Reverse Genetics System for Rabbit vesivirus.

Most caliciviruses are refractory to replication in cell culture and only a few members of the family propagate in vitro. Rabbit vesivirus (RaV) is unique due to its ability to grow to high titers in several animal and human cell lines. This outstanding feature makes RaV an ideal candidate for reverse genetics studies, an invaluable tool to understand the molecular basis of virus replication, the biological functions of viral genes and their roles in pathogenesis. The recovery of viruses from a cDNA clone is a prerequisite for reverse genetics studies. In this work, we constructed a RaV infectious cDNA clone using a plasmid expression vector, under the control of bacteriophage T7 RNA-polymerase promoter. The transfection of permissive cells with this plasmid DNA in the presence of T7 RNA-polymerase, provided in trans by a helper recombinant poxvirus, led to de novo synthesis of RNA transcripts that emulated the viral genome. The RaV progeny virus produced the typical virus-induced cytopathic effect after several passages of cell culture supernatants. Similarly, infectious RaV was recovered when the transcription step was performed in vitro, prior to transfection, provided that a 5'-cap structure was added to the 5' end of synthetic genome-length RNAs. In this work, we report an efficient and consistent RaV rescue system based on a cDNA transcription vector, as a tool to investigate calicivirus biology through reverse genetics.

Influence of sows' parity on performance and humoral immune response of the offspring.

BACKGROUND: Primiparous sows (PP) have higher nutrient requirements, fewer piglets born with lower birth weight and growth performance than multiparous sows (MP). The aim of the current study was to investigate the effect of parity of sow (PP or MP) on the growth performance and humoral immune response of piglets. A total of 10 PP and 10 MP (3rd to 5th parity) sows were used. There were 4 treatments in a 2 × 2 factorial arrangement, with piglets from PP sows suckled by PP or MP sows, and piglets from MP sows suckled by PP or MP sows. Average daily gain (ADG) of piglets during the lactation period, and ADG, average daily feed intake (ADFI) and gain:feed ratio (G:F) from weaning to 144 days of age were controlled, and concentrations of immunoglobulins G (IgG) and major acute phase protein (Pig-MAP) were measured as markers of humoral immune response throughout the study. RESULTS: Total ADG was higher in piglets born from MP than in those born from PP (669 vs. 605 g/day; standard error of the mean (SEM) = 15.5, n = 5; P = 0.001) and in piglets suckled by MP than in piglets suckled by PP (655 vs. 620 g/day; SEM = 15.5, n = 5, P = 0.037). Total ADFI was higher for pigs born from MP than for those born from PP (1592 vs. 1438 g/d, SEM = 42.2, n = 5, P < 0.001). Total G:F tended to be higher for pigs suckled by MP than for those suckled by PP (0.43 vs. 0.41, SEM = 0.006, n = 5, P = 0.076). At weaning, IgG serum concentration was higher (30.0 vs. 17.8 mg/mL, SEM = 4.98, n = 15, P = 0.013) in pigs suckled by MP than in piglets suckled by PP. However, IgG concentrations were higher for pigs born from PP than for pigs born from MP on days 116 (P < 0.001) and 144 (P = 0.088). Pig-MAP tended to be lower in pigs suckled by MP than in pigs suckled by PP on days 40 and 60 of age (P < 0.10). CONCLUSIONS: The research indicates that the growth performance and humoral immune response of the offspring of PP is improved by cross-fostering with MP. These results open the possibility of an interesting strategy for improving the growth of litters from PP, that is easier to apply than current programs based on parity segregation, which implies a separate building site to house gilts, first parity sows and their offspring.

Surgical castration with pain relief affects the health and productive performance of pigs in the suckling period.

BACKGROUND: Surgical castration is still practiced in many EU countries to avoid undesirable aggressive behavior and boar taint in male pigs. However, evidence shows that castration is painful and has a detrimental influence on pig health. This study investigated the clinical and productive effects of surgical castration in the suckling period. A total of 3696 male pigs, 3 to 6 days old, comprising of 721 litters from two different farms were included in the study. Within each litter, half of the males were kept as intact males (IM) and half were surgically castrated (CM). Surgical castration was conducted by a trained farmer. Average daily gain (ADG), body weight at weaning (BWW), percentage of pre-weaning mortality (PWM) and antibiotic usage were measured. Pig major acute phase protein (PigMAP) serum concentrations were analyzed prior to castration, and on days 1 and 10 after castration. Productive performance data were analyzed using a linear mixed model. Mortality and percentage of pigs treated with antibiotics were analyzed using the Fisher's exact test. RESULTS: No overall differences in BWW and ADG were observed between the two groups. However, differences were observed when the same effects were analyzed in the 25% lightest, 50% medium and 25% heaviest pigs at birth. PWM was higher in CM than in IM groups (6.3% vs 3.6%; p < 0.001), especially in the light (12.2% vs 6.2%; p = 0.02) and in the medium (5.5% vs 2.7%; p = 0.04) weight groups. In the heaviest pigs group PWM was not affected by castration, but IM tended to show higher ADG (p = 0.06) and showed higher BWW (8.0 kg vs 7.8 kg; p = 0.05) than CM. There were no differences in percentage of pigs treated with antibiotics between the two groups (5.8% vs 5.8%; p = 0.98) in this study. Furthermore, PigMAP was increased in CM the day after castration (0.944 mg/ml vs 0.847 mg/ml; p = 0.025), but there was no difference between CM and IM groups at day 10. CONCLUSIONS: Surgical castration has a negative impact on production in the suckling period because it causes an increase in PWM, especially in pigs in the three lower quartiles for body weight, and negatively affects the BWW in pigs born in the highest quartile for body weight.

RTP801 immunoreactivity in retinal ganglion cells and its down-regulation in cultured cells protect them from light and cobalt chloride.

RTP801, a stress-related protein, is activated by adverse environmental conditions and inhibits the activity of mammalian target of rapamycin (mTOR) in promoting oxidative stress-dependent cell death. RTP801 exists both in the mammalian retina and the lens of the eye. Here, we observed RTP801 immunoreactivity in some retinal ganglion cells. Intravitreal injection of cobalt chloride (CoCl2) to mimick hypoxia influenced retinal GFAP (glial fibrillary acidic protein) and heme oxygenase-1 (HO-1) levels, but did not affect RTP801 immunoreactivity or mRNA content relative to GAPDH. However, RTP801 mRNA was elevated when compared with Brn3a mRNA, suggesting that RTP801 is activated in stressed Brn3a retinal ganglion cells. In cultures of RGC-5 cells, RTP801 immunoreactivity was located in the cytoplasm and partly present in the mitochondria. An insult of blue light or CoCl2 increased RTP801 expression, which was accompanied by cell death. However, in cultures where RTP801 mRNA was down-regulated, the negative influence of blue light and CoCl2 was blunted. Rapamycin nullified the CoCl2-induced up-regulation of RTP801 and attenuated cell death. Moreover, rapamycin was non-toxic to RGC-5 cells, even at a high concentration (10µM). The protective effect of rapamycin on RGC-5 cells caused by the inhibition of RTP801 suggests that rapamycin might attenuate retinal ganglion cell death in situ, as in glaucoma.

Light might directly affect retinal ganglion cell mitochondria to potentially influence function.

Visible light (360-760 nm) entering the eye impinges on the many ganglion cell mitochondria in the non-myelinated part of their axons. The same light also disrupts isolated mitochondrial function in vitro and kills cells in culture with the blue light component being particularly lethal whereas red light has little effect. Significantly, a defined light insult only affects the survival of fibroblasts in vitro that contain functional mitochondria supporting the view that mitochondrial photosensitizers are influenced by light. Moreover, a blue light insult to cells in culture causes a change in mitochondrial structure and membrane potential and results in a release of cytochrome c. Blue light also causes an alteration in mitochondria located components of the OXPHOS (oxidative phosphorylation system). Complexes III and IV as well as complex V are significantly upregulated whereas complexes I and II are slightly but significantly up- and downregulated, respectively. Also, blue light causes Dexras1 and reactive oxygen species to be upregulated and for mitochondrial located apoptosis-inducing factor to be activated. A blue light detrimental insult to cells in culture does not involve the activation of caspases but is known to be attenuated by necrostatin-1, typical of a death mechanism named necroptosis.

Role of annexin A2 in cellular entry of rabbit vesivirus.

The mechanisms of calicivirus attachment and internalization are not well understood, mainly due to the lack of a reliable cell-culture system for most of its members. In this study, rabbit vesivirus (RaV) virions were shown to bind annexin A2 (ANXA2) in a membrane protein fraction from HEK293T cells, using a virus overlay protein-binding assay and matrix-assisted laser desorption/ionization time-of-flight analysis. A monoclonal anti-ANXA2 antibody and small interfering RNA-mediated knockdown of ANXA2 expression in HEK293T cells reduced virus infection significantly, further supporting the role of ANXA2 in RaV attachment and/or internalization.

Structural and functional analysis of virus factories purified from Rabbit vesivirus-infected Vero cells.

Rabbit vesivirus infection induces membrane modifications and accumulation of vesicular structures in the cytoplasm of infected Vero cells. Crude RaV replication complexes (RCs) have been purified and their structural and functional properties have been characterized. We show that calnexin, an ER-resident protein, RaV non-structural proteins 2AB-, 2C-, 3A-, 3B- and 3CD-like as well as viral RNAs co-localize within membranous structures which are able to replicate the endogenous RNA templates. The purified virus factories protected their viral RNA contents from microccocal nuclease degradation and were inaccessible to exogenously added synthetic transcripts. In addition, we have shown that RCs can be used to investigate uridylylation of native endogenous VPg. In contrast to the observation that the virus factories were inaccessible to RNAs, RCs were accessible to added recombinant VPg which was subsequently nucleotidylylated. Nevertheless no elongation of an RNA chain attached to native or recombinant VPg could be demonstrated.