking. The purpose of this pediatric study was to prospectively uncover viral pathogens that may promote the emergence of petechiae and to analyze the correlation with the clinical characteristics and course. METHODS:: We conducted a prospective study in children (0 to 18 years) presenting with petechiae and signs or symptoms of infection at the emergency department between November 2009 and March 2012. In nasopharyngeal aspirates the following viruses were analyzed by q-PCR: Cytomegalovirus, Epstein-Barr virus, parvovirus B19, Influenza A and B, parainfluenza viruses, human respiratory syncytial virus A and B, human metapneumovirus, rhinovirus, enterovirus, adenovirus, human coronavirus OC43, 229E, NL63 and human bocavirus. RESULTS:: A viral pathogen was identified in 67% of the analyzed 58 cases with petechial rash. Virus positive patients showed a significantly higher incidence of lower respiratory tract infections. Forty-one percent were viral co-infections, which were significantly younger than virus negative patients, had a higher leukocyte count and were hospitalized for a longer time. CONCLUSIONS:: A petechial rash is frequently associated viral single- and co-infections and can rapidly be identified via q-PCR.

Targets of emerging therapies for viral hepatitis B and C

Viral hepatitis B and C, structurally two completely different viruses, commonly infect human hepatocytes and cause similar clinical manifestations. Since their discovery, IFN has been a pillar in the treatment. However, because of the different natures of the viruses, therapeutic approaches diverge and new treatment targets are tailored specifically for each virus. Herein, the authors analyse therapeutic approaches for hepatitis B virus (HBV) and hepatitis C virus (HCV) and focus on emerging concepts that are under clinical evaluation. In particular, promising viral inhibitors for HBV and HCV are reviewed and the current status of research for gene therapy for HCV is described. Immune therapy is a fast-moving field with fascinating results which include therapeutic vaccines and toll-like receptor agonists that could improve tomorrow's treatment approaches.

Viral etiology of acute respiratory infections with cough in infancy: a community-based birth cohort study

BACKGROUND: Acute respiratory infections (ARI) are a major cause of morbidity in infancy worldwide, with cough and wheeze being alarming symptoms to parents. We aimed to analyze in detail the viral aetiology of ARI with such symptoms in otherwise healthy infants, including rhinoviruses and recently discovered viruses such as human metapneumovirus (HMPV), coronavirus NL63 and HKU1, and human bocavirus (HBoV). METHODS: We prospectively followed 197 unselected infants during their first year of life and assessed clinical symptoms by weekly standardized interviews. At the first ARI with cough or wheeze, we analyzed nasal swabs by sensitive individual real time polymerase chain reaction assays targeting 16 different respiratory viruses. RESULTS: All 112 infants who had an ARI had cough, and 39 (35%) had wheeze. One or more respiratory viruses were found in 88 of 112 (79%) cases. Fifteen (17%) dual and 3 (3%) triple infections were recorded. Rhino- (23% of all viruses) and coronaviruses (18%) were most common, followed by parainfluenza viruses (17%), respiratory syncytial virus (RSV) (16%), HMPV (13%), and HBoV (5%). Together rhinoviruses, coronaviruses, HMPV, and HBoV accounted for 60% (65 of 109) of viruses. Although symptom scores and need for general practitioner (GP) consultations were highest in infants infected with RSV, they were similar in infants infected with other viruses. Viral shedding at 3 weeks occurred in 20% of cases. CONCLUSIONS: Rhinoviruses, coronaviruses, HMPV, and HBoV are common pathogens associated with respiratory symptoms in otherwise healthy infants. They should be considered in the differential diagnosis of the aetiology of ARI in this age group.

Models with Plants, Microorganisms and Viruses for Basic Research in Homeopathy

Most criticism about homeopathy concerns the lack of a scientific basis and theoretical models. In order to be accepted as a valid part of medical practice, a wellstructured research strategy for homeopathy is needed. This is often hampered by methodological problems as well as by gross underinvestment in the required academic resources. Fundamental research could make important contributions to our understanding of the homeopathic and high dilutions mechanisms of action. Since the pioneering works of Kolisko on wheat germination (Kolisko, 1923) and Junker on growth of microorganisms (paramecium, yeast, fungi) (Junker, 1928), a number of experiments have been performed either with healthy organisms (various physiological aspects of growth) or with artificially diseased organisms, which may react more markedly to homeopathic treatments than healthy ones. In the latter case, the preliminary stress may be either abiotic, e.g. heavy metals, or biotic, e.g. fungal and viral pathogens or nematode infection. Research has also been carried out into the applicability of homeopathic principles to crop growth and disease control (agrohomeopathy): because of the extreme dilutions used, the environmental impact is low and such treatments are well suited to the holistic approach of sustainable agriculture (Betti et al., 2006). Unfortunately, as Scofield reported in an extensive critical review (Scofield, 1984), there is little firm evidence to support the reliability of the reported results, due to poor experimental methodology and inadequate statistical analysis. Moreover, since there is no agricultural homeopathic pharmacopoeia, much work is required to find suitable remedies, potencies and dose levels.

Has "Omnics" Gone Viral?

Discussion on viruses from mild to wild and how using "omnics" (such as genomes, mRNA, proteins, metabolites) can help uncover unexpected biology.

[Cryptosporidiose (C. parvum) in a foal with diarrhea]

The protozoon parasite Cryptosporidium parvum is an important cause of diarrhea in farm animals, but it can also infect other animals and humans. In this case report, oocysts of Cryptosporidium spp. were microscopically detected by modified Ziehl-Neelsen staining in the feces of a 9 day old Arabian colt presented with yellowish, foul smelling, diarrhea and fever of 40 degrees C. PCR and sequencing of the isolate revealed C. parvum (bovine genotype). Hemato-chemical analysis of the foals blood revealed a marked hypogammaglobulinaemia (IgG 108mg/dl). The colt responded well to a supportive therapy and administration of plasma (until a gammaglobulin-concentration of 620 mg/dl was reached) and was released in good health from the clinic after 10 days. Follow-up testing for Cryptosporidium oocycsts remained negative. Cryptosporidiosis with life-threatening diarrhea is a rare diagnosis in foals in Switzerland. Immunodeficiency increases the risk for cryptosporidiosis. We hypothesize that the low concentration of gammaglobulins together with the weak INF-gamma response normally observed in young foals may have favored the clinical manifestation with diarrhea. Foals with diarrhea should be screened for cryptosporidia with specific tests.

Laribacter hongkongensis: a potential cause of infectious diarrhea

In this study, we describe the isolation of Laribacter hongkongensis, a recently described genus and species of bacterium, in pure culture on charcoal cefoperazone deoxycholate agar from the stool of six patients with diarrhea. Three patients were residents of Hong Kong, and three of Switzerland. In none of the stool samples obtained from these six patients was Salmonella, Shigella, enterohemorrhagic Escherichia coli, Vibrio, Aeromonas, Plesiomonas, or Campylobacter recovered. Rotavirus antigen detection, electron microscopic examination for viruses, and microscopic examinations for ova and cysts were all negative for the stool samples obtained from the three patients in Hong Kong. Enterotoxigenic E. coli was recovered from one of the patients in Hong Kong. Unlike L. hongkongensis type strain HKU1, all the six strains were motile with bipolar flagellae. Sequencing of the 16S ribosomal RNA genes of the six strains showed that they all had sequences with only 0-2 base differences to that of the type strain. Pulsed field gel electrophoresis of the SpeI digested genomic DNA of the six isolates and that of the type strain revealed that the seven isolates were genotypically unrelated strains. More extensive epidemiologic studies should be carried out to ascertain the causative association between L. hongkongensis and diarrhea and to define the reservoir and modes of transmission of L. hongkongensis.

Antibody Response to Inactivated Viral Vaccines Administered to Calves at Weaning

Respiratory disease in beef calves has been associated with the stress of weaning. Management practices commonly delay vaccination of calves to this time, and weaning stress could potentially suppress the immune response. To reduce this stress we have been experimenting with a procedure termed “pasture weaning” in which the dams are removed and the calves remain on pasture. Observation suggests that calves weaned with this approach adapt to the weaned state much better than those held in drylot. Consequently, one would expect less stress-mediated effects including those on the immune system. Calves were weaned and assigned to groups that were pasture or drylot weaned, and calves within the groups were vaccinated with one of two inactivated virus vaccines by either the intramuscular or subcutaneous route. Weaning placement did not affect antibody responses to the viruses included in the vaccines. The route of administration did not influence responses with subcutaneous injection inducing responses equivalent to the intramuscular site. Utilization of this route for vaccination could be advantageous because it precludes the tissue damage and hidden abscessation that sometimes results from intramuscular injections. A distinct difference was noted in the immunogenicity of the vaccines with the Vira Shield product yielding significantly better responses to all viral entities.

Bovine Respiratory Syncytial Virus Infection in McNay Farm Calves

Clinical respiratory disease occurs almost every year in fall calves in the McNay Farm herd. Diagnostic procedures have implicated Haemophilus somnus (H. somnus) and bovine respiratory syncyial virus (BRSV) as the infectious agents primarily associated with this disease. Therefore, the 1995 calves were closely monitored after weaning and during the course of a respiratory disease. Serologic evidence indicated the involvement of the same two agents in the pathogenesis of the disease. Also, experimental evidence suggested a role for a preexisting immediate hypersensitivity to H. somnus and the development of this type of response to BRSV. We theorize that the pathogenesis of the clinical disease involved infection with H. somnus, establishment of immediate hypersensitivity in the lungs, viral infection with associated pathologic lesions, and viral exacerbation of the immediate hypersensitivity reaction with resultant clinical signs and tissue damage.

Genetic Characterization of a New U.S. Bovine Rotavirus Isolate

We have identified a new group A rotavirus associated with diarrheic calves in the field. The VP7 gene of this virus (designated VMRI-29), appears to differ genetically from that of the reference strain NCDV-Lincoln. Studies are underway to determine the importance of this genetic variant in the etiology of rotavirus-induced calf diarrhea.

Infectious Bovine Rhinotracheitis Virus Antibody Responses to Vaccines Used in Combination

The objective of this experimentation was to determine if circulating antibody titers to parainfluenza type-3 (PI-3) and infectious bovine rhinotracheitis (IBR) viruses could be enhanced by a combination of vaccines. The vaccines utilized were a modified live virus vaccine administered by the intranasal route and an inactivated virus vaccine injected intramuscularly. Virus neutralization tests were conducted on sera obtained at intervals before and following vaccination. Unfortunately, the calves were apparently exposed naturally to PI-3 virus, and the responses to that virus were inconclusive. However, antibody responses to IBR virus were dramatically enhanced by the combination of the two vaccines.

Antibody Responses of Young Calves to Inactivated Viral Vaccines

Three commercial inactivated virus vaccines were evaluated for immunogenicity in young calves with residual maternal antibodies. Groups of 30 calves were administered each of the vaccines at the start of the experimentation and were administered a second dose 32 days later. Serum was obtained from these calves and 30 calves in a nonvaccinated control group prior to vaccination and at 32, 61, 99 and 125 days thereafter. Antibody responses to viruses in two of the vaccines were extremely limited. The third vaccine overcame suppression by maternal antibodies and elicited responses clearly differentiated from antibody levels in the control group of calves.