Evaluation of Fluorescence Microsphere Immunoassay for Detection of Antibodies to Rift Valley Fever Virus Nucleocapsid Protein and Glycoproteins.

Rift Valley fever virus (RVFV) is a mosquito-borne, zoonotic virus that infects ruminants, including cattle, sheep, goats, camels, and buffalo. Multiplexing diagnostic assays that can simultaneously detect antibodies against multiple RVFV antigens offer a high-throughput test for disease surveillance and vaccine evaluations. We describe the improvement and evaluation of a previously developed fluorescence microsphere immunoassay (FMIA) for the detection of IgG and IgM antibodies against the RVFV glycoprotein (Gn) and the immunogenic nucleocapsid protein (Np). Well-characterized vaccinated and experimentally infected ruminant sera were used for the evaluation of the assay. Recombinant viral proteins were produced and then coupled to polystyrene magnetic beads for analysis using the Luminex MAGPIX system with xMAP technology. The FMIA was performed in parallel with virus neutralization tests. Our results revealed the highest median fluorescence intensity (MFI) values for the detection of IgG antibodies against RVFV Np, indicating that this antigen would be a good candidate for a screening assay. The Np and Gn targets could differentiate infected animals from animals vaccinated with a candidate subunit vaccine formulation based on the RVFV Gn and Gc proteins. The results presented in this report demonstrate that FMIA provides a rapid and robust serological diagnostic tool for the detection of antibodies against RVFV. The targets developed in this assay provide the basis for the development of a companion diagnostic test for an RVFV Gn/Gc subunit vaccine that is capable of differentiating infected from vaccinated animals (DIVA), as well as a multiplex serodiagnostic assay that can simultaneously screen for several ruminant diseases.

Dnmt3a regulates T-cell development and suppresses T-ALL transformation.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic neoplasm resulting from the malignant transformation of T-cell progenitors, and comprises ~15% and 25% of pediatric and adult ALL cases, respectively. It is well-established that activating NOTCH1 mutations are the major genetic lesions driving T-ALL in most patients, but efforts to develop targeted therapies against this pathway have produced limited success in decreasing leukemic burden and come with significant clinical side effects. A finer detailed understanding of the genetic and molecular mechanisms underlying T-ALL is required identify patients at increased risk for treatment failure and the development of precision medicine strategies. Generation of genetic models that more accurately reflect the normal developmental history of T-ALL are necessary to identify new avenues for treatment. The DNA methyltransferase enzyme DNMT3A is also recurrently mutated in T-ALL patients, and we show here that inactivation of Dnmt3a combined with Notch1 gain-of-function leads to an aggressive T-ALL in mouse models. Moreover, conditional inactivation of Dnmt3a in mouse hematopoietic cells leads to an accumulation of immature progenitors in the thymus, which are less apoptotic. These data demonstrate that Dnmt3a is required for normal T-cell development, and acts as a T-ALL tumor suppressor.

Molecular evolution of epizootic hemorrhagic disease viruses in North America based on historical isolates using motif fingerprints.

Epizootic hemorrhagic disease virus (EHDV) is an orbivirus of the Reoviridae family that has significant impact on wild and captive white-tailed deer. Although closely related to bluetongue virus that can cause disease in sheep and cattle, North American EHDV historically has not been associated with disease in cattle or sheep. Severe disease in cattle has been reported with other EHDV strains from East Asia and the Middle East. To understand the potential role of viral genetics in the epidemiology of epizootic hemorrhagic disease, a molecular characterization of North American EHDV strains from 1955 to 2012 was conducted via conventional phylogenetic analysis and a new classification approach using motif fingerprint patterns. Overall, this study indicates that the genetic make-up of EHDV populations in North America have slowly evolved over time. The data also suggested limited reassortment events between serotypes 1 and 2 and introduces a new analysis tool for more detailed sequence pattern analysis.

Lesser-known bunyavirus infections.

This paper reviews less well-known or less widely distributed viruses of the Bunyaviridae family that are nonetheless of significant veterinary and public health concern. These include: Cache Valley fever, Main Drain, Ingwavuma, Bhanja and Heartland viruses. A description of the agents, clinical signs of infection, epidemiology, and insect transmission is provided for each, and the authors discuss current diagnostic strategies plus the lack of control measures.

West Nile virus.

This review covers the basic biology of the West Nile virus and the host-vector-pathogen interactions that result in significant disease in wild birds, horses and humans. The review describes the basic properties of the virus, cellular infection and the pathogenesis of the disease, and the ecology of virus maintenance, amplification and transmission. Disease epidemiology and risk estimation strategies that are currently in use are also examined, and host immune responses and vaccination practices described. The principles of vector control, exposure control and long-term risks caused by climatic and habitat factors are also included.

Diagnostic approaches for Rift Valley fever.

Disease outbreaks caused by arthropod-borne animal viruses (arboviruses) resulting in significant livestock and economic losses world-wide appear to be increasing. Rift Valley fever (RVF) virus is an important arbovirus that causes lethal disease in cattle, camels, sheep and goats in Sub-Saharan Africa. There is concern that this virus could spread because of global warming, increased animal trade or through bioterrorism. This paper discusses the current and developing approaches to diagnosis of RVF. Diagnostic assays are available for RVF, but availability can be limited and there is a need for global harmonization. Continued improvement of standard serological and viral genome amplification approaches, including new embedded/syndromic testing, biosensor, emerging virus detection and characterization technologies is needed.

Detection of a novel reassortant epizootic hemorrhagic disease virus (EHDV) in the USA containing RNA segments derived from both exotic (EHDV-6) and endemic (EHDV-2) serotypes.

Epizootic hemorrhagic disease virus (EHDV) is a Culicoides-transmitted orbivirus that infects domestic and wild ruminants and is provisionally thought to be distributed throughout Africa, North America, Australia, East Asia and the Middle East. Historically, of the seven proposed serotypes of EHDV, only EHDV-1 and EHDV-2 have been reported from North America. In 2006, EHDV isolates were recovered from moribund or dead white-tailed deer (Odocoileus virginianus) in Indiana and Illinois that could not be identified as either EHDV-1 or EHDV-2 by virus neutralization tests or by serotype-specific RT-PCR. Additional serological and genetic testing identified the isolates as EHDV-6, a serotype that, although originally described from Australia, has recently been recognized as an emerging pathogen of cattle in Morocco, Algeria and Turkey. In 2007 and 2008, EHDV-6 was isolated again from white-tailed deer, this time in Missouri, Kansas and Texas, suggesting that the virus is capable of overwintering and that it may become, or already is, endemic in a geographically widespread region of the USA. Genetic characterization of the virus indicates that it is a reassortant, such that the outer capsid proteins determining serotype specificity (VP2 and VP5) are derived from exotic EHDV-6, whilst the remaining structural and non-structural proteins are apparently obtained from indigenous EHDV-2 (Alberta).

Dilated common bile ducts mimicking choledochal cysts in ketamine abusers.

Substance abuse is a major health and social problem among Hong Kong youth and ketamine is the drug most commonly abused. Ketamine abuse is associated with a series of side-effects that include hallucination, nausea, vomiting, elevation of blood pressure, and urinary bladder dysfunction. Here we report three cases of ketamine abuse in which the abusers presented with recurrent epigastric pain and dilated common bile ducts that mimicked choledochal cysts on imaging. The dilated biliary tree may occur more frequently than was once assumed.

Ruptured gastroduodenal artery pseudoaneurysm as the initial presentation of chronic pancreatitis.

Gastroduodenal artery pseudoaneurysm is a rare but life threatening complication of pancreatitis. Diagnosis and management of it remain challenging. Surgical treatment was associated with a high mortality. Percutaneous transarterial embolization of bleeding artery has recently been advocated as a definitive therapy and can be attempted as the initial measure to control bleeding. We herein report a case of chronic pancreatitis presented with ruptured pseudoaneurysm of gastroduodenal artery which was successfully controlled with transarterial embolisation.

The NS3 proteins of global strains of bluetongue virus evolve into regional topotypes through negative (purifying) selection.

Comparison of the deduced amino acid sequences of the genes (S10) encoding the NS3 protein of 137 strains of bluetongue virus (BTV) from Africa, the Americas, Asia, Australia and the Mediterranean Basin showed limited variation. Common to all NS3 sequences were potential glycosylation sites at amino acid residues 63 and 150 and a cysteine at residue 137, whereas a cysteine at residue 181 was not conserved. The PPXY and PS/TAP late-domain motifs were conserved in all but three of the viruses. Phylogenetic analyses of these same sequences yielded two principal clades that grouped the viruses irrespective of their serotype or year of isolation (1900-2003). All viruses from Asia and Australia were grouped in one clade, whereas those from the other regions were present in both clades. Each clade segregated into distinct subclades that included viruses from single or multiple regions, and the S10 genes of some field viruses were identical to those of live-attenuated BTV vaccines. There was no evidence of positive selection on the S10 gene as assessed by reconstruction of ancestral codon states on the phylogeny, rather the functional constraints of the NS3 protein are expressed through substantial negative (purifying) selection.

Laparoscopic versus open hepatectomy for liver tumours: a case control study.

OBJECTIVE: To evaluate the benefits of laparoscopic versus open resection of liver tumours. DESIGN: Case control study. SETTING: Tertiary teaching hospital, Hong Kong. PATIENTS: Data from 25 patients who underwent laparoscopic resections for liver tumours from 2003 to 2006 were compared to a retrospective series of 25 patients who underwent open hepatectomy in a pair-matched design. MAIN OUTCOME MEASURES: Duration of operation, operative morbidity and mortality, blood loss, tumour resection margin, analgesics usage, days to return to an oral diet, duration of postoperative hospital stay, and survival of patients with malignancy. RESULTS: The demographic data and the tumour characteristics were comparable in the two patient groups, as were mortality (0% in both groups) and morbidity rates (4% in both groups). Two (8%) of the patients having laparoscopic resections were converted to open surgery. There was no statistically significant difference between the two groups in terms of operating time or resection margins. However, the laparoscopically treated patients experienced significantly less blood loss (median, 100 vs 250 mL), had shorter hospital stays (median, 4 vs 7 days), were prescribed less analgesia (median morphine dosage, 0.16 vs 0.83 mg per kg body weight), and resumed oral diet earlier (median, 1 vs 2 days). For patients with malignant tumours, there was no significant difference between the two groups in terms of actuarial and disease-free survival. CONCLUSION: Compared to open hepatectomy, in selected patients laparoscopic liver resection delivers the benefits of decreased blood loss, shorter hospital stay, lesser requirement for analgesics, and an earlier return to an oral diet, without evidence of compromised oncological clearance.

Is regular follow-up scan for giant liver haemangioma necessary?

OBJECTIVES: To review the reliability of radiological diagnosis and need of regular scans for giant liver haemangioma, in terms of long-term outcome and management options. DESIGN: Retrospective study. SETTING: Division of Hepato-biliary and Pancreatic Surgery, Prince of Wales Hospital, Hong Kong. PATIENTS: Patients with giant liver haemangioma noted on initial imaging from February 1996 to July 2006. MAIN OUTCOME MEASURES: Patient demographics, clinical assessments, management, and outcomes. RESULTS: There were 42 female and 22 male patients with a median age of 49 (range, 27-84) years with a suspected haemangioma. The median maximal diameter of the lesions was 5.5 cm (range, 4.0-20.3 cm). They were first detected by ultrasonography (n=45), contrast-enhanced computed tomographic scan (n=18), or magnetic resonance imaging (n=1). Besides regular follow-up scans, 22 patients were investigated further to confirm the diagnosis/exclude malignancy. Finally, 63 patients had a haemangioma and one had a hepatocellular carcinoma. Regarding the patients with haemangiomas, two were operated on for relief of pain and the rest were managed conservatively. The median duration of follow-up was 34 months. Most (54%) of the patients were asymptomatic, but in 17% the haemangioma enlarged to exceed its original size by more than 20%. There were no haemangioma-associated complications. CONCLUSIONS: Majority of patients having giant liver haemangioma are asymptomatic and do not suffer complications. If the diagnosis is uncertain, selective further investigations may be necessary. Lesions with a confirmed diagnosis tend to remain static in size; performing regular scans for asymptomatic giant liver haemangiomas may not be necessary.

Safe modification of the liver-hanging maneuver by endoscopic-assisted dissection of the retrohepatic tunnel.

As a safer approach to right hepatectomy, Belghiti et al. (J Am Coll Surg 193:109-11, 2001) described a liver-hanging maneuver. However, this procedure is performed blind, with the risks of damaging the small retrohepatic veins and consequential bleeding. To overcome this problem, we modified the procedure so that, instead of performing blind dissection using a long vascular clamp, we use a flexible choledochoscope to dissect the retrohepatic space filled by loose alveolar tissue anterior to the inferior vena cava (IVC). The avascular path is identified by a combination of saline irrigation and gentle movement of the tip of the choledochoscope. Cotton tape can then be passed around the liver parenchyma to elevate the liver away from the anterior surface of the IVC. This modification of Belghiti's liver-hanging maneuver allows direct vision along the plane anterior to the IVC, thus avoiding injury to the retrohepatic veins.

Midgut and salivary gland transcriptomes of the arbovirus vector Culicoides sonorensis (Diptera: Ceratopogonidae).

Numerous Culicoides spp. are important vectors of livestock or human disease pathogens. Transcriptome information from midguts and salivary glands of adult female Culicoides sonorensis provides new insight into vector biology. Of 1719 expressed sequence tags (ESTs) from adult serum-fed female midguts harvested within 5 h of feeding, twenty-eight clusters of serine proteases were derived. Four clusters encode putative iron binding proteins (FER1, FERL, PXDL1, PXDL2), and two clusters encode metalloendopeptidases (MDP6C, MDP6D) that probably function in bloodmeal catabolism. In addition, a diverse variety of housekeeping cDNAs were identified. Selected midgut protease transcripts were analysed by quantitative real-time PCR (q-PCR): TRY1_115 and MDP6C mRNAs were induced in adult female midguts upon feeding, whereas TRY1_156 and CHYM1 were abundant in midguts both before and immediately after feeding. Of 708 salivary gland ESTs analysed, clusters representing two new classes of protein families were identified: a new class of D7 proteins and a new class of Kunitz-type protease inhibitors. Additional cDNAs representing putative immunomodulatory proteins were also identified: 5' nucleotidases, antigen 5-related proteins, a hyaluronidase, a platelet-activating factor acetylhydrolase, mucins and several immune response cDNAs. Analysis by q-PCR showed that all D7 and Kunitz domain transcripts tested were highly enriched in female heads compared with other tissues and were generally absent from males. The mRNAs of two additional protease inhibitors, TFPI1 and TFPI2, were detected in salivary glands of paraffin-embedded females by in situ hybridization.

Antigen capture competitive enzyme-linked immunosorbent assays using baculovirus-expressed antigens for diagnosis of bluetongue virus and epizootic hemorrhagic disease virus.

Bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) are orbiviruses that infect both livestock and wild ruminants. Antigenic cross-reactivity between BTV and EHDV often results in serologic misdiagnosis. Competitive enzyme-linked immunosorbent assays (c-ELISAs) show increased sensitivity and specificity for the identification of these viral diseases; however, the preparation of cell culture-derived viral antigen for these tests is laborious and variable from batch to batch, and the resulting antigen may be infectious. To overcome these problems, the genes coding for a structural protein, VP7, of BTV and EHDV were cloned into baculovirus and the recombinant proteins were expressed in Sf9 cultured insect cells. Recombinant viral proteins released into the baculovirus-infected Sf9 cell culture supernatant were used in antigen capture c-ELISAs (Ag Cap c-ELISA) tests that specifically detected antibody in the serum of cattle experimentally infected with BTV and EHDV. The diagnostic utility of the Ag Cap c-ELISA was demonstrated by comparison with a commercial c-ELISA. The Ag Cap c-ELISA offers the advantages of using an easily produced, easily standardized, noninfectious antigen that does not require further purification or concentration.

Molecular investigations of orbivirus/vector interactions.

Defining predictors for insect-transmitted virus (arbovirus) disease cycles requires an understanding of the molecular interactions between the virus and vector insect. Studies of orbiviruses from numerous geographic regions have indicated that virus genes are affected by insect population differences. Therefore, the authors have initiated genetic studies of Culicoides sonorensis, isolating cDNAs for characterisation of differential insect gene expression, as well as a gene discovery project. Previous work identified insect transcripts elevated in orbivirus-infected female midguts at one day post infection (pI). Here, we report cDNAs that were more abundant in midguts two days following an epizootic haemorrhagic disease virus feeding, as well in head/salivary glands at three days pI. Of the cDNAs identified in midguts at two days pI, three encode translational machinery components, and three encode components that affect cellular structural features. Of the differentially expressed salivary gland cDNAs, only one was homologous to a previously identified gene, a putative odorant binding protein.

Field-deployable real-time polymerase chain reaction detection of bluetongue and epizootic haemorrhagic disease viral ribonucleic acid.

Nucleic acid sequence information from molecular evolution studies of bluetongue virus (BTV) and related epizootic haemorrhagic disease virus (EHDV) strains has resulted in a large database of genomic information. Published sequence data and sequence data from our laboratory were used to design real-time field-deployable reverse transcriptase-polymerase chain reaction assays for the detection of BTV or EHDV viral RNA. The assays used standard RNA extraction and TaqMan chemistries and the entire process was completed in

Differentially expressed midgut transcripts in Culicoides sonorensis (Diptera: ceratopogonidae) following Orbivirus (reoviridae) oral feeding.

Understanding the vector insect's gene expression response to a virus infection may aid design of control measures for arbovirus diseases. Culicoides sonorensis is a vector of several agriculturally important pathogens, such as epizootic haemorrhagic disease virus (EHDV) that causes disease in ruminants. Two approaches, differential display and suppression subtractive hybridization, were used to identify 400+ Culicoides transcripts that were more abundant in midguts 1 day following an oral meal containing EHDV. Of these, quantitative PCR confirmed seven to be more abundant in virus-fed midguts than controls. One such transcript encodes a putative RNA editase, CsRED1, induced by dsRNA. Transcripts encoding putative receptors involved in cell differentiation included CsLAR, a protein tyrosine phosphatase, and CsFZ2, homologous to the wingless receptor in D. melanogaster. Transcripts encoding putative translation machinery components included CseIF3, CseIF5A and CsRPS6. Overall, the cDNA fragments identified in this study increased in the midgut at one day postfeeding; by 2 days postfeeding, increases in transcript levels shifted from the midgut to the remainder of the infected midge.

Verification of bluetongue virus S9 segment nucleotide sequences.

During the course of our bluetongue virus (BTV) nucleic acid sequence investigations, conflicts among United States (US) prototype BTV S9 genome segment sequences deposited in GenBank were noted. In order to rectify these inter-laboratory discrepancies, the S9 segments of Arthropod-borne Animal Diseases Research Laboratory (ABADRL)-stored US prototype BTV 2, BTV 10, BTV 11, BTV 13, and BTV 17 isolates were resequenced. Our S9 sequences, determined by direct sequencing of full-length reverse transcriptase-polymerase chain reaction (RT-PCR) generated amplicons, shared 99% or greater nucleotide identity with one or more respective S9 sequences previously reported. Possible sources of remaining unsupported US prototype BTV S9 sequences were evaluated by amplifying and sequencing the S9 segments of BTV 2 Ona A strain, South African (SA) prototype BTV 1, BTV 2, and BTV 4 strains, and the North American (NA) prototype epizootic hemorrhagic disease virus (EHDV) serotype 2 (Alberta) strain. Comparative analysis using these S9 sequences, as well as sequences of US BTV 2 field isolates, identified potential contributors to inter-laboratory sequence disagreements.

Relationship between exposure to community violence and psychological distress: linear or curvilinear?

This paper presents data testing the hypothesis that the relationship between exposure to recurring community violence during high school years and later experiencing of psychological distress symptoms is a quadratic curvilinear one. Data were collected between 1994-1996 by self-administered questionnaires using multi-item scales; the sample (N = 452) comprises older urban adolescents. Although exposure to community violence was positively and linearly related to psychological distress, no statistically significant quadratic curvilinear relationship was found between community violence and psychological distress.