This retrospective study was aimed to assessing clinical, paraclinical symptoms of 340 patients between 16-45 years old, with Dengue haemorrhagic fever. Patients were divided into 2 groups with and without Dengue Shock Syndrome (DSS). The results showed that clinical symptoms including sudden high fever, headache, orbital pain, congested skin, petechie, and positive tourniquet test. Paraclinical symptoms included thrombocytopenia increased haematocrite, leukopenia, increased serum transaminases. This disease often occurred in October and November. DSS can occure at 4th to 7th day with symptoms vomiting, sudden subsided fever, cold clammy skin, pleural or pericardial effusion, ascites, rapid increased haematocrite, thrombocytopenia, leukopenia. Dengue haemorrhagic fever can be cured in short time with infusion of isotonic fluid and Ringer lactate fluid
Dengue ; Hemorrhagic Disease Virus, Rabbit ; Dengue Hemorrhagic Fever ; diagnosis

Between 2000 and 2001, there were 143 cases of placenta previa (1.95%) at Institue for Protection of Mothers and Newborns. The potential causes were abortion many times, cesar section, and multi delivery... Diagnosis is accurate and early by using good pregnant management system and ultrasound. Monitoring and treatment before delivery were improved. In cases of central placenta previa, Cesar section could help decreasing the risk of haemorrhage
Placenta Previa ; Hemorrhagic Disease Virus, Rabbit ; Pregnant Women ; Therapeutics

In this study, to understand the pathogenesis of new rabbit hemorrhagic disease virus (RHDVa) serotype, we carried out to administrate RHDVa to rabbits, and to examine sequential electron microscopic changes and relationship between pathogenesis and apoptosis. TUNEL-positive cells began to be observed from 24 hours after inoculation (HAI) and the number of positive cells was slightly increased with the course of time. Whereas marked increase of positive cells was seen in the liver from the rabbits died acutely. Typical viral particles with cup-like projections and a diameter of 30~40 nm were detected in homogenized liver samples and tissues at 36 and 48, and 48 HAI, respectively. Ultrastructurally, glycogen deposition was observed from the first stage of hepatocellular degeneration by RHDVa infection and then, swelling and disruption of cristae of mitochondria by viral particles, swelling of smooth endoplasmic reticulum, vacuoles and vesicles were detected. Condensation, margination and fragmentation of chromatin were observed in degenerative hepatocytes at 36 and 48 HAI, indicating apoptotic bodies. These data offer that hepatocytic apoptosis by RHDV infection could be closely related with mitochondrial impairment in the hepatocytes.
Apoptosis ; Chromatin ; Electrons ; Endoplasmic Reticulum, Smooth ; Glycogen ; Hemorrhagic Disease Virus, Rabbit ; Hepatocytes ; Liver ; Mitochondria ; Rabbits ; Sprains and Strains ; Vacuoles ; Virion

Rabbit haemorrhagic disease virus (RHDV) and myxoma virus (MYXV), are two pathogens that have harmful effect on rabbit breeding and population decline of European rabbits in their native range, causing rabbit haemorrhagic disease (rabbit fever) and myxomatosis, respectively. The capsid protein VP60 of the RHDV represents the major antigenic protein. To develop a recombinant bivalent vaccine candidate that can simultaneously prevent these two diseases, we used the nonessential gene TK (thymidine kinase) of MYXV as the insertion site to construct a recombinant shuttle vector p7.5-VP60-GFP expressing the RHDV major capsid protein (VP60) and the selectable marker GFP. Then the shuttle vector p7.5-VP60-GFP was transfected into rabbit kidney cell line RK13 which was previously infected with MYXV. After homologous recombination, the recombinant virus expressing GFP was screened under a fluorescence microscope and named as rMV-VP60-GFP. Finally, the specific gene-knock in and expression verification of the vp60 and gfp genes of the recombinant virus was confirmed by PCR and Western blotting. The results showed that these two genes were readily knocked into the MYXV genome and also successfully expressed, indicating that the recombinant MYXV expressing the vp60 of RHDV was generated. Protection against MYXV challenge showed that the recombinant virus induced detectable antibodies against MYXV which would shed light on development of the effective vaccine.
Animals ; Blotting, Western ; Caliciviridae Infections/veterinary* ; Hemorrhagic Disease Virus, Rabbit/immunology* ; Rabbits ; Vaccines, Synthetic/immunology* ; Viral Structural Proteins/genetics*

Rabbit hemorrhagic disease is a highly acute and fatal viral disease caused by rabbit hemorrhagic disease virus (RHDV). Since first outbreak in Korea 1987, RHDV has been continually affected in the country, but the pattern of outbreak seem to be changed. In this study, to understand the pathogenesis of the new RHDVa serotype, we therefore carried out to inoculate RHDVa to rabbits, and to examine the sequential histopathologic changes and viral distribution. Macroscopically, various sized dark red or white spots or appearance were observed in the liver, lung, kidney uterus and ureter. In euhanized rabbits, significant pathologic findings such as infiltration of heterophils and mononuclear cells were observed at 24 hours after inoculation (HAI), and these were sequentially extended periportal to centrilobular area. However, in dead rabbits, severe hepatic degeneration and/or necrosis with relatively weak inflammatory responses were observed. RHDV antigens began to detect in liver, spleen, and lung from 12 HAI by PCR. Immunohistochemically, RHDV positive cells were seen in only liver from 24 HAI, and the degree of immunogen reactivity was stronger in dead rabbits than in euthanized ones. In conclusion, RHDVa caused the subacute or chronic infection accompanying low mortality and moderate to severe inflammatory reaction in rabbits, suggesting the possibility that RHD could become endemic.
Dental Caries ; Hemorrhagic Disease Virus, Rabbit ; Kidney ; Korea ; Liver ; Lung ; Necrosis ; Polymerase Chain Reaction ; Rabbits ; Spleen ; Sprains and Strains ; Ureter ; Uterus ; Virus Diseases

To provide an efficient and safe technology platform for studying the replication and pathogenesis mechanisms of RHDV, the interaction between the RHDV and its host cells, a replicon system of RHDV, was constructed based on the infectious cDNA clone of RHDV, in which VP60 gene encoding the capsid protein was deleted, but all the necessary protease coding regions and non-coding regions were retained. Results from RT-PCR, IFA and qRT-PCR confirmed that the replicon RNA could efficiently replicate in RK-13 cells. Besides, the results also suggested that the capsid protein which is the structural protein of RHDV is necessary for maintaining the viral infectivity.
Animals ; Capsid Proteins ; physiology ; Fluorescent Antibody Technique ; Hemorrhagic Disease Virus, Rabbit ; genetics ; RNA, Viral ; biosynthesis ; Rabbits ; Replicon ; Reverse Transcriptase Polymerase Chain Reaction

The apoptosis of RK13 cells induced by RHDV was investigated with DAPI staining, DNA ladder, Caspase 3 activity and flow cytometry, etc. The results showed that nuclear staining of infected cells with DAPI showed gradually morphological changes of the nuclei. As shown in the paper, a canonic oligonucleosome-sized DNA ladder was observed in cells harvested at 24h, 48h and 72h post-infection, confirming that DNA fragmentation was induced by RHDV infection. The results of flow cytometry showed that about 63% of cells were in apoptosis at 48h post-infection. Besides, we also demonstrated that the activation of Caspase 3 occurred during the infection process. In conclusion, our results showed that apoptosis in RHD might be determinant in the development of the pathogenesis of RHD.
Animals ; Apoptosis ; Caliciviridae Infections ; genetics ; physiopathology ; veterinary ; virology ; Caspase 3 ; metabolism ; Cell Line ; Cell Nucleus ; genetics ; virology ; DNA Fragmentation ; Hemorrhagic Disease Virus, Rabbit ; physiology ; Rabbits

Rabbit hemorrhagic disease was described in China in 1984 and can cause hemorrhagic necrosis of the liver within two or three days after infection. The etiological agent, rabbit hemorrhagic disease virus (RHDV), belongs to the Lagovirus genus in the Caliciviridae family. Compared to other calicivirus, such as rNV and SMSV, the structure of Lagovirus members is not well characterized. In this report, structures of two types of wild RHDV particles, the intact virion and the core-like particle (CLP), were reconstructed by cryo-electron microscopy at 11 &0A and 17 &0A, respectively. This is the first time the 3D structure of wild caliciviruses CLP has been provided, and the 3D structure of intact RHDV virion is the highest resolution structure in Lagovirus. Comparison of the intact virion and CLP structures clearly indicated that CLP was produced from the intact virion with the protrusion dissociated. In contrast with the crystal structures of recombinant Norovirus and San Miguel sea lion virus, the capsomers of RHDV virion exhibited unique structural features and assembly modes. Both P1 and P2 subdomains have interactions inside the AB capsomer, while only P2 subdomains have interaction inside CC capsomer. The pseudo atomic models of RHDV capsomers were constructed by homology modeling and density map fitting, and the rotation of RHDV VP60 P domain with respect to its S domain, compared with SMSV, was observed. Collectively, our cryo-electron microscopic studies of RHDV provide close insight into the structure of Lagovirus, which is important for functional analysis and better vaccine development in the future.
Amino Acid Sequence ; Animals ; Caliciviridae Infections ; virology ; China ; Cryoelectron Microscopy ; Hemorrhagic Disease Virus, Rabbit ; ultrastructure ; Molecular Sequence Data ; Rabbits ; Sequence Alignment ; Viral Structural Proteins ; chemistry ; Virion ; ultrastructure

The capsid protein (VP60) gene of RHDV was subcloned into the Pichia expressin vector pPICZ B to express the VP60 protein intracellularly. The recombinant plasmid was initially transformed into a E. coli strain TOP10 F'. After verification of the construct by sequencing, the recombinant plasmid was linearized by Sac I in the 5' AOX1 region and then transformed into Pichia pastoris strain GS115 using the Pichia EasyComp Kit. After selecting and verifing for the insertion of VP60 gene in the genome, two clones of Pichia transformants were select for expression test. The recombinant clones were first inoculate with BMGY in baffled flask at 28-30 degrees C in a shaking incubator (250-300 r/min) until culture reaches an OD600 = 2-6, then resuspend the cell pellet to an OD6oo of 1.0 in BMMY medium to induce expression for 5 days by methanol at a concentration of 0.5% in a 1 liter baffled flask covered with 2 layers of sterile gauze. Collect the cell pellets and break it by acid-washed 0.5 mm glass beads. The expression of recombinant Pichia strains was detected by SDS-PAGE and Western analysis with a polyclonal serum which showed a specific protein band of 60kD. Theses results indicates that the recombinant VP60 produced in Pichia was antigenically similar to the viral polypeptide. Electron microscopic observation of the recombinant Pichia-derived protein revealed the presence of virus-like particles similar in size and appearance to native virus capsids. In the haemagglutination test, the recombinant VLPs, like the native RHDV, also agglutinated human blood type O erythrocytes and could be inhibited by the anti-RHDV polyclonal serum.
Animals ; Capsid ; metabolism ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; metabolism ; Hemorrhagic Disease Virus, Rabbit ; genetics ; Pichia ; genetics ; metabolism ; Rabbits ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Viral Structural Proteins ; biosynthesis ; genetics ; isolation & purification

Rabbit hemorrhagic disease (RHD) is contagious and highly lethal. Commercial vaccines against RHD are produced from the livers of experimentally infected rabbits. Although several groups have reported that recombinant subunit vaccines against rabbit hemorrhagic disease virus (RHDV) are promising, application of the vaccines has been restricted due to high production costs or low yield. In the present study, we performed codon optimization of the capsid gene to increase the number of preference codons and eliminate rare codons in Spodoptera frugiperda 9 (Sf9) cells. The capsid gene was then subcloned into the pFastBac plasmid, and the recombinant baculoviruses were identified with a plaque assay. As expected, expression of the optimized capsid protein was markedly increased in the Sf9 cells, and the recombinant capsid proteins self-assembled into virus-like particles (VLPs) that were released into the cell supernatant. Rabbits inoculated with the supernatant and the purified VLPs were protected against RHDV challenge. A rapid, specific antibody response against RHDV was detected by an ELISA in all of the experimental groups. In conclusion, this strategy of producing a recombinant subunit vaccine antigen can be used to develop a low-cost, insect cell-derived recombinant subunit vaccine against RHDV.
Animals ; Antigens, Viral/genetics/metabolism ; Caliciviridae Infections/prevention & control/*veterinary/virology ; Capsid Proteins/*genetics/metabolism ; Cell Culture Techniques/*methods ; Codon/genetics/metabolism ; Enzyme-Linked Immunosorbent Assay/veterinary ; *Gene Expression Regulation, Viral ; Hemorrhagic Disease Virus, Rabbit/*genetics/immunology ; *Rabbits ; Recombinant Proteins/genetics/metabolism ; Sf9 Cells ; Spodoptera ; Viral Structural Proteins/*genetics/metabolism ; Viral Vaccines/genetics/immunology