Xunzhang Wang

Autographa californica multiple nucleopolyhedrovirus ac66 is required for the efficient egress of nucleocapsids from the nucleus, general synthesis of preoccluded virions and occlusion body formation.

Although orf66 (ac66) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is conserved in all sequenced lepidopteran baculovirus genomes, its function is not known. This paper describes generation of an ac66 knockout AcMNPV bacmid mutant and analyses of the influence of ac66 deletion on the virus replication in Sf-9 cells so as to determine the role of ac66 in the viral life cycle. Results indicated that budded virus (BV) yields were reduced over 99% in ac66-null mutant infected cells in comparison to that in wild-type virus infected cells. Optical microscopy revealed that occlusion body synthesis was significantly reduced in the ac66 knockout bacmid-transfected cells. In addition, ac66 deletion interrupted preoccluded virion synthesis. The mutant phenotype was rescued by an ac66 repair bacmid. On the other hand, real-time PCR analysis indicated that ac66 deletion did not affect the levels of viral DNA replication. Electron microscopy revealed that ac66 is not essential for nucleocapsid assembly, but for the efficient transport of nucleocapsids from the nucleus to the cytoplasm. These results suggested that ac66 plays an important role for the efficient exit of nucleocapsids from the nucleus to the cytoplasm for BV synthesis as well as for preoccluded virion and occlusion synthesis.

vlf-1 Deletion Brought AcMNPV to Defect in Nucleocapsid Formation

Recent studies have provided direct evidence that the baculovirus very late factor 1 (VLF-I) of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) was essential for BV production. To elucidate how vlf-1 deletion blocks BV production we generated a vlf-1 knockout bacmid by ET-recombination technology on AcMNPV bacmid propagated in Escherichia coli. Bacmid DNA transfection and supernatant passage assay revealed that the vlf-1 knockout bacmid was unable to replicate in cell culture, while vlf-1 repair bacmid, which was generated by transposition of the vlf-1 ORF under control of its native promoter into polyhedrin gene locus of vlf-1 knockout bacmid, resumed viral replication ability at wildtype levels. Results of these assays proved the correct construction of the vlf-1 knockout bacmid. Subsequent electron microscopy revealed that the vlf-1 knockout bacmid failed to form nueleocapsid in the nuclei of the transfected cells. Instead, intensely electron-dense virogenic stroma characteristic of viral DNA synthesis were observed. Thus, it is demonstrated for the first time that vlf-1 knockout blocked nucleocapsid formation and the defective nucleocapsid formation resulted in the abolishment of BV and ODV production. Possible roles of vlf-1 in genome processing are suggested and discussed.

Genome-Based Phylogeny of Poxvirus

A comprehensive phylogenetic analysis of Poxviridae was performed in this study. Phylogenetic trees were reconstructed from whole genomic information including gene order, gene content, and all conserved gene protein sequences. Gene content and gene order were the methods used for the first time in cladogram reconstruction of poxvirus. The results of the different methods finally shared mainly consistent tree topologies, which were supported by the classic taxonomy of Poxviridae. According to these topologies, the Entomopoxvirinae and Chordopoxvirinae subfamilies are divergent from each other. Subfamily Chordopoxvirinae could be divided into four main groupings, which could be further divided into subgroups. Parapoxvirus and Molluscipoxvirus have been considered distinct from other Chordopoxvirus. Results from our study consistently supported this suggestion. In our results, Parapoxvirus and Molluscipoxvirus were the most closely related genera and formed a group. This group could be the one that branched after the divergence of Avipoxviruses. Additionally, rabbitpox virus and vaccinia virus steadily formed clusters with high bootstrap support in all the cladograms.

Cladistic analysis of iridoviruses based on protein and DNA sequences.

Summary. Cladograms of iridoviruses were inferred from bootstrap analysis of molecular data sets comprising all published protein and DNA sequences of the major capsid protein, ATPase and DNA polymerase genes of members of the Iridoviridae family Iridovirus. All data sets yielded cladograms supporting the separation of the Iridovirus, Ranavirus and Lymphocystivirus genera, and the cladogram based on data derived from major capsid proteins further divided both the Iridovirus and Ranavirus genera into two groups. Tests of alternative hypotheses of topological constraints were also performed to further investigate relationships between infectious spleen and kidney necrosis virus (ISKNV), an unclassified fish iridovirus for which the complete genome sequence data is available, and other iridoviruses. Cladograms inferred and results of Shimodaira–Hasegawa tests indicated that ISKNV is more closely related to the Ranavirus genus than it is to the other genera of the family.

Short communication: PTreeRec: Phylogenetic Tree Reconstruction based on genome BLAST distance

Phylogenetic Tree Reconstruction (PTreeRec) is a web-based tool for automatic phylogeny inferences from whole-genome sequences, which accepts files of DNA sequences in the FASTA format and allows users to save the output tree file, and displays the inferred tree through an applet in a web browser. PTreeRec involves three basic steps. First, regions of maximal segment pairs (MSPs) based on an all-against-all pairwise comparison of genomes are located. Second, a distance matrix is calculated from MSP scores or coverage. Finally, a phylogenetic tree is reconstructed by the neighbor-joining method.

High-level expression, purification, and characterization of porcine somatotropin in Pichia pastoris

Porcine somatotropin (pST) significantly improves the growth rate, carcass composition, and growth efficiency of pigs while reducing feed consumption and fat deposition. Pichia pastoris was used as a host to efficiently express the pST gene in this study. Up to 90% of the recombinant protein was secreted into the culture medium, yielding about 900 mg/L rpST in shake-flask cultures. SDS-PAGE and Western blot analyses showed that rpST migrated as a single band with a molecular weight of approximately 25 kDa, and had the same immunoreactivity as native pST. The culture supernatant of our rpST expression strain, X-33/pPICZalphaA-pST/9, was purified to greater than 95% homogeneity with 71.4% recovery using ammonium sulfate precipitation, Sephadex G-25 Fine desalting, and Q Sepharose High Performance Ion Exchange chromatography. MALDI-TOF-MS demonstrated a molecular mass of 21,771Da for rpST, close to its predicted size. Isoelectric focusing electrophoresis results from three batches of purified rpST consistently showed a pI value between 4.55 and 5.2. Purified rpST was able to promote Nb2 cell proliferation and reduce feed intake of crossbred gilts, a type of pig breed, with no decrease in body weight gain when administered by injection. These results indicate that the P. pastoris expression system will be useful for production of bioactive rpST at commercially relevant levels.

Characterization of Ac M NPV with a deletion of me53 gene

The Autographa Californica multiple nucleopolyhedrovirus (AcMNPV) me53 gene, which was previously reported as one of the major early-transcribed genes, was deleted through homologous recombination from an AcMNPV genome propagated as a bacmid DNA in E. coli, generating a me53 gene knockout bacmid. Green fluorescent protein (GFP) expression analysis and supernatant passage assay revealed that the me53 knockout bacmid was unable to replicate in cell culture, while me53 repair bacmid, which was generated by reinsertion of the me53 gene into the mutant at a different locus (the gentamicin locus) with ET-recombination technique, resumed viral replication ability at wild-type levels, indicating that the defective phenotype of the me53 knockout virus was directly due to the deletion of the me53-ORF. Subsequent electron microscopy revealed that the me53 knockout bacmid failed to form nucleocapsid in the nuclei of the transfected cells, though viral infection seemed to be initiated. Meanwhile, real-time PCR analysis based on SYBR Green fluorescence indicated abolishment of the viral DNA replication by me53 gene inactivation. Thus, it is demonstrated for the first time that me53 knockout blocked viral DNA replication, nucleocapsid formation, and consequent BV and ODV production.

Ac109 is required for the nucleocapsid assembly of autographa californica multiple nucleopolyhedrovirus.

ORF109 (Ac109) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved gene in all sequenced baculovirus genomes, but its function is not known. This paper describes generation of an ac109 knockout virus (Ac-ac109-KO-GP) and analyses of the influence of ac109 deletion on the virus replication in Sf-9 cells so as to investigate the role of ac109 in the viral life cycle. Results revealed that budded virus (BV) yields and occlusion body synthesis were completely blocked in cells infected with the mutant virus. Electron microscopy demonstrated that ac109 deletion blocked nucleocapsid formation, though infection was initiated and electron-dense bodies associated with the virogenic stroma appeared. The mutant phenotype was rescued by an ac109 rescue virus. On the other hand, real-time PCR analysis indicated that ac109 is not required for viral DNA replication. Thus, these results suggested that ac109 plays an important role in AcMNPV nucleocapsid formation.

Serologic and genetic characterization analyses of a highly pathogenic influenza virus (H5N1) isolated from an infected man in Shenzhen.

Highly pathogenic avian influenza (HPAI) H5N1 virus caused a wave of outbreaks in China during 2005–2006, resulting in a total of 20 cases of human infection in 14 provinces of China. On June16, 2006, a case of H5N1 human infection was confirmed in Shenzhen. The virus isolated from the patient, A/Guangdong/2/06, was characterized genetically and the relationship between the tracheal virus load and the antibody titer of the infected man was analyzed. Serological analysis confirmed that the patients neutralizating antibodies had been generated 2 weeks after the onset of symptoms. The patients serum antibodies could efficiently neutralize A/Guandong/2/06 infectivity in vitro. Phylogenetic analysis showed that the H5N1 virus of Shenzhen belonged to subclade 2.3.4, which contained viruses that were mainly responsible for the outbreaks in domestic poultry and in the cases of human infection in southern China. Homology and molecular characterization analysis revealed that all the segments of Shenzhen H5N1 virus still belonged to avian segments. Several specific amino acid residue mutations were detected. J. Med. Virol. 80:1058–1064, 2008.

An efficient method for precise gene substitution in the AcMNPV genome by homologous recombination in E. coli

The RecA-mediated homologous recombination method was improved and used to direct gene replacement in baculoviruses. With this method, the p74 gene in the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) genome was substituted precisely by the p74 gene of Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV). In the recombinant bacmid, the AcMNPV p74 gene promoter controlled directly the expression of SpltMNPV p74 gene. Results of RT-PCR showed transcription of SpltMNPV p74 gene in the recombinant, implying the potential use of this easy and efficient method in baculovirus gene function research.