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.

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.

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.

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.

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.

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.

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.

Sequence analysis of the non-structural protein 2 from epizootic hemorrhagic disease viruses.

The non-structural protein 2 (NS2) of epizootic hemorrhagic disease virus serotype 1 (EHD-1) was cloned and sequenced. The NS2 gene was found to be 1185 bp containing a single open reading frame that encodes a 376 amino acid protein. A 97% nucleic acid identity was found between EHD-1 and a previously published NS2 sequence of EHD-2. Only a 60% nucleic acid identity was found between EHD and the bluetongue virus (BTV) serogroup. Comparison of the deduced amino acid sequences revealed 97% identity within the EHD serogroup, and less than or equal to 43% identity between serogroups.

Molecular comparison of VP3 from bluetongue and epizootic hemorrhagic disease viruses.

The complete nucleic acid sequence of gene 3 from epizootic hemorrhagic disease of deer (EHD) virus serotype 1 was determined. The 2768 bp sequence encodes a single protein that contains 899 amino acids and has a molecular weight of 103 kDa. The predicted protein sequence has 94.7% identity with EHD virus serotype 2 and greater than 77% identity with the related bluetongue viruses serotypes 1, 10 and 17 VP3 proteins. The relevance of these data to studies of recombinant DNA diagnostics and genetic relatedness is discussed.

Development of a nested-PCR test based on sequence analysis of epizootic hemorrhagic disease viruses non-structural protein 1 (NS1).

Two orbiviruses, epizootic hemorrhagic disease (EHD) and bluetongue (BTV) viruses, cause disease in domestic and wild ruminant species. The gene that encodes non-structural protein 1 (NS1) of EHD virus, serotype 1, was sequenced and compared to EHD and BTV NS1 sequences. The NS1 gene was found to be more conserved than the VP3 gene, and was selected as a target for polymerase chain reaction (PCR) amplification. The NS1 genes of several BTV viruses and another orbivirus, African horse sickness (AHS), were compared to the EHD NS1 genes. This information was used to develop a capture nested-PCR for detection and differentiation of EHD from BTV viral RNA.

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.

Geographical genetic variation in the gene encoding VP3 from the Alberta isolate of epizootic hemorrhagic disease virus.

The complete nucleic acid and deduced amino acid sequences of gene segment 3 and the encoded VP3 from the North American, Alberta isolate of epizootic hemorrhagic disease virus serotype 2 (EHDV-2) are reported. Complementary DNA corresponding to segment 3 was 2768 nucleotides in length with an open reading frame of 2697 base pairs which encoded a VP3 polypeptide of 899 amino acid residues. Sequence comparison with genome segment 3 and VP3 from the Australian strain of EHDV-2 indicated genotypic and phenotypic homologies of 79% and 94%, respectively. Two North American field isolates of EHDV-2, as well as EHDV-1 (New Jersey isolate), had virtually identical homology to the Alberta isolate. Sequence analysis delineated North American EHDV strains as members of a genetically homologous and geographically distinct group of orbiviruses (topotype). The data support the hypothesis that geographic isolation between North American and Australian orbiviruses has permitted the viral topotypes to maintain their genetic distinctness.

Complete nucleotide sequence of RNA segment 3 of bluetongue virus serotype 2 (Ona-A). Phylogenetic analyses reveal the probable origin and relationship with other orbiviruses.

The nucleotide sequence of the RNA segment 3 of bluetongue virus (BTV) serotype 2 (Ona-A) from North America was determined to be 2772 nucleotides containing a single large open reading frame of 2703 nucleotides (901 amino acid). The predicted VP3 protein exhibited general physiochemical properties (including hydropathy profiles) which were very similar to those previously deduced for other BTV VP3 proteins. Partial genome segment 3 sequences, obtained by polymerase chain reaction (PCR) sequencing, of BTV isolates from the Caribbean were compared to those from North America, South Africa, India, Indonesia, Malaysia and Australia, as well as other orbiviruses, to determine the phylogenetic relationships amongst them. Three major BTV topotypes (Gould, A.R. (1987) Virus Res. 7, 169-183) were observed which had nucleotide sequences that differed by approximately 20%. At the molecular level, geographic separation had resulted in significant divergence in the BTV genome segment 3 sequences, consistent with the evolution of distinct viral populations. The close phylogenetic relationship between the BTV serotype 2 (Ona-A strain) from Florida and the BTV serotypes 1, 6 and 12 from Jamaica and Honduras, indicated that the presence of BTV serotype 2 in North America was probably due to an exotic incursion from the Caribbean region as previously proposed by Sellers and Maaroof ((1989) Can. J. Vet. Res. 53, 100-102) based on trajectory analysis. Conversely, nucleotide sequence analysis of Caribbean BTV serotype 17 isolates suggested they arose from incursions which originated in the USA, possibly from a BTV population distinct from those circulating in Wyoming.

The smallest gene of the orbivirus, epizootic hemorrhagic disease, is expressed in virus-infected cells as two proteins and the expression differs from that of the cognate gene of bluetongue virus.

The smallest gene (S10) of the virus of epizootic hemorrhagic disease of deer (EHD, serotype 2) is expressed as two proteins in virus-infected cells. By contrast, the non-structural proteins (NS3 and NS3A) encoded in the smallest gene of bluetongue (BT) viruses are difficult to detect in virus-infected cells. The nucleotide sequence of S10 of EHDV-2 contains two in-frame initiation codons which allow for translation of proteins of mol. wt. 25503 and 23921 analogous to NS3 and NS3A of BT viruses. The S10 genes of BT viruses are highly conserved (82%-99%); the nucleotide sequence similarity of S10 of EHDV-2 and BT viruses is about 64%. Some structural features of NS3 and NS3A are conserved in the two viruses, despite the divergence in the amino acid sequences of the proteins. The hydrophobic domains of the proteins and the putative transmembrane sequences are conserved, as are potential glycosylation sites in the proteins. A cluster of proline residues, which is conserved at residues 36-50 in all of the published sequences of NS3 of BT viruses, is conserved exactly in the alignment of the sequence of NS3 of EHDV-2 with that of the BT viruses. An explanation for the differences in expression of NS3/NS3A in EHD and BT viruses was not evident in comparing the nucleotide sequences of S10 of the viruses.

Phylogenetic relationships of bluetongue viruses based on gene S7.

Previous phylogenetic analyses based on bluetongue virus (BTV) gene segment L3, which encodes the inner core protein, VP3, indicated a geographical distribution of different genotypes. The inner core protein, VP7, of BTV has been identified as a viral attachment protein for insect cell infection. Because the inner core proteins are involved with infectivity of insect cells, we hypothesized that certain VP7 protein sequences are preferred by the insect vector species present in specific geographic locations. We compared the gene segment S7, which encodes VP7, from 39 strains of BTV isolated from Central America, the Caribbean Basin, the United States, South Africa and Australia. For comparison, the S7 sequences from strains of the related orbiviruses, epizootic hemorrhagic disease virus (EHDV) and African horse sickness virus (AHSV) were included. The S7 gene was highly conserved among BTV strains and fairly conserved among the other orbiviruses examined. VP7 sequence alignment suggests that the BTV receptor-binding site in the insect is also conserved. Phylogenetic analyses revealed that the BTV S7 nucleotide sequences do not unequivocally display geographic distribution. The BTV strains can be separated into five clades based on the deduced VP7 amino acid sequence alignment and phylogeny but evidence for preferential selection by available gnat species for a particular VP7 clade is inconclusive. Differences between clades indicate allowable variation of the VP7 binding protein.

Development and optimization of a hybridization assay for epizootic hemorrhagic disease viruses.

Hybridization assays for the double-stranded RNA (dsRNA) orbiviruses causing bluetongue and epizootic hemorrhagic disease are more labor-intensive and less sensitive than enzyme-linked immunosorbent assay (ELISA) and immunoperoxidase assay. Cell-culture EHD virus amplification, rapid extraction, and optimization of RNA blotting conditions were examined to increase sensitivity and decrease labor. Aldehyde RNA denaturations and nylon hybridization membranes resulted in stronger positive hybridization signals. Treatment of infected cells with protease did not increase the yield of viral RNA target. Because RNA extraction is a tedious process, a simple non-phenolic diethyl pyrocarbonate extraction procedure was developed. The sensitivity, versatility, and the reproducibility of hybridization assays are addressed.