The power of human cytomegalovirus (HCMV) hijacked UL/b' functions lost in vitro.

Viruses have evolved sophisticated strategies to subvert immunity to benefit overall viral fitness. Human cytomegalovirus (HCMV, ß-herpesvirus) represents a paradigm of very effective hijacking of gene functions that imitate host encoded immunomodulatory proteins. This co-evolution with the host immune system allowed for establishment of lifelong persistence. The HCMV infection is largely asymptomatic in healthy persons; however, it can induce serious disease in immunocompromised individuals. For this reason, great attention is paid to the development of therapeutics based on HCMV immunomodulatory 'tricks' as well as to the search for active vaccine against HCMV. While comparing the HCMV clinical isolates with extensively passaged laboratory strains, the unique long (UL) b' locus was commonly found to be deleted in HCMV genome while adapted to replication in human fibroblasts in vitro. This missing region, called UL/b' region, encodes up to 22 canonical genes with different functions, as of targeting cellular tropism (e.g. UL133-UL138); viral entry and assembly (e.g. UL128, UL130, UL131A); regulation of immunological synapses (e.g. UL135); inhibition of NK and T cell function (e.g. UL141, UL142, UL148, UL144), ablating activity (e.g. UL146, UL147), but mainly aimed at manipulating the host immune response. Moreover, the presence of UL/b' genomic region dramatically correlates with adverse effects in vaccinated persons, indicating that viral genes in this region play a significant role in controlling virulence. Here, we review how HCMV shapes our immunity by hijacked genes originated from UL/b' locus, discuss their impact in immunomodulation mechanism and how this knowledge may translate to clinical applications. Keywords: immunomodulation; HCMV genes; UL/b' locus; NK cell function; HCMV vaccine; immunity; immunotherapeutics.

Chemokine-binding proteins encoded by herpesviruses.

To establish infection, a wide variety of pathogens, including viruses, have evolved a number of strategies to avoid immune elimination. Viruses have acquired and optimized molecules that interact with the host chemokine network in order to disrupt immune surveillance and defense of vertebrates, helping to promote cell entry, facilitating dissemination of infected cells, and evasion the immune response. Viral immunomodulators include ligands, chemokine receptors and chemokine-binding proteins (vCKBPs) functioning as either cell surface receptor mimics, ligand mimics, or secreted chemokine-binding proteins. vCKBPs specifically modulate chemokine gradient formation and ligand-receptor recognition when they have a potential to even completely block chemokine-mediated responses to viral infection. Members of only two virus families (Herpesviridae and Poxviridae) encode vCKBPs capable of sequestering host chemokines through either the chemokine receptor, GAG-binding pocket, or both, which may result in the inhibition of chemotaxis in vivo. Here, we focused on vCKBPs encoded by α-, ß-, and γ-herpesviruses, of which several have been experimentally used as anti-inflammatory or anti-immune reagents in animal models. Current results suggest that vCKBPs could be used to regulate the activity of both chemokines and chemokine receptors for the treatment of infections such as AIDS, diseases such as arthritis, neurotrauma, inflammatory CNS disorders, atherosclerosis, transplant rejection, and metastatic spread and angiogenesis. Better understanding of vCKBPs biology will help evaluate, which human diseases related to chemokine network dysregulation might be effectively treated with these novel promising immunomodulatory drugs to enable the manipulation of chemokine functions and leukocyte trafficking. Keywords: herpesviruses; chemokine-binding proteins; chemokines; immunomodulation viral infection, chemokines and viral immunomodulators.

Clinical Neuroscience in Practice: An Experiential Learning Course for Undergraduates Offered by Neurosurgeons and Neuroscientists.

Many pre-health students pursue extracurricular shadowing opportunities to gain clinical experience. The Virginia Tech School of Neuroscience introduced a formal course that provides a clinical experience superior to that received by many medical students. This course is composed of weekly 75-minute seminars that cover diseases affecting the nervous system, their diagnosis and treatment, complemented by weekly half-day intensive clinical experiences with unprecedented access to a team of neurosurgeons (in hospital operating rooms, Intensive Care Units, emergency room, angiographic suites, and wards). In the operating rooms, students routinely "scrub-in" for complex surgeries. On hospital rounds, students experience direct patient care and receive in-depth exposure to modern nervous system imaging. Students participate in two 24-hour "on-call" experiences with team providers. After call, students participate in cognitive and psychological studies to assess physiological and psychological effects of call-related sleep deprivation. Students prepare weekly essays on challenging socioeconomic and ethical questions, exploring subjects such as the cost of medicine and inequalities in access to health care. Towards the end of the course, students meet with the admission dean of the Virginia Tech Carilion medical school; they prepare a personal statement for medical school/graduate school applications, and attend a half-day block of mock medical school/graduate school interviews delivered by experienced clinicians. In lieu of a final exam, each student presents to the entire neurosurgery department, an in-depth clinical analysis of a case in which they participated. We provide details on implementation, challenges and outcomes based on experiences from three semesters with a total enrollment of approximately 60 students.

Bovine serum albumin-sodium alkyl sulfates bioconjugates as drug delivery systems.

Precipitation of bovine serum albumin (BSA) by anionic surfactants with alkyl chains of increasing lengths (octyl, decyl, dodecyl sulfates) was studied at room temperature, at pH 3.0, in isotonic sodium chloride solution. The particle size of albumin, the zeta potential, the surface charge and fluorescent properties of BSA-surfactant composites were investigated concerning addition of increasing amount of surfactant. The thermal stability of the systems was monitored by calorimetric analysis (DSC). The formation of the well-ordered structure in the self-assembly process in liquid phase was studied by XRD measurement. The structure of the precipitated BSA-surfactant nanocomposites was characterized by small-angle X-ray scattering (SAXS). Finally, ibuprofen (IBU) molecules were enclosed in BSA-surfactant bioconjugate systems and the release properties of the drug were investigated. It has been found out that, as a consequence to the increasing number of carbon atoms in the alkyl chains of the surfactant, the structure and the fluorescent properties of the aggregates formed can be controlled due to the increase in the hydrophobicity of BSA-surfactant composites. The bioconjugates are well applicable as carrier to realize controlled release of drug molecules.

BSA/polyelectrolyte core-shell nanoparticles for controlled release of encapsulated ibuprofen.

Bovine serum albumin (BSA) based core-shell nanoparticles were developed as carrier systems for drug transportation. At pH=3, the oppositely charged polyelectrolytes: poly(sodium-4-styrene)sulphonate (PSS) and the chitosan (Chit) bind to the positively charged protein via electrostatic interactions. We applied ibuprofen (IBU) as model molecule which has low solubility. The changes in the BSA's secondary structure during the steps of the synthesis were inspected by FT-IR measurements. The size and the zeta potential were determined by dynamic light scattering (DLS). The changes in the structure and in the size were investigated by small angle X-ray scattering (SAXS) too, for each composite. The release of the ibuprofen was studied by vertical diffusion cell (Franz cell) at pH 7.4 at 25 and 37.5°C. The structure of the core-shell nanoparticles have significantly changed as the pH has risen from 3.0 to 7.4. Kinetic models were used to describe the release mechanism. The experimental results demonstrated that the BSA has an ordered structure at pH=3 which will become random coil by adding ibuprofen. The first shell restores the ordered structure of the protein. The controlled release was carried out; the IBU release decreased by 40% in the case of two-layered composites compared with the "naked" BSA.

Isolation of bovine adenovirus serotype 6 from a calf in the United Kingdom.

Two viruses, designated 99-8130(C) and 99-8130(I), were isolated in calf testis cells from the colon and ileum, respectively, of a suckled beef calf which had developed dysentery and died. Electron microscopy indicated that the mean (sd) size of the viral particles, 83 (2.5) nm, and their morphology were consistent with their being members of the family Adenoviridae. They were confirmed as adenoviruses by PCR when products of the expected size (608 bp) were amplified from both isolates by using a primer pair specific for members of the genus Atadenovirus. A comparison of the sequence of a 567 bp segment of the 99-8130(C) amplicon with that of other prototype bovine adenovirus (BAdV) strains of atadenoviruses identified the isolate as BAdV serotype 6 (BAdV-6), which had 99.3 per cent and 100 per cent identities at the nucleotide and amino acid levels, respectively, with the prototype BAdV-6 strain 671130. A virus neutralisation test was developed and indicated a high prevalence of antibody to BAdV-6 in Northern Irish cattle. There was no evidence of adenoviral inclusions in tissues from the affected calf and no antigen was detected when the tissues were stained by an immunoperoxidase technique, using a homologous antiserum raised in rabbits. The two viruses were the third reported isolation of BAdV-6, and the first from a clinically ill bovine animal.

Molecular evolution of adenoviruses.

New advances in the field of genetic characterization of adenoviruses originating from different animal species are summarized. Variations seen in the host range and specificity, pathogenicity, genomic arrangement or gene complement are much wider than expected based on previous studies of human adenoviruses. Several exceptional adenoviruses from the two traditional conventional genera are now removed, and proposed to form at least two new genera. The eventual host origin of the new genera, however, is not clarified. Novel results from the genomic and phylogenetic analyses of adenoviruses originating from lower vertebrate species (including reptiles, amphibians and fish) seem to imply that probably five major clusters of adenoviruses exist corresponding to the five major classes of Vertebrata. Adenoviruses, which are now suspected to have common origin with enterobacterium phages from the family Tectiviridae, are perhaps very ancient indeed, and may have undergone a co-evolution with vertebrate hosts.

Characterisation of the fiber gene and partial sequence of the early region 4 of bovine adenovirus 2 (short communication).

The full sequence of the fiber gene and partial sequence of the putative 17 kD protein gene of bovine adenovirus-2 (BAdV-2) were determined. The size of the fiber gene of BAdV-2 proved to be 561 amino acids, of which the amino acids 37 to 385 form a typical shaft domain of 22 repetitive motifs. On the complementary strand, a gene homologous to the 17 kD protein coded in the E4 region of several human adenoviruses was found. The sequence analysis seems to confirm the presence of an intron in the sequenced part of the E4 region.

DNA sequence of a small, unidentified plasmid isolated from a Haemophilus somnus strain: short communication.

One of the plasmids present in a Haemophilus somnus strain isolated from nasal discharge of a cattle with respiratory disease was purified and cloned for DNA sequencing. The plasmid was found to be 1065 base pairs long with 39.2% G + C content, and showed no homology to any DNA sequenced so far. It has no capacity to code any protein longer than 43 residues. It is not clear yet if this plasmid codes Haemophilus somnus specific factors.

Four new inverted terminal repeat sequences from bovine adenoviruses reveal striking differences in the length and content of the ITRs.

The inverted terminal repeat (ITR) of the genome of four bovine adenovirus (BAdV) types have been sequenced, analysed and compared to the ITRs of other adenoviruses. The length of ITRs of the examined BAdVs ranged between 59 and 368 base pairs, thus the presently known longest adenovirus ITR sequence is from BAdV-10. The conserved motifs and characteristic sequence elements of the ITRs providing different binding sites for replicative proteins of viral and cellular origin seemed to be distributed according to the proposed genus classification of BAdVs. The ITRs of BAdV-10 share similarity with the members of the genus Mastadenovirus, while the ITRs of the other three sequenced serotypes (BAdV-4, 5 and strain Rus) which are candidate members of the genus Atadenovirus are very short and contain NFI and Sp1 binding sites only. The analysis of the new ITRs implied that the nucleotide sequence of the so-called core origin is highly preserved within the mastadenovirus genus only.

Identification of fish-parasitic Myxobolus (Myxosporea) species using a combined PCR-RFLP method.

Polymerase chain reaction (PCR) with primers specific for the family Myxobolidae was used to amplify a part of the 18S ribosomal RNA gene of Myxobolus species. The length of the amplified fragments was approximately 1600 base pairs. Six Myxobolus species identified on the basis of morphological features were compared using a combined PCR-RFLP method. The cleavage patterns generated by 2 frequent cutter restriction enzymes (HinfI and MspI) were suitable for the differentiation of the examined Myxobolus species.

The role of egg drop syndrome virus in acute respiratory disease of goslings.

An outbreak of severe acute respiratory disease characterized by tracheitis and bronchitis was observed in young goslings on a large-scale goose farm in Hungary. Histological examination revealed amphophilic intranuclear inclusion bodies in the superficial epithelial cells of the trachea and bronchi. Adenovirus-like particles were detected by electron microscopy, and the virus isolated from the trachea and the lungs was identified as egg drop syndrome (EDS) virus by serological and genomic examination. The clinical and pathological signs were reproduced by intratracheal administration of the virus isolate to 1-day-old goslings free of EDS antibodies. The presence of EDS virus DNA in different organs of the naturally and experimentally infected goslings was detected by polymerase chain reaction. This is the first report on the involvement of EDS virus in severe respiratory disease of geese.

Identification and sequence analysis of the core protein genes of bovine adenovirus 2.

The DNA sequence of the genome of bovine adenovirus type 2 (BAdV-2) was determined between map units 42.5 and 50. By sequence analysis and homology search, the genes of five structural proteins were identified within this region: the penton base protein (III; partial sequence), the major core protein precursor (pVII), the minor core protein (V), the mu core protein precursor (pX) and the hexon associated protein precursor (pVI; partial sequence). The putative polypeptides were compared to their known counterparts from other adenoviruses. The existence of protein V and the presence and structure of certain protease cleavage recognition sites confirmed BAdV-2 as a member of the genus Mastadenovirus.

DNA sequencing and phylogenetic analysis of the protease gene of ovine adenovirus 3 suggest that adenoviruses of sheep belong to two different genera.

Until now, the only published ovine adenovirus DNA sequence was the complete genome of ovine adenovirus isolate 287 (OAV287) which, compared to other mammalian adenoviruses, possesses strikingly unique genomic organisation and should properly be classified into a new adenovirus genus. The protease gene sequence of ovine adenovirus type 3 (OAdV-3) was determined and analysed. The results of phylogenetic analysis of the 205 residue long protein demonstrated that OAdV-3 belongs to the genus Mastadenovirus, and is surprisingly closely related to bovine adenovirus type 2. In spite of the common host origin, the evolutionary distance between OAdV-3 and OAV287 proved to be great suggesting that sheep, similarly to cattle and fowl, might be infected by distantly related adenoviruses belonging to different genera.

Comparison of the genome of ovine adenovirus types 1 through 5 by restriction enzyme analysis and DNA hybridisation.

The DNA of the prototype strains of ovine adenovirus (OAdV) 1 through 5 was analysed by restriction enzyme (RE) digestion. The RE patterns generated by HindIII and PstI enzymes were characteristic of the examined strains. OAdV-2 and 3 resembled each other the most, and their EcoRI and HindIII patterns seemed to be identical. Considering the number of comigrating fragments, serotypes OAdV-2, 3, 4 and 5 looked more closely related to each other than to OAdV-1. This finding was strengthened by Southern blot hybridisations probed with random HindIII clones of OAdV-3. The estimated genome size of the examined OAdV types ranged between 31.9 and 32.8 kilobase pairs. The results supported the new genus classification of OAdVs.

Detection of adenovirus hexon sequence in a cat by polymerase chain reaction (short communication).

Adenoviral nucleic acid was detected by polymerase chain reaction (PCR) in pharyngeal and rectal swab samples of a cat seropositive for adenovirus and suffering from transient hepatic failure. The samples were taken at a one-year interval, and both faecal samples as well as the second pharyngeal sample were positive in PCR performed with general adenovirus primers. The size of the amplified products corresponded to that of the positive control. The identity of the amplicons was also confirmed by DNA sequencing. The 301 bp long hexon gene fragment was very similar to but distinguishable from the corresponding hexon sequence of human adenovirus type 2. This result suggests the possibility of persistent carrier status and shedding of adenovirus in cats.