Clinical course and pathogenicity of variant rabbit haemorrhagic disease virus in experimentally infected adult and kit rabbits: Significance towards control and spread.

RHDVb has become the dominant RHDV on the Iberian Peninsula. A better understanding of its pathogenicity is required to aid control measures. Thus, the clinical course, humoral immune response, viraemia and kinetics of RHDV-N11 (a Spanish RHDVb isolate) infection in different tissues at both viral RNA and protein levels were studied in experimentally infected young and adult rabbits. The case fatality rate differed between the two age groups, with 21% of kits succumbing while no deaths were observed in adults. Fever and viremia were strongly associated with death, which occurred 48 h post infection (PI) too fast for an effective humoral immune response to be mounted. A significant effect on the number of viral RNA copies with regard to the variables age, tissue and time PI (p < 0.0001 in all cases) was detected. Histological lesions in infected rabbits were consistently more frequent and severe in liver and spleen and additionally intestine in kits, these tissues containing the highest levels of viral RNA and protein. Although no adults showed lesions or virus antigen in intestine, both kits and adults maintained steady viral RNA levels from days 1 to 7 PI in this organ. Analysis revealed the fecal route as the main dissemination route of RHDV-N11. Subclinically infected rabbits had detectable viral RNA in their faeces for up to seven days and thus may play an important role spreading the virus. This study allows a better understanding of the transmission of this virus and improvement of the control strategies for this disease.

ELISA for detection of variant rabbit haemorrhagic disease virus RHDV2 antigen in liver extracts.

The emergence and rapid spread of variant of the rabbit hemorrhagic disease virus (RHDV2) require new diagnostic tools to ensure that efficient control measures are adopted. In the present study, a specific sandwich enzyme-linked immunosorbent assay (ELISA) for detection of RHDV2 antigens in rabbit liver homogenates, based on the use of an RHDV2-specific monoclonal antibody (Mab) 2D9 for antigen capture and an anti-RHDV2 goat polyclonal antibody (Pab), was developed. This ELISA was able to successfully detect RHDV2 and RHDV2 recombinant virions with high sensitivity (100%) and specificity (97.22%). No cross-reactions were detected with RHDV G1 viruses while low cross-reactivity was detected with one of the RHDVa samples analyzed. The ELISA afforded good repeatability and had high analytical sensitivity as it was able to detect a dilution 1:163,640 (6.10ng/mL) of purified RHDV-N11 VLPs, which contained approximately 3.4×108molecules/mL particles. The reliable discrimination between closely related viruses is crucial to understand the epidemiology and the interaction of co-existing pathogens. In the work described here we design and validate an ELISA for laboratory based, specific, sensitive and reliable detection of RHDVb/RHDV2. This ELISA is a valuable, specific virological tool for monitoring virus circulation, which will permit a better control of this disease.

Fast specific field detection of RHDVb.

This work describes a simple and rapid test for field detection of the emerging rabbit pathogen RHDVb. The assay is specific for RHDVb, showing no cross-reactivity with other RHDV types giving a specific result in under 10 min using rabbit liquid exudates or liver homogenate samples taken at necropsy.

Conventional and real time RT-PCR assays for the detection and differentiation of variant rabbit hemorrhagic disease virus (RHDVb) and its recombinants.

Since its emergence, variant RHDV (RHDVb/RHDV2) has spread throughout the Iberian Peninsula aided by the apparent lack of cross protection provided by classic (genogroup 1; G1) strain derived vaccines. In addition to RHDVb, full-length genome sequencing of RHDV strains has recently revealed the circulation of recombinant viruses on the Iberian Peninsula. These recombinant viruses contain the RHDVb structural protein encoding sequences and the non-structural coding regions of either pathogenic RHDV-G1 strains or non-pathogenic (np) rabbit caliciviruses. The aim of the work was twofold: firstly to validate a diagnostic real time RT-PCR developed in 2012 for the detection of RHDVb strains and secondly, to design a conventional RT-PCR for the differentiation of RHDVb strains from RHDVb recombinants by subsequent sequencing of the amplicon.

Illegal discharges in Spanish waters. Analysis of the profile of the Alleged Offending Vessel.

There is at present a growing concern, on an international level, over environmental offences caused by oil discharges into the sea from vessels. The objective of the Spanish Maritime Administration is to prevent the illegal discharges of polluting substances in Spanish maritime waters by vessels in transit. To combat such discharges, since 2007 Spain has reinforced its means of response with the use of aircrafts that provide services of maritime surveillance, identifying the Alleged Offending Vessels and acting as a deterrent. The objective of the present study is both to introduce the concept and to analyze certain aspects of the so-called "Alleged Offending Vessel" (AOV) that have been detected within Spanish Search and Rescue (SAR) jurisdiction waters in the period 2008-2012, in order to build a profile of such a vessel. For this purpose, an analysis methodology is formalized based on the GINI index and Lorenz curves, associated with certain aspects of vessels: type, flag and sailing area.

Heterologous expression and functional characterization of thioredoxin from Fasciola hepatica.

The full thioredoxin coding sequence from Fasciola hepatica has been cloned into the pGEX-2T expression vector and produced in Escherichia coli as a fusion protein. The recombinant protein proved to be biologically active, using an insulin reduction assay, and was also able to activate thioredoxin peroxidase from F. hepatica. These observations suggest that this protein could participate in a redox cascade involved in the maintenance of cell homeostasis as well as in parasite protection against reactive oxygen species produced by the host.

Identification of the amino acid residue involved in rabbit hemorrhagic disease virus VPg uridylylation.

The virus genome-linked protein (VPg) coding region from rabbit hemorrhagic disease virus (RHDV) (isolate AST/89) was expressed in Escherichia coli by using a glutathione S-transferase-based vector. The recombinant polypeptide could be purified in good yields and was uridylylated in vitro from [alpha-32P]UTP in a reaction catalyzed by the recombinant RNA-dependent RNA polymerase from RHDV in the absence of added template RNA. The use of deletion and point mutants allowed the identification of Tyr-21 as the residue involved in uridylylation and consequently in the linkage between VPg and the viral genome. These data constitute the first report on the identity of the amino acid residue involved in VPg uridylylation in a member of the Caliciviridae family.

Characterisation of the RNA-dependent RNA polymerase from Rabbit hemorrhagic disease virus produced in Escherichia coli.

All positive-strand RNA viruses encode a RNA-dependent RNA polymerase which in most cases has been only identified on the basis of its sequence conservation. Catalytic activity has been experimentally demonstrated in only a handful of these viral proteins, including that from Rabbit hemorrhagic disease virus. Studies from our laboratory have reported that RHDV RNA polymerase produced in Escherichia coli was enzymatically active showing poly(A)-dependent poly(U) polymerase as well as RNA polymerase activity on heteropolymeric substrates. In this work, we have investigated the in vitro activity of the recombinant 3Dpol from RHDV, including ion requirements, resistance to inhibitors, substrate specificity as well as data on the initiation mechanism of the template-linked products derived from heteropolymeric RNA substrates. Our study demonstrates that in an in vitro reaction recombinant RHDV RNA polymerase generated the minus strand of the heteropolymeric RNA substrates by a "copy-back" mechanism that initiated at the template 3'-terminal OH.

Fasciola hepatica: heterologous expression and functional characterization of a thioredoxin peroxidase.

A Fasciola hepatica cDNA clone of 779 bp was isolated from an adult worm cDNA expression library by immunological screening using a rabbit serum against the excretory-secretory antigens. The nucleotide sequence of the cDNA revealed the presence of an open reading frame of 582 bp which encoded a 194-amino-acid-residue polypeptide (M(r) 21,723 Da) showing a high degree of homology to thioredoxin peroxidases. This putative antioxidant protein gene was expressed in Escherichia coli as a GST fusion protein. The recombinant fusion protein showed in vitro antioxidant properties and protected rabbit muscle enolase and E. coli glutamine synthetase from inactivation by nonenzymatic Fe(3+)/O(2)/DTT or Fe(3+)/O(2)/ascorbate metal-catalyzed oxidation systems.

Cloning and expression in Escherichia coli of a Fasciola hepatica gene encoding a calcium-binding protein.

A Fasciola hepatica cDNA clone of 994 bp was isolated from an adult worm cDNA expression library using a rabbit serum against the excretory-secretory antigens. The nucleotide sequence of the cDNA clone revealed the presence of an open reading frame of 572 bp which encoded a 22 kDa polypeptide (Fh22) showing putative EF-hand domains. This gene was expressed in Escherichia coli and the recombinant protein used for the production of specific antibodies. Immunoblotting studies using the anti-Fh22 serum showed the presence of a polypeptide of similar molecular mass in the excretory-secretory extract of the adult parasite. The recombinant Fh22 polypeptide showed calcium-dependent electrophoretic mobility (decreased with Ca2(+)-ions and increased with EGTA). The observed behaviour of recombinant Fh22 in gel filtration experiments also suggested calcium-induced conformational changes.

Macrophage tropism of rabbit hemorrhagic disease virus is associated with vascular pathology.

To delineate the interactions between rabbit hemorrhagic disease virus (RHDV) and host cells, organ and cellular targets of infection were identified in vivo. Viral specific antigens were detected by immunohistochemistry in liver, lung, spleen and lymph nodes cells. Also, intravascular infected cells were detected in most organs including kidneys, myocardium, thymus and central nervous system. To further characterize infected target cells, viral proteins and cell-specific surface antigens were identified simultaneously in double labeling experiments. Numerous lymphoid organ macrophages, from the splenic red pulp, circulating monocytes, alveolar macrophages and Kupffer cells were double labeled, demonstrating that cells of the mononuclear phagocyte lineage are major hosts for RHDV. Double labeling for other specific cell markers were negative. The distribution of viral antigens in these tissues coincided with those areas where cells presented morphology of apoptosis. Association of intravascular monocyte infection and apoptosis, could represent a possible mechanism to develop disseminated intravascular coagulation.

Immunization with potato plants expressing VP60 protein protects against rabbit hemorrhagic disease virus.

The major structural protein VP60 of rabbit hemorrhagic disease virus (RHDV) has been produced in transgenic potato plants under the control of a cauliflower mosaic virus 35S promoter or a modified 35S promoter that included two copies of a strong transcriptional enhancer. Both types of promoters allowed the production of specific mRNAs and detectable levels of recombinant VP60, which were higher for the constructs carrying the modified 35S promoter. Rabbits immunized with leaf extracts from plants carrying this modified 35S promoter showed high anti-VP60 antibody titers and were fully protected against the hemorrhagic disease.

Hypoderma lineatum: expression of enzymatically active hypodermin C in Escherichia coli and its use for the immunodiagnosis of hypodermosis.

The cDNA coding for the mature hypodermin C from first instars of Hypoderma lineatum was cloned by reverse transcription and PCR amplification of total larval RNA using specific oligonucleotide primers. This cDNA was expressed in Escherichia coli as a glutathione S-transferase fusion protein. Mature hypodermin C was released from the GST-fusion after glutathione-Sepharose 4B affinity chromatography and proteolytic cleavage using factor Xa. The purified recombinant protein showed enzymatic activity in gelatin-polyacrylamide gels and when azocoll was used as substrate. An enzyme-linked immunosorbent assay was developed using the recombinant antigen. Positive/negative cutoff values were calculated using the mean OD percentage (1.74%) of 113 negative sera plus three standard deviations. Sensitivity and specificity according to the resulting cutoff (10.74%) were 85 and 98.2% respectively.

Expression of enzymatically active rabbit hemorrhagic disease virus RNA-dependent RNA polymerase in Escherichia coli.

The rabbit hemorrhagic disease virus (RHDV) (isolate AST/89) RNA-dependent RNA-polymerase (3Dpol) coding region was expressed in Escherichia coli by using a glutathione S-transferase-based vector, which allowed milligram purification of a homogeneous enzyme with an expected molecular mass of about 58 kDa. The recombinant polypeptide exhibited rifampin- and actinomycin D-resistant, poly(A)-dependent poly(U) polymerase. The enzyme also showed RNA polymerase activity in in vitro reactions with synthetic RHDV subgenomic RNA in the presence or absence of an oligo(U) primer. Template-size products were synthesized in the oligo(U)-primed reactions, whereas in the absence of added primer, RNA products up to twice the length of the template were made. The double-length RNA products were double stranded and hybridized to both positive- and negative-sense probes.

Programmed cell death in the pathogenesis of rabbit hemorrhagic disease.

Rabbit hemorrhagic disease is a rapidly lethal infection caused by a calicivirus, characterized by acute liver damage and disseminated intravascular coagulation (DIC). Following morphological criteria and using a specific in situ labeling technique, we have found that liver cell death induced upon infection is due to apoptosis, and that programmed cell death is a constant feature in rabbits experimentally infected with RHDV. The process affected mainly hepatocytes, but also macrophages and endothelial cells presented morphologic hallmarks of apoptosis, expressing all these cell types viral antigens as determined by immunohistochemistry. The occurrence of programmed cell death was correlated with the appearance of the RHDV induced pathology in tissues by DNA fragmentation detection in situ. Hepatocyte apoptosis produced extensive parenchymal destruction causing a lethal, acute fulminant hepatitis that is characteristic of RHD. Apoptosis of intravascular monocytes and endothelial cell was observed together with fibrin thrombi in blood vessels. Since apoptotic cells are known sites of enhanced procoagulant activity, apoptosis of these cell populations might constitute a first step in the pathogenesis of DIC and a common pathway to other viral hemorrhagic fevers. In conclusion, apoptosis in RHD may be determinant in the development of the pathogenesis of this disease.

A single dose immunization with rabbit haemorrhagic disease virus major capsid protein produced in Saccharomyces cerevisiae induces protection.

The gene coding for the major capsid protein (VP60) from rabbit haemorrhagic disease virus was expressed in Saccharomyces cerevisiae under the phosphoglycerate kinase promoter. The recombinant VP60 produced in yeast was antigenically similar to the viral polypeptide as determined with a polyclonal serum. Electron microscopic observation of the recombinant yeast-derived antigen revealed the presence of virus-like particles similar in size and appearance to native capsids. Subcutaneous vaccination of rabbits with a single dose of this antigen in the absence of commercial adjuvants conferred complete protection against the haemorrhagic disease.

Processing of rabbit hemorrhagic disease virus polyprotein.

Expression of rabbit hemorrhagic disease virus (RHDV) cDNAs in vitro with rabbit reticulocyte lysates and in Escherichia coli have been used to study the proteolytic processing of RHDV polyprotein encoded by ORF1. An epitope tag was used for monitoring the gene products by a specific antibody. We have identified four gene products with molecular masses of 80, 43, 73, and 60 kDa, from the amino to the carboxy terminus of the polyprotein. The amino-terminal sequences of the 43- and 73-kDa products were determined and indicated that RHDV 3C proteinase cleaved Glu-Gly peptide bonds.

Immunogenic properties of rabbit haemorrhagic disease virus structural protein VP60 expressed by a recombinant baculovirus: an efficient vaccine.

We have constructed a recombinant baculovirus containing the gene encoding the structural protein VP60 from the Spanish field isolate AST/89 of rabbit haemorrhagic disease virus (RHDV). Infection of cultured Spodoptera frugiperda Sf9 cells with this recombinant virus resulted in the production of high yields of VP60 protein which did not seem to assemble to form virus like particles, but was antigenically similar to the corresponding viral protein obtained from purified virions. A VP60-dose study showed that the recombinant protein was able to elicit a protective response in rabbits against a nasal challenge with 100 LD50 of RHDV. The effective dose able to protect 50% of the animals in the absence of adjuvant was found to be 10-25 micrograms of recombinant VP60.

Membrane protein molecules of transmissible gastroenteritis coronavirus also expose the carboxy-terminal region on the external surface of the virion.

The binding domains of four monoclonal antibodies (MAbs) specific for the M protein of the PUR46-MAD strain of transmissible gastroenteritis coronavirus (TGEV) have been located in the 46 carboxy-terminal amino acids of the protein by studying the binding of MAbs to recombinant M protein fragments. Immunoelectron microscopy using these MAbs demonstrated that in a significant proportion of the M protein molecules, the carboxy terminus is exposed on the external surface both in purified viruses and in nascent TGEV virions that recently exited infected swine testis cells. The same MAbs specifically neutralized the infectivity of the PUR46-MAD strain, indicating that the C-terminal domain of M protein is exposed on infectious viruses. This topology of TGEV M protein probably coexists with the structure currently described for the M protein of coronaviruses, which consists of an exposed amino terminus and an intravirion carboxy-terminal domain. The presence of a detectable number of M protein molecules with their carboxy termini exposed on the surface of the virion has relevance for viral function, since it has been shown that the carboxy terminus of M protein is immunodominant and that antibodies specific for this domain both neutralize TGEV and mediate the complement-dependent lysis of TGEV-infected cells.

Molecular cloning, sequencing and expression in Escherichia coli of the capsid protein gene from rabbit haemorrhagic disease virus (Spanish isolate AST/89).

We describe the cloning, nucleotide sequencing and expression in Escherichia coli of the major capsid component (VP60) from the Spanish field isolate AST/89 of rabbit haemorrhagic disease virus (RHDV). The sequence of the 3'-terminal 2483 nucleotides of the genome was found to be 95.4% identical to the German RHDV strain, showing ten changes in the deduced VP60 amino acid sequence. The gene coding for this structural polypeptide has been expressed in bacteria as a beta-galactosidase fusion protein or using a T7 RNA polymerase-based system. The VP60 fusion protein showed only partial antigenic similarity with native VP60 and did not confer protective immunity. The recombinant VP60 produced in the T7 RNA polymerase-based system was antigenically similar to the viral polypeptide as determined using polyclonal and monoclonal antibodies. When used to immunize rabbits the recombinant VP60 was able to protect the animals against a lethal challenge using purified RHDV.