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*

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

To develop a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for rapid and sensitive detection of Norwalk GII. 4 primers which recognized 6 distinct regions on the RNA-dependent RNA polymerase gene of Norwalk GII were designed and used for LAMP assay. Norwalk GII RNA was amplified under isothermal conditions (65 degrees C) for 120 min, and LAMP results were then judged with naked eye, SYBR Green I staining, electrophoretic analysis and restriction digestion. To evaluate the specificity of the RT-LAMP, 48 fecal specimens of Norwalk GII and 12 fecal specimens of group A rotaviruses were tested. To compare the sensitivity of the RT-LAMP with that of conventional RT-PCR, Norwalk GII RNA was serially diluted and amplified by RT-LAMP and RT-PCR, respectively. With 46 fecal specimens of Norwalk GII, observation with naked eyes, SYBR Green I staining and electrophoretic analysis were able to detect the PCR products in the RT-LAMP assay. The specificity of RT-LAMP products was also confirmed by digestion of the RT-LAMP products with restriction enzymes. No RNA amplification was observed in 2 fecal specimens of Norwalk GII and 12 fecal specimens of group A rotaviruses. The specificity of the RT-LAMP assay with regard to RT-PCR were 100% for Norwalk GII. The detection limits of RT-LAMP was 15.6 pg/tube for Norwalk GII and similar to that of a RT-PCR assay. Compared to RT-PCR, the RT-LAMP assay has been proven to be a rapid, sensitive, specific and accurate method for detection of the Norwalk GII in fecal specimens, and that RT-LAMP assay is potentially useful for the rapid detection of Norwalk GII from fecal specimens in outbreaks of infectious diarrhea.
Caliciviridae Infections ; virology ; Feces ; virology ; Humans ; Norwalk virus ; genetics ; isolation & purification ; RNA Replicase ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Viral Proteins ; genetics

Caliciviridae Infections ; epidemiology ; Child, Hospitalized ; Child, Preschool ; China ; epidemiology ; Female ; Gastroenteritis ; epidemiology ; virology ; Humans ; Infant ; Male ; Sapovirus ; isolation & purification

OBJECTIVETo investigate the epidemiological features of human calicivirus( HuCV) infection in children with diarrhea in a hospital in Guangzhou.

METHODSStool specimens were collected from children with viral diarrhea diagnosed between October, 2003 and January, 2004 and between October, 2004 and January, 2005. HuCV was detected by means of RT-PCR and sequence analysis of the PCR products.

RESULTSEighty specimens positive for Norwalk-like viruses (NLVs) were identified from 648 stool specimens, with a positivity rate of 12.35%, and sapporo-like viruses (SLVs) were identified in 2 specimens (0.31%). The monthly NLV positivity rate was 11.74% (Oct.), 14.16% (Nov.), 9. 09% (Dec.) and 13.95% (Jan.), respectively, showing no significant variation in these months. NLVs mainly infected children below 2 years old. Twenty-two strains of NLVs were sequenced and analyzed, and 21 of them were identified as GII and the genotype of 1 strain could not be determined. The prevalent viral population were GII-3 and GII-4 in 2003 and was GII-4 in 2004, and both of the SLVs belong to GI-1.

CONCLUSIONNLVs is one of the important pathogens causing sporadic acute gastroenteritis in children admitted in the hospital in Guangzhou, and the prevalent strains are GII-3 and GII-4 , but different prevalent strains are possible in different periods.

Caliciviridae ; classification ; genetics ; isolation & purification ; Caliciviridae Infections ; epidemiology ; virology ; Child, Preschool ; China ; epidemiology ; Diarrhea ; epidemiology ; virology ; Diarrhea, Infantile ; epidemiology ; virology ; Feces ; virology ; Female ; Genotype ; Hospitals ; statistics & numerical data ; Humans ; Infant ; Male ; Molecular Epidemiology ; methods ; Molecular Sequence Data ; Norwalk virus ; genetics ; isolation & purification ; Phylogeny ; Prevalence ; RNA, Viral ; genetics ; Seasons ; Sequence Analysis, DNA

Feline calicivirus (FCV) is an important and highly prevalent pathogen of cats that causes feline respiratory disease. The reverse genetic systems for FCV have been established in national and international laboratories since 1995. This technique has been used widely in FCV basic research and good progress has consequently been made to determine the relationship between viral genome structures and the function of their proteins, the expression of foreign proteins, virus-host interactions, and viral pathogenic mechanisms. In this article,we review the state of progress with regards to the establishment and application of the FCV reverse genetic operating system,which will provide a useful reference tool for future related research.
Animals ; Caliciviridae Infections ; veterinary ; virology ; Calicivirus, Feline ; genetics ; metabolism ; Cat Diseases ; virology ; Cats ; Reverse Genetics ; methods ; trends ; Viral Proteins ; genetics ; metabolism

Caliciviridae Infections ; prevention & control ; therapy ; Disease Outbreaks ; prevention & control ; Humans ; Norovirus ; Practice Guidelines as Topic

Caliciviridae Infections ; epidemiology ; China ; epidemiology ; Disease Outbreaks ; Female ; Humans ; Male ; Norovirus ; Schools

Caliciviridae Infections ; epidemiology ; virology ; Gastroenteritis ; virology ; Genetic Variation ; Genotype ; Humans ; Norovirus ; classification ; genetics