A glycoprotein I- and glycoprotein E-deficient mutant of infectious laryngotracheitis virus exhibits impaired cell-to-cell spread in cultured cells

Summary. In alphaherpesviruses, glycoprotein I (gI) and glycoprotein E (gE) form a heterodimer that functions in cell-to-cell spread of the virus. Generally, alphaherpesvirus mutants that lack these glycoproteins are replication competent in cell culture but show a reduced capacity for cell-to-cell spread and hence smaller plaque sizes. Infectious laryngotracheitis virus (ILTV), or Gallid herpesvirus 1, is an alphaherpesvirus that causes respiratory disease in chickens. The roles of gI and gE in ILTV have not been investigated previously. In this study, a glycoprotein I and glycoprotein E deletion mutant of ILTV (gI/gE-ve ILTV) was generated by replacing the region of the ILTV genome coding for the adjacent gI and gE genes with the gene for enhanced green fluorescent protein (eGFP). This gI/E-ve ILTV was readily propagated in cell culture in the presence of wildtype ILTV (wt ILTV). However, in the absence of wt ILTV the propagation of gI/gE-ve ILTV was severely impaired. Infection of permissive cell cultures with gI/gE-ve ILTV failed to produce plaques but single infected cells could be identified by fluorescence microscopy. This suggests that gI/gE has a more significant role in the cell-to-cell spread of ILTV in vitro than in many other alphaherpesviruses.     

Infectious laryngotracheitis virus growth,DNA replication,and protein synthesis

Summary The polypeptides associated with infection of primary chicken kidney (CK) cells with infectious laryngotracheitis virus (ILTV) were examined by metabolic labelling with [³⁵S]methionine and SDS-PAGE. Polypeptide synthesis was followed over the first 24 h post-infection (p.i.) as this was shown to be the period of viable virus production. A total of 16 ILTV encoded or induced polypeptides were identified using metabolic labelling. The use of inhibitors of protein and DNA synthesis in conjunction with metabolic labelling and viral DNA replication studies enabled a cascade pattern of gene expression, similar to that of herpes simplex virus type 1 (HSV-1), to be established for ILTV. Representatives of alpha, beta, gamma 1 and gamma 2 classes of genes were identified. In contrast to infection with HSV types 1 and 2, which leads to a rapid inhibition of total host cell polypeptide synthesis, ILTV infection of CK cells did not result in a complete inhibition of cellular protein synthesis, with only a small number of host cell polypeptides absent from infected cells.     

Strand specific quantitative real-time PCR to study replication of hepatitis C virus genome

Qualitative detection of negative hepatitis C virus (HCV) RNA has been used widely to demonstrate HCV replication. However, relative quantitation of both positive and negative HCV RNA strands has never been reported for studying viral genome replication. A strand specific real-time PCR carried out in the highly conserved 5'-non-coding region of HCV genome and monitored either by the DNA binding dye SYBR Green I or by molecular beacons is described. Using these techniques, it was found that negative HCV RNA strand was a 100-1000 times less abundant than the positive strand in the liver of HCV infected patients.     

Reverse restriction fragment length polymorphism (RRFLP): A novel technique for genotyping infectious laryngotracheitis virus (ILTV) live attenuated vaccines

A novel technique, the reverse restriction fragment length polymorphism (RRFLP) assay, was developed as a means of detecting specific informative polymorphic sites in the infectious laryngotracheitis virus (ILTV) genome. During the RRFLP procedure, DNA is digested with restriction enzymes targeting an informative polymorphic site and then used as template in a real-time polymerase chain reaction (PCR) with primers flanking the informative region. The analysis of the ΔC_t values obtained from digested and undigested template DNA provides the genotype of the DNA. In this study, the RRFLP assay was applied as a method to differentiate between the two types of infectious laryngotracheitis virus attenuated live vaccines. Sequence analysis of ILTV vaccines revealed an informative polymorphic site in the 5′-non-coding region of the infected cell protein (ICP4) gene. Unique AvaI and AlwI restriction enzyme sites were identified in the tissue culture origin and chicken embryo origin attenuated vaccines, respectively. These two informative polymorphic sites were used in a RRFLP assay to genotype rapidly and reproducibly ILTV attenuated live vaccines.     

Genome type analysis of Brazilian adenovirus strains of serotypes 1,2,3,5, and 7 collected between 1976 and 1995.

A collection of 92 epidemiologically unrelated isolates of Ad1 (n = 14), Ad2 (n = 29), Ad3 (n = 19), Ad5 (n = 16), and Ad7 (n = 14) collected in the cities of Belem do Pará (1 degree S 48 degrees W) and Rio de Janeiro (23 degrees S 43 degrees W) between 1976 and 1995 from patients with respiratory disease and conjunctivitis were characterized by restriction enzyme analysis of genomic DNA. Among the strains of subgenus B, two different genome types of serotype 7, 7b and 7e, were identified. The analysis of their temporal distribution throughout the study period suggested an alternating appearance of these two DNA variants. Only one genome type of Ad3, 3p, was detected during the sampling period. Further analysis with Xba I, Bcl I, and Hpa I indicated that it is a p1-like genome type. Both previously described and new genomic variants were identified among subgenus C strains. Genome types D1, D7, D10, and one not previously described were identified among the 14 Ad1 strains analyzed. Genome types D2, D5, D25, and 13 new DNA variants were identified among the 29 Ad2 isolates. Genome type D38 and 5 new variants were found among the 16 strains of Ad5. In spite of the relatively small size of the sample analyzed, the results of this study confirm the important genetic variability previously observed for members of subgenus C by other authors.     

Interactions of plus and minus strand leader RNAs of the New Jersey serotype of vesicular stomatitis virus with the cellular La protein

The New Jersey serotype of vesicular stomatitis virus (VSV-NJ) was found to synthesize a minus strand leader RNA of 44-46 bases long and a plus strand leader RNA of 47-50 bases long in infected cells. The minus strand leader RNA of VSV-NJ was found associated with the host cell La protein in infected cells by immunoprecipitation with antisera from patients with systemic lupus erythematosus. These results differ from those reported previously (J. Wilusz , M. G. Kurilla , and J. D. Keene (1983). Proc. Natl. Acad. Sci. USA 80, 5827-5831) for the similarly sized species of minus strand leader RNA made by the Indiana serotype of VSV (VSV-IND). Despite sequence differences between the 3' ends of the plus strand leader RNAs of the two serotypes, the plus strand leader RNA of VSV-NJ was found to have a pattern of La protein accumulation similar to that reported previously for the plus strand leader RNA of VSV-IND. These results provide additional support for a role for La protein in VSV replication and help further delineate the sequence requirements for La protein binding to VSV leader RNAs.     

Sequence polymorphism in the E3 7.7K ORF of subspecies B1 human adenoviruses

Sequences corresponding to the 7.7K open-reading frame (ORF) of the E3 region of subspecies B1 adenoviruses (Ads) were compared with prototype strains of Ad3, Ad7, Ad16, Ad21, and Ad50 and field isolates representing a variety of genome restriction types of Ad3 and Ad7 to better assess the extent of genetic variation in this intriguing region of the viral genome encoding a product whose function is still unknown. Alignment of 55 species B1 Ad sequences revealed a marked polymorphism in the 7.7K ORF and allowed the identification of eight distinct sequence profiles (SPs) characterized by (1) deletions that retain or change the reading frame, (2) single-base mutations (SBMs) that change the start codon (ATG to ATT or ATC), and (3) other SBMs. mRNAs of expected size for the observed sequence polymorphisms were identified by RT-PCR from DNAse I-treated total RNA extracts of infected cells. Predicted proteins ranged from 0 to 94 amino acids corresponding to molecular masses of 0-11 K. Together with the hypervariable regions of the hexon gene, the E3 7.7K ORF appears to be another area of the Ad genome in which genetic diversity may be generated by illegitimate recombination.     

Genetic susceptibility of chicken macrophages to in vitro infection with infectious laryngotracheitis virus

Chicken macrophages are susceptible to infection with infectious laryngotracheitis virus (ILTV), the level of infection being dependent, in part, on the genotype of the host cell. Following infection in vitro a greater proportion of macrophages from the ILTV-resistant J1(B113/113) and N1(B114/114) inbred lines of chickens were found to be positive for ILTV antigens, than macrophages from the ILTV-susceptible M1(B15/15) chickens. The proportion of ILTV-positive macrophages was found to be genetically regulated, in part by the chicken major histocompatibility complex (MHC), although alloantisera to class I and class II MHC antigens did not reduce the number of macrophages infected. Similarly, the phagocytic activity of the cultured macrophages from chickens of different MHC genotype did not correlate with their susceptibility to infection with ILTV.     

Biosynthesis of reovirus-specified polypeptides: the reovirus s1 mRNA encodes two primary translation products

Reovirus serotypes 1 (Lang strain) and 3 (Dearing strain) code for a hitherto unrecognized low-molecular-weight polypeptide of Mr approximately 12,000. This polypeptide (p12) was synthesized in vitro in L-cell-free protein synthesizing systems programmed with either reovirus serotype 1 mRNA, reovirus serotype 3 mRNA, or with denatured reovirus genome double-stranded RNA, and in vivo in L-cell cultures infected with either reovirus serotype. The synthesis of p12 in vivo was insensitive to actinomycin D, and occurred at similar times after infection as the previously identified reovirus encoded lambda, mu, and sigma polypeptides. Pulse-chase experiments in vivo, and the relative kinetics of synthesis of p12 in vitro, indicate that it is a primary translation product. Fractionation of reovirus mRNAs by velocity sedimentation and translation of separated mRNAs in vitro suggests that p12 is coded for by the s1 mRNA, which also codes for the previously recognized sigma 1 polypeptide. Synthesis of both p12 and sigma 1 in vitro in L-cell-free protein synthesizing systems programmed with denatured reovirus genome double-stranded RNA also suggests that these two polypeptides can be coded by the same mRNA species. The Mr approximately 12,000 polypeptide was not a detectable structural component of purified virions, and antiserum prepared against purified reovirions did not immunoprecipitate p12. It is proposed that the Mr approximately 12,000 polypeptide encoded by the S1 genome segment be designated sigma 1bNS, and that the polypeptide previously designated sigma 1 be renamed sigma 1a.     

基于转录组测序分析微重力环境下MC3T3-E1成骨细胞miRNA/mRNA表达谱及功能变化

目的探讨微重力环境对MC3T3-E1成骨细胞分化的影响。方法采用转录组测序技术(RNA-seq)观察微重力培养前后MC3T3-E1成骨细胞miRNA/mRNA表达谱变化,测序结果采用q-PCR验证。采用生物信息学方法进一步研究差异性表达的miRNA/mRNA。结果与对照组(CON)相比,模拟微重力组(SMG)共有160条miRNA和1 912个mRNAs发生显著改变;根据生物信息学结果,筛选出10个关键性基因(3条miRNA、7个mRNA),其中miR-9_6666-5p为微重力敏感miRNA,可能在微重力环境下成骨细胞分化过程中起到重要的作用。结论在微重力环境下,成骨细胞分化受到抑制可能与miRNA/mRNA表达谱的改变有关。研究结果将有助于深入理解miRNA与mRNA在微重力环境下调控成骨分化与骨生成的分子机制。