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Baculoviruses are insect pathogens that are characterized by assembling the viral dsDNA into two different enveloped virions during an infective cycle: occluded virions (ODVs; immersed in a protein matrix known as occlusion body) and budded virions (BVs). ODVs are responsible for the primary infection in midgut cells of susceptible larvae thanks to the per os infectivity factor (PIF) complex, composed of at least nine essential viral proteins. Among them, P74 is a crucial factor whose activity has been identified as virus-specific. In this work, the p74 gene from AcMNPV was pseudogenized using CRISPR/Cas9 technology and then complemented with wild-type alleles from SeMNPV and HearSNPV species, as well as chimeras combining the P74 amino and carboxyl domains. The results on Spodoptera exigua and Rachiplusia nu larvae showed that an amino terminal sector of P74 (lacking two potential transmembrane regions but possessing a putative nuclear export signal) is sufficient to restore the virus infectivity whether alone or fused to the P74 transmembrane regions of the other evaluated viral species. These results provide novel information about the functional role of P74 and delimit the region on which mutagenesis could be applied to enhance viral activity and, thus, produce better biopesticides.

Vi-rút gâybệnh đốm trắng (WSSV) là một trong những vi-rút gây bệnh nguy hiểm trên tôm và là mối đe dọa lớn cho nghề nuôi trồng thủy sản.Tuy nhiên, chưa có biện pháp hữu hiệu để điều trị bệnh đốm trắng. Hiện nay nhiều nghiên cứu chỉ ra rằng protein VP28 và VP19 là những protein giúp tạo vắc-xin protein đề kháng lại sự xâm nhiễm của WSSV cho tôm. Phương thức lan truyền bệnh qua đường miệng (peroral infectivity) luôn luôn cần sự hiện diện của các nhân tố truyền bệnh qua đường miệng (peroral infectivity factor ? PIFs). Các nhân tố này không chỉ hiện diện ở Baculovirus mà hiện diện trên hầu hết các loài vi-rút có vật chất di truyền là ADN của động vật không xương sống. Những nhân tố truyền bệnh này có thể là những yếu tố thay thế tiềm năng trong quá trình can thiệp miễn dịch giúp động vật kháng lại sự nhiễm bệnh. Trong nghiên cứu này, protein tái tổ hợp được biểu hiện trong tế bào vi khuẩn E. coli bằng cách nối gen mã hóa cho protein P74 (WSSV ORF 72) trên WSSV với vector biểu hiện pET28?. Để thu được lượng protein nhiều nhất cho quá trình tinh sạch tiếp theo, mức độ biểu hiện của protein được chuẩn hóa qua các thông số khác nhau để đạt được mức độ cao nhất. Sau khi tinh sạch, protein P74 được gửi đi tạo kháng thể đa dòng kháng lại protein P74 của WSSV trên thỏ. Protein WSSV-P74 có kích thước là 108 kDa. Trong thời gian tạo kháng thể, WSSV được tăng sinh trên tôm thẻ chân trắng Penaeus vannamei (P. vannamei). Dịch chiết vi-rút được sử dụng cho các thí nghiệm nhận diện và xác định vị trí của protein P74 trên WSSV. Kết quả đã xác định sự hiện diện của protein P74 trên WSSV. Mặc dù vị trí và chức năng của protein cần nghiên cứu thêm nhưng kết quả này cũng tạo tiền đề cho các nghiên cứu kế tiếp trong việc tạo ra các loại vắc-xin protein hiệu quả trong việc phòng bệnh đốm trắng trên tôm.

Baculovirus occlusion-derived viruses (ODVs) and budded viruses (BVs) are morphologically and functionally distinct. ODVs are responsible for primary infection in insect hosts because of their high per os infectivity. On the contrary, BVs poorly infect endothelial gut cells, but propagate the infection in the tissues of insects with a high efficiency. P74 is one of the most important proteins from ODVs, and it participates in the attachment of this viral phenotype to endothelial cells in the midgut. We evaluated the possibility of pseudotyping BVs of Autographa californica multiple nucleopolyhedrovirus with two versions of P74 and its effect on their oral infectivity. Both recombinant BVs contained P74 and replicated similarly to wild-type viruses. Nevertheless, the presence of P74 on the BV's surface does not enhance the oral infectivity of this phenotype, suggesting that the presence of P74 in the membrane of budded virions interferes with their mechanism of infecting midgut cells. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: Dione juno and Agraulis vanillae are very common butterflies in natural gardens in South America, and also bred worldwide. In addition, larvae of these butterflies are considered as pests in crops of Passiflora spp. For these reasons, it is important to identify and describe pathogens of these species, both for preservation purposes and for use in pest control. Baculoviridae is a family of insect viruses that predominantly infect species of Lepidoptera and are used as bioinsecticides. Larvae of D. juno and A. vanillae exhibiting symptoms of baculovirus infection were examined for the presence of baculoviruses by PCR and transmission electron microscopy. Degenerate primers were designed and used to amplify partial sequences from the baculovirus p74, cathepsin, and chitinase genes, along with previously designed primers for amplification of lef-8, lef-9, and polh. Sequence data from these six loci, along with ultrastructural observations on occlusion bodies isolated from the larvae, confirmed that the larvae were infected with nucleopolyhedroviruses from genus Alphabaculovirus. The NPVs from the two different larval hosts appear to be variants of the same, previously undescribed baculovirus species. Phylogenetic analysis of the sequence data placed these NPVs in Alphabaculovirus group I/clade 1b. [ABSTRACT FROM AUTHOR]

Abstract: To develop complementary baculovirus-based tools for gene delivery and display technologies, the interaction of occlusion-derived baculovirus (ODV) with human cells, and the functionality of the P74 ODV envelope protein for display of the IgG-binding Z domains (ZZP74) were evaluated. The cellular binding of ODV was concentration-dependent and saturable. Only minority of the bound virions were internalized at both 37 and 4°C, suggesting usage of direct membrane fusion as the entry mode. The intracellular transport of ODV was confined in vesicular structures peripheral to the plasma membrane, impeding subsequent nuclear entry and transgene expression. Transduction of ODV was not rescued by mimicking the preferred alkaline environment and lowered temperature of the ODV infective entry, or following treatment with the microtubule depolymerizing agent nocodazole or with the histone deacetylase inhibitor sodium butyrate. Similar to unmodified P74, the ZZP74 chimera localized in the intranuclear ring zone, and was enriched in virus-induced microvesicles. However, Western blotting of ODV and budded virions (BV), as well as viral envelope and nucleocapsid fractions combined with functional infection/transduction studies revealed incorporation of the ZZP74 fusion protein into viral nucleocapsids. The ZZP74 BV preserved normal infectivity, polypeptide profile, and morphology, but became incapable of entering and transducing human cells. [Copyright &y& Elsevier]

P74 is a protein encoded in the genome of baculoviruses, associated with the envelopes of occluded virus. Its presence proved to be essential for per os infection. In first place, in this work we designed two universal primers to amplify a sequence region of the p74 ORF in baculoviruses from different classification groups. Then, by the use of these amplicons we obtained the complete sequence of the p74 locus from two isolates of AgMNPV, 2D (Brazil) and SF (Argentina). In the flanking regions we determined the complete sequence of p10 gene and a portion of p26 gene. Comparing both p74 sequence data (ORFs of 1935 bp) we found fifteen nucleotide changes that result in six amino acid changes. Comparisons of AgMNPV p74s with other baculovirus homologous genes indicate a close relationship with other group I Nucleopolyhedrovirus, in particular CfDEFNPV. These results were based on ORF sequence, amino acid sequence and gene order. The predictive studies about secondary structure and hydrophobic index point at six regions potentially associated to its function or native conformation. Finally, the detection of p74 mRNA after virus DNA replication confirms a late expression pattern. [ABSTRACT FROM AUTHOR]

In order to study the function of the envelope protein P74 of Heliothis armigera single nucleocapsid nucleopolyhedrovirus (HaSNPV), a p74-null recombinant baculovirus, rHa-gfΔp74, was constructed by inserting gfp driven by the polyhedrin promoter into the p74 locus of HaSNPV genome. The resulting p74-inactivation occlusion-derived viruses (ODV) failed to infect its natural host larvae per os. However, its inability of oral infectivity was rescued using the purified P74 protein expressed by Bac-to-Bac system in Hz-AM1 cells. Feeding the purified P74 protein along with the p74 deletion mutant virus rHa-gfpΔp74 to H. armigera larvae resulted in the rescue of oral infectivity in a dose dependent manner. The P74 protein was expressed in-frame with GFP to create a P74-GFP chimera for studying the localization of P74, the GFP portion of the chimera facilitating the visualization of the trafficking of P74 in cells. The P74-GFP chimeric proteins localized in the intranuclear ring zone and accumulated into microvesicles. In addition, the specific and saturable binding of P74 protein to its host brush border membrane vesicles (BBMVs) was involved in the invasion of virus. Further investigations (pull-down assay) showed that an about 30 kDa protein in the BBMVs was involved in the specific binding. These results demonstrated that the P74 protein is essential for oral infectivity of ODV and plays a role in midgut attachment and fusion. [Copyright &y& Elsevier]

This article deeply researched into the existent five problems and four main “bottlenecks” in the high-technologization of marine industry in China on the basis of analyzing the new trends in international marine problems and the necessity of implementing the strategy of developing China based on marine. This article brought up specific measures to the five “bottlenecks” according to the situations, and pointed out that new marine industry should be high-technologization and the traditional marine industry should be reformed by high-technique. The research results may provide the scientific basis for realizing the high-technologization of marine industry in China. [ABSTRACT FROM AUTHOR]

In order to investigate the genomic organization of the Trichoplusia ni Single Capsid Nucleopolyhedrovirus (TnSNPV), a 2,966 basepairs (bp) genomic fragment was sequenced. The fragment was found to contain five open reading frames (ORFs) homologous to baculovirus genes, including p26, fibrillin ( p10), AcMNPV ORF-29, late expression factor 6 ( lef-6) and the C-terminal portion of p 74, on either strand of DNA. Predicted amino acid sequences for the ORFs were compared and identity values of between 12% and 54% were observed. TnSNPV has previously been tentatively identified as a member of the Group II NPVs. Clustering and arrangement of the TnSNPV genes were similar to the clustering reported for SeMNPV, confirming TnSNPV as a Group II NPV. [ABSTRACT FROM AUTHOR]

Baculoviruses are insect pathogens that are characterized by assembling the viral dsDNA into two different enveloped virions during an infective cycle: occluded virions (ODVs; immersed in a protein matrix known as occlusion body) and budded virions (BVs). ODVs are responsible for the primary infection in midgut cells of susceptible larvae thanks to the per os infectivity factor (PIF) complex, composed of at least nine essential viral proteins. Among them, P74 is a crucial factor whose activity has been identified as virus-specific. In this work, the p74 gene from AcMNPV was pseudogenized using CRISPR/Cas9 technology and then complemented with wild-type alleles from SeMNPV and HearSNPV species, as well as chimeras combining the P74 amino and carboxyl domains. The results on Spodoptera exigua and Rachiplusia nu larvae showed that an amino terminal sector of P74 (lacking two potential transmembrane regions but possessing a putative nuclear export signal) is sufficient to restore the virus infectivity whether alone or fused to the P74 transmembrane regions of the other evaluated viral species. These results provide novel information about the functional role of P74 and delimit the region on which mutagenesis could be applied to enhance viral activity and, thus, produce better biopesticides. [ABSTRACT FROM AUTHOR]