Establishment of a new cell line susceptible to Cyprinid herpesvirus 3 (CyHV‐3) and possible latency of CyHV‐3 by temperature shift in the cells

A new cell line named CCF‐K104 predominantly consisting of fibroblastic cells showed optimal growth at temperatures from 25 °C to 30 °C. Serial morphological changes in the cells induced by Cyprinid herpesvirus 3 (CyHV‐3) included cytoplasmic vacuolar formation, cell rounding and detachment. Mature virions were purified from CyHV‐3‐infected CCF‐K104 cells by sucrose gradient ultracentrifugation and had a typical herpesvirus structure on electron microscopy. Infectious CyHV‐3 was produced stably in CCF‐K104 cells over 30 viral passages. Our findings showed that CCF‐K104 is a useful cell line for isolation and productive replication of CyHV‐3. A temperature shift from 25 °C to 15 °C or 35 °C did not allow serial morphological changes as observed at 25 °C for 14 days. Under the same conditions, real‐time PCR showed that CyHV‐3 was present with low viral DNA loads, suggesting that CyHV‐3 may establish latent infection in CCF‐K104 cells. Amplification of the left and right terminal repeat sequences of the CyHV‐3 genome arranged in a head‐to‐tail manner was detected by nested PCR following an upshift in temperature from 25 °C to 35 °C. The PCR results suggested that the circular genome may represent a latent form of CyHV‐3.     

Validation of multi-frequency inversion method by using dummy scatterers of zooplankton

ABSTRACT:   High- and multi-frequency acoustic measurement systems and the multi-frequency inversion (MFI) method have been used to measure spatial distributions and abundances of zooplankton by size. In this study, the calibration method for high- and multi-frequency systems was developed and the validation of MFI method was carried out by scatterer measurement. The standard sphere calibration method that has not been applied to such high- and multi-frequencies was applied to calibrate our high- and multi-frequency system, TAPS-6 (Tracor Acoustic Profiling System, BAE Systems). An optimum size of standard sphere of tungsten carbide of 1 mm radius was derived to have a small target strength variation for the six frequencies of TAPS-6, and the practicability and precision of the standard sphere calibration method was confirmed for those frequencies. A school or cluster of dummy scatterers of zooplankton with small tungsten carbide spheres were designed to validate the MFI method, and volume back-scattering strength values were measured by the multi-frequency system. By comparing the result of the inversion with their real composition, the features of the MFI method could be validated and examined.     

Genetic and phylogenetic analysis of glycoprotein of rabies virus isolated from several species in Brazil

Genetic and phylogenetic analyses of the region containing the glycoprotein (G) gene, which is related to pathogenicity and antigenicity, and the G-L intergenic region were carried out in 14 Brazilian rabies virus isolates. The isolates were classified as dog-related rabies virus (DRRV) or vampire bat-related rabies virus (VRRV), by nucleoprotein (N) analysis. The nucleotide and amino acid (AA) homologies of the area containing the G protein gene and G-L intergenic region were generally lower than those of the ectodomain. In both regions, nucleotide and deduced AA homologies were lower among VRRVs than among DRRVs. There were AA differences between DRRV and VRRV at 3 antigenic sites and epitopes (IIa, WB+ and III), suggesting that DRRV and VRRV can be distinguished by differences of antigenicity. In a comparison of phylogenetic trees between the ectodomain and the area containing the G protein gene and G-L intergenic region, the branching patterns of the chiropteran and carnivoran rabies virus groups differed, whereas there were clear similarities in patterns within the DRRV and VRRV groups. Additionally, the VRRV isolates were more closely related to chiropteran strains isolated from Latin America than to Brazilian DRRV. These results indicate that Brazilian rabies virus isolates can be classified as DRRV or VRRV by analysis of the G gene and the G-L intergenic region, as well as by N gene analysis.     

Ripening-related defense proteins in Annona fruit

In order to obtain a better understanding of the active defense strategy of cherimoya (Annona cherimola Mill.) fruit, hydrolytic and antifungal activity, as well as expression of proteins functionally and immunogenically related to the pathogenesis-related proteins chitinase (PR-Q) and 1,3-β-glucanase (PR-2), were estimated in fruit at different ripening stages. Increase in expression of the 27 kDa constitutive chitinase and the induction of two new proteins, a 26 kDa chitinase and a 51 kDa 1,3-β-glucanase were associated with enhanced in vitro hydrolytic and antifungal activity of the acidic protein extract in ripe fruit. Ripening modified the expression of constitutive basic isoenzymes, with a sharp decrease in both relative accumulation and hydrolytic activity. Likewise, a new basic 33 kDa chitinase was induced in the over-ripe fruit, concomitant with accumulation of a basic constitutive 76 kDa 1,3-β-glucanase. At this stage, the basic protein extract modified in vitro growth inhibition of Botrytis cinerea. Short-term high CO₂ treatment delayed fruit ripening and maintained a similar distribution of activity and isoenzymatic pattern in both protein fractions to that in unripe fruit. These results indicate that the changes in the pattern of defense proteins and hydrolytic activity in cherimoyas appear to be associated with ripening. Moreover, unlike the constitutively expressed isoenzymes, only the transitorily induced chitinases and 1,3-β-glucanases were associated with an active defense-related response.     

Chicken peripheral blood CD3+CD4-CD8- cells are regulated by endocrine and nerve systems

The existence of CD3(+)CD4(-)CD8(-) T cells in thymus and spleen has already been known. However, because of the presence of large amounts of thrombocytes in peripheral blood (PB), the proportion of CD3(+)CD4(-)CD8(-) T cells in PB has yet to be investigated. Therefore, the proportion of peripheral T cell-subsets was investigated in 6-week-old chickens. The percentage of CD3(+) cells, CD4(+) cells, CD8 alpha(+) cells, CD8 beta(+), and CD3(+)CD4(-)CD8(-) cells was 76%, 41%, 14%, 5%, and 15%, respectively. The proportion of CD3(+)CD4(-)CD8(-) cells in PB increased during egg-laying periods and in chickens treated with an analog of estrogen, while it decreased with age and in response to restraint stress. All of the CD3(+)CD4(-)CD8(-) cells expressed TCR1, and did not have NK activity. CD3(+)CD4(-)CD8(-) cells represent about 60% of peripheral TCR1(+) cells. These findings indicate that the proportion of CD3(+)CD4(-)CD8(-) cells is regulated by the endocrine and nerve systems.     

Gentian Kobu-sho-associated virus: a tentative, novel double-stranded RNA virus that is relevant to gentian Kobu-sho syndrome

A virus-like dsRNA of about 23 kbp was detected in gentian plants showing Kobu-sho syndrome including stunting, shortened internodes, and tumors on stems, nodes and roots. Nucleotide sequence analysis has suggested that this dsRNA is related to Pestivirus species but not to any other plant viruses. It was protected from externally added RNase, suggesting that the dsRNA is encapsidated. The dsRNA was co-extracted in a crude homogenate of glutaraldehyde-fixed tissue with the virus-like particles that have been associated previously with Kobu-sho syndrome in gentian (Usugi et al. Jpn J Phytopathol 76:21–24, 2010). The RNA sequence was detected in more than 99 % of Kobu-sho gentian individuals but in less than 20 % of apparently healthy gentian individuals from fields affected with Kobu-sho. Thus, we propose naming the virus Gentian Kobu-sho-associated virus.     

Molecular epidemiological analysis of bat rabies viruses in Brazil

A molecular epidemiological analysis was performed in 19 rabies viruses (RVs) isolated from haematophagous, frugivorous and insectivorous bats, in Sao Paulo, Brazil. The authors carried out RT-PCR for amplification of the RV nucleoprotein (N) gene, and determined 1,335 nucleotide sequences of N gene by direct sequencing method. Phylogenetic analysis, which was based on the N gene of Brazilian RV isolates identified presently and previously, revealed that RVs isolated from bats were genetically divided into four lineages had a tendency to depend on the host bat species. The first lineage consisted mainly of haematophagous bat (Desmodus rotundus) isolates, including frugivorous bat (Artibeus spp.) isolates. Other three lineages consisted of insectivorous bat isolates; mainly Eptesicus spp., Molossus spp. and Nyctinomops spp. isolates, respectively. These results indicate a possibility of that there are bat species-specific RV variants in Brazil.     

不同光质对嫁接砧木下胚轴生长的影响

近年随着蔬菜生产规模化、工厂化发展的不断扩大和蔬菜嫁接技术的广泛推广,研制蔬菜自动嫁接机已成为蔬菜工厂化生产发展的必然趋势.而要实现嫁接装备高效、精准完成蔬菜幼苗的自动化嫁接作业,首先必须克服蔬菜幼苗生长的不一致性,因此培育健壮、整齐、均匀一致的嫁接蔬菜用苗也是实现自动化嫁接作业的一个重要前提.蔬菜幼苗的生长发育是一个十分复杂的过程,而光源是影响植物生长的一个重要因素.为此,对不同光源(红、蓝、红蓝)照射对3种瓜科砧木幼苗下胚轴生长的影响进行了研究.研究结果表明,红光处理的幼苗下胚轴最高,茎较细;红蓝光处理的砧木幼苗下胚轴最高,茎较粗.对于嫁接砧木幼苗,红光处理具有光合活性,有利于砧木幼苗积累的能量,促进幼苗下胚轴的生长.     

Antimicrobial activity of Viola tricolor herb

The antimicrobial activity of infusion, decoction, ethanol extract and fractions obtained by successive extraction of Viola tricolor herb with dichloromethane, ethyl acetate and methanol was evaluated. The infusion, decoction and ethanol extract were found to be most effective against the tested microorganisms.