Mutation of the first ATG ORF-3 of Chicken Anemia Virus into ACG completely abolish the apoptin production

To test the effect of one point mutation on the first initiation codon of Chicken Anemia Virus (CAV) open reading frame-3 (ORF-3), an apoptin knocked out expressor plasmid pCLS-VP3(-) and a CAV apoptin knocked out plasmid pCAV/Ap(-) were constructed. In both plasmids, the first ATG in ORF-3 was mutated into ACG by a reversed long PCR. No apoptin detected in COS-1 cells transfected with pCLS-VP3(-) using western blotting and immunofluorescence assay, while apoptin was detected in COS-1 cells transfected with pCLS wild type. After released from pCAV/Ap(-), the complete genome of CAV/Ap(-) was ligated to form the replicative form. The apoptin production was completely abolished in MDCC-MSB1 cells transfected with replicative form of CAV/Ap(-). The apoptin production was fully regained after a reverse mutation into CAV/Ap(-)RM. These data shows the first evidence that mutation of the first ATG of ORF-3 into ACG could completely abolish the production of apoptin. Key words: Chicken Anemia Virus, Circovirus, Gyrovirus, Apopti

Prevalence of Anti-Borna Disease Virus Antibody in Horses and Their Caretakers in Bangladesh

To elucidate the spread of Borna disease virus (BDV) in Asian countries, we surveyed 48 normal horses in Bangladesh and their 26 caretakers for the BDV antibody by electrochemiluminescence immunoassay. Eleven horses (23%) were found positive. None of the 5 horses at the age of < 1 year was positive. Seven of 23 horses (30%) at the age of 1 year were positive, as well as 4 of 16 horses (25%) at the age of 3 years. The geometric average of the ECLIA titer of the antibody positive horses at the age of 1 year, 3041, was significantly lower than that found at the age of 3 years, 6887, by the Mann-Whitney test (P = 0.012). Sexual preference in the prevalence of anti-BDV was not evident. None of the 26 male horse caretakers between the ages of 12 to 54 years was positive, including those who were taking care of the antibody positive horses. Total RNA extracted from the peripheral blood nucleated cells was tested by polymerase chain reaction coupled with reverse transcription capable of detecting 200 molecules of BDV p40 RNA per reaction. None of the 11 seropositive horses and the 5 randomly selected seronegative horses was positive. The results showed that BDV is penetrating the Bangladeshi labor horse population with similar levels reported in Germany, Iran and Japan, although the viral genome in the blood was not detected

Three Spliced mRNAs of TT Virus Transcribed from a Plasmid Containing the Entire Genome in COS1 Cells

A permuted whole-genome construct of a TT virus (TTV), named VT416, had 3,852 nucleotides (nt) 98.2% similar to the prototype TA278 genome. To allow the transcription of TTV from the internal promoter, pBK*VT416(1.3G), carrying 1.3 units of VT416, was constructed. The poly(A)(+) RNAs expressed in COS1 cells 48 h posttransfection contained three TTV mRNA species 3.0, 1.2, and 1.0 kb in length, which were recovered in the 13 DNA clones from a λ phage cDNA library. These mRNAs in the antigenomic orientation possessed in common the 3′ terminus downstream of a poly(A) signal (A(3073)ATAAA) and the 5′ terminus downstream of a cap site (C(98)ACTTC). A common splicing to join nt 185 with nt 277 was detected in all mRNAs. The coding region of the largest open reading frame (ORF) was maintained in 3.0-kb mRNA, because this splicing was located upstream of its initiation codon (A(589)TG). The second splicing was detected in 1.2-kb mRNA to join nt 711 with nt 2374 and in 1.0-kb mRNA to bind nt 711 to nt 2567. They linked a proposed ORF2 to another ORF for creating new ORFs over nt 2374 to 2872 in frame 2 and nt 2567 to 3074 in frame 3. The donor and acceptor sites of all three splicings matched the consensus sequence and were conserved in most of the 16 TTVs of distinct genotypes retrieved from the database. The observed transcription profile is unique to TTV among known members in the family Circoviridae

Replication of chicken anemia virus (CAV) requires apoptin and is complemented by VP3 of human torque teno virus (TTV)

AbstractTo test requirement for apoptin in the replication of chicken anemia virus (CAV), an apoptin-knockout clone, pCAV/Ap(−), was constructed. DNA replication was completely abolished in cells transfected with replicative form of CAV/Ap(−). A reverse mutant competent in apoptin production regained the full level of DNA replication. DNA replication and virus-like particle (VLP) production of CAV/Ap(−) was fully complemented by supplementation of the wild-type apoptin. The virus yield of a point mutant, CAV/ApT108I, was 1/40 that of the wild type, even though its DNA replication level was full. The infectious titer of CAV was fully complemented by supplementing apoptin. Progeny virus was free from reverse mutation for T108I. To localize the domain within apoptin molecule inevitable for CAV replication, apoptin-mutant expressing plasmids, pAp1, pAp2, pAp3, and pAp4, were constructed by deleting amino acids 10–36, 31–59, 59–88 and 80–112, respectively. While Ap1 and Ap2 were preferentially localized in nuclei, Ap3 and Ap4 were mainly present in cytoplasm. Although complementation capacity of Ap3 and Ap4 was 1/10 of the wild type, neither of them completely lost its activity. VP3 of TTV did fully complement the DNA replication and VLP of CAV/Ap(−). These data suggest that apoptin is inevitable not only for DNA replication but also VLP of CAV. The common feature of apoptin and TTV-VP3 presented another evidence for close relatedness of CAV and TTV

The Organization of a Summer Camp for Children

Cílem práce je zpracovat metodický materiál pro vedoucí a pořadatele táborů a dětských ozdravných akcí. Je zde zpracován přesný postup při přípravě a samotné organizaci dětského tábora. V teoretické části se práce zabývá takovými pojmy, jako je volný čas, výchova mimo vyučování a střediska volného času. V praktické části podrobně popisuji, jak lze optimálně postupovat při přípravě a organizaci tábora. Získané informace by měly posloužit jako metodická pomoc pro všechny vedoucí a pořadatele dětských táborů. Klíčová slova: Dětský tábor, zotavovací akce, hlavní vedoucí, zdravotník, volný čas, Dům dětí a mládeže, výchova mimo vyučování, bezpečnost práceCílem práce je zpracovat metodický materiál pro vedoucí a pořadatele táborů a dětských ozdravných akcí. Je zde zpracován přesný postup při přípravě a samotné organizaci dětského tábora. V teoretické části se práce zabývá takovými pojmy, jako je volný čas, výchova mimo vyučování a střediska volného času. V praktické části podrobně popisuji, jak lze optimálně postupovat při přípravě a organizaci tábora. Získané informace by měly posloužit jako metodická pomoc pro všechny vedoucí a pořadatele dětských táborů. Klíčová slova: Dětský tábor, zotavovací akce, hlavní vedoucí, zdravotník, volný čas, Dům dětí a mládeže, výchova mimo vyučování, bezpečnost práceÚstav profesního rozvoje pracovníků ve školstvíPedagogická fakultaFaculty of Educatio

Molecular Epidemiology of a Hepatitis C Virus Outbreak in a Leprosy Sanatorium in Japan▿

The hepatitis C virus (HCV) outbreak that occurred between 1940 and 1999 in a closed leprosy sanatorium located on a small island in Japan was analyzed. The analysis of 318 nucleotides in the NS5B region of HCV allowed us to establish the existence of at least three different HCV strains in this sanatorium

Additional file 5: of Finding the superior allele of japonica-type for increasing stem lodging resistance in indica rice varieties using chromosome segment substitution lines

Table S3. Heading date (date after sowing) of parent lines and reciprocal CSSLs of chromosome 5 in 2016. (DOCX 19 kb