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Abstract: Autographa californica multiple nucleopolyhedrovirus (AcMNPV) p48 (ac103) is a highly conserved baculovirus gene of unknown function. In the present study, we generated a knockout of the p48 gene in an AcMNPV bacmid and investigated the role of P48 in baculovirus life cycle. The p48-null Bacmid vAcP48-KO-PH-GFP was unable to propagate in cell culture, while a ‘repair’ Bacmid vAcP48-REP-PH-GFP was able to replicate in a manner similar to a wild-type Bacmid vAcPH-GFP. Titration assays and Western blotting confirmed that vAcP48-KO-PH-GFP was unable to produce budded viruses (BVs). qPCR analysis showed that p48 deletion did not affect viral DNA replication. Electron microscopy indicated that P48 was required for nucleocapsid envelopment to form occlusion-derived viruses (ODVs) and their subsequent occlusion. Confocal analysis showed that P48 prominently condensed in the centre of the nucleus. Our results demonstrate that P48 plays an essential role in BV production and ODV envelopment in the AcMNPV life cycle. [Copyright &y& Elsevier]

During baculovirus infection, most viral proteins must be imported to the nucleus to support virus multiplication. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf34 ( ac34 ) is an alphabaculovirus unique gene that is required for optimal virus production. Ac34 distributes in both the cytoplasm and the nuclei of virus-infected Sf9 cells, but contains no conventional nuclear localization signal (NLS). In this study, we investigated the nuclear targeting domains in Ac34. Transient expression assays showed that Ac34 localized in both the cytoplasm and the nuclei of Sf9 cells, indicating that no viral protein is required for Ac34 nuclear localization. Subcellular localization analysis of Ac34 truncations and internal deletions fused with green fluorescent protein in plasmid-transfected Sf9 cells identified that the 91-205 amino acid (aa) region is required for Ac34 nuclear localization. Mutations in a potential C3H zinc finger (aa 116-131) in Ac34 resulted in exclusive cytoplasmic distribution of GFP:Ac34, suggesting that the zinc finger is required for Ac34 nuclear localization. To assess the functional importance of Ac34 in the nucleus during virus replication, recombinant AcMNPV bacmids containing a series of Ac34 truncations, internal deletions, or site mutations fused with HA tags were constructed. Subcellular localization analysis showed that Ac34 with internal deletions in aa 91-205 or site mutations in the potential zinc finger was predominantly distributed in the cytoplasm. Viral plaque assays and virus growth curves indicated that disruption of Ac34 nuclear localization significantly impaired virus replication. Taken together, our findings demonstrated that the nuclear localization of Ac34 requires the 91-205 aa region and its nuclear localization is essential for optimal virus replication. [ABSTRACT FROM AUTHOR]

• The two CnmeGV isolates came from the same region and were collected 35 years apart. • CnmeGV hr4 presents a nested repeating pattern. • Tandem repeats were the only highly variable region of CnmeGV during replication. • CnmeGV obtained a non-coding sequence from its host. Cnaphalocrocis medinalis granulovirus (CnmeGV), belonging to Betabaculovirus cnamedinalis , can infect the rice pest, the rice leaf roller. In 1979, a CnmeGV isolate, CnmeGV-EP, was collected from Enping County, China. In 2014, we collected another CnmeGV isolate, CnmeGV-EPDH3, at the same location and obtained the complete virus genome sequence using Illumina and ONT sequencing technologies. By combining these two virus isolates, we updated the genome annotation of CnmeGV and conducted an in-depth analysis of its genome features. CnmeGV genome contains abundant tandem repeat sequences, and the repeating units in the homologous regions (hrs) exhibit overlapping and nested patterns. The genetic variations within EPDH3 population show the high stability of CnmeGV genome, and tandem repeats are the only region of high genetic variation in CnmeGV genome replication. Some defective viral genomes formed by recombination were found within the population. Comparison analysis of the two virus isolates collected from Enping showed that the proteins encoded by the CnmeGV-specific genes were less conserved relative to the baculovirus core genes. At the genomic level, there are a large number of SNPs and InDels between the two virus isolates, especially in and around the bro genes and hrs. Additionally, we discovered that CnmeGV acquired a segment of non-ORF sequence from its host, which does not provide any new proteins but rather serves as redundant genetic material integrated into the viral genome. Furthermore, we observed that the host's transposon piggyBac has inserted into some virus genes. Together, dsDNA viruses could acquire non-coding genetic material from their hosts to expand the size of their genomes. These findings provide new insights into the evolution of dsDNA viruses. [ABSTRACT FROM AUTHOR]

Night shift is a common work schedule. This study aimed to analyze the interaction between age and frequency of night shift on the hypertension prevalence. A census questionnaire was conducted in 512 medical institutions in 11 cities of Hebei Province. One lakh twenty‐one thousand nine hundred three female nurses were included in this study. Binary Logistic regression analysis was done by SPSS Version 26.0. The youngest age group without night shift was used as the reference group. The odds ratio was calculated by different combinations of interaction items. Interaction coefficients were calculated by an Excel table designed by Andersson. Compared with the 18–25 year old ones without night shift, there existed an additive interaction between the age of 36–45 and more than 5–10 night shifts per month on hypertension prevalence. Odds ratio, the relative excess risk of interaction, the attributable proportion of interaction, and the synergy index and their 95% confidence intervals were 2.923(2.292‐3.727), 0.631(0.309‐0.954), 0.216(0.109‐0.323), 1.488(1.158‐1.913). Additive interaction was also found between the age of 36–45 and more than 10 night shifts per month. OR, RERI, API, SI, and their 95% confidence intervals were 3.430(2.273‐5.175) 1.037(0.061‐2.013), 0.303(0.089‐0.516), and 1.746(1.093‐2.788). There also existed an additive interaction between the age of 46–65 and more than 5–10 night shifts per month on hypertension prevalence. OR, RERI, API, SI, and their 95% confidence intervals were 7.398(5.595‐9.781) 1.809(0.880‐2.739), 0.245(0.148‐0.341), and 1.394(1.199‐1.622).There existed interaction between specific age groups and night shift frequency on the prevalence of hypertension among female nurses. [ABSTRACT FROM AUTHOR]

• pkip is required for optimal budded virus production during AcMNPV replication. • The deletion of pkip impairs nucleocapsid assembly. • PKIP is distributed in both the cytoplasm and nuclei of viruses-infected cells. • PKIP is associated with the nucleocapsid of BV. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf24 (pkip) is a unique Alphabaculovirus gene. A previous study showed that a temperature-sensitive mutant of AcMNPV with a mutation in pkip displayed severe defects in progeny budded virion (BV) production and very late gene transcription, however, the underlying mechanism has not been determined. To investigate the function of pkip in the baculovirus replication cycle, we constructed a pkip -knockout AcMNPV bacmid in this study. Our results showed that deletion of pkip led to significant reduction of BV production, while the synthesis of viral DNA and the transcription of early and late genes were not affected. Further examination by transmission electron microscopy analysis showed that deletion of pkip resulted in the formation of massive electron-lucent tubular structures in the nucleus of the infected cells, along with some normal electron‐dense nucleocapsids. The pkip -encoded protein PKIP could be detected at late phase during infection and was distributed in both the cytoplasm and nuclei of viruses-infected cells, with a ring pattern near the inner nuclear membrane and punctate distribution in the virogenic stroma area. Biochemical fractionation of virions into nucleocapsid and envelop components showed that PKIP was associated with the nucleocapsid fraction of BV. Taken together, our results indicated that PKIP is associated with nucleocapsids of BV and involved in nucleocapsid assembly, which contributes to the optimal production of BV. [ABSTRACT FROM AUTHOR]

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac110 gene is especially conserved in lepidopteran-specific baculoviruses and is uncharacterized. To investigate the role of ac110 in the baculovirus life cycle, an ac110- knockout (vAc110KO) and a repair (vAc110:HA) viruses were constructed in this study. Budded virion production and occlusion body formation were unaffected in vAc110KO-transfected or infected Sf9 cells. Intrahemocoelic injection of budded virions of vAc110KO killed Trichoplusia ni larvae as efficiently as the repair or the wild-type viruses. However, per os inoculation of occlusion bodies of vAc110KO failed to establish infection in T. ni larvae, while the repair virus was as efficient as the wild-type virus. Treatment with calcofluor white, a reagent that damages the peritrophic membrane, did not rescue the oral infectivity of vAc110KO. These results suggested that Ac110 is a new per os infectivity factor that may play a role after occlusion-derived virions pass through the peritrophic membrane during oral infection. [ABSTRACT FROM AUTHOR]

• Psilogramma increta granulovirus is a new member of the genus Betabaculovirus. • Four betabaculoviruses, ClasGV-B, ChfuGV, ErelGV, and PsinGV, contain a horizontally transferred p10-2. • PsinGV isolate harbors abundant variants. The complete genome of Psilogramma increta granulovirus (PsinGV), isolated from P. increta (Lepidoptera: Sphingidae), was ultra-deep sequenced with a Novaseq PE150 platform and de novo assembled and annotated. The PsinGV genome is a circular double-stranded DNA, 103,721 bp in length, with a G+C content of 33.0%, the third lowest G+C content in present sequenced baculoviruses. It encodes 123 putative open reading frames, including 38 baculovirus core genes, 42 lepidopteran baculovirus conserved genes, 38 betabaculovirus conserved genes, and 5 genes unique to PsinGV. Meanwhile, 3 homologous repeated regions with the core sequence TTGCAA and 3 direct repeated sequences were identified within the PsinGV genome. Kimura two-parameters distance analysis confirmed that Psilogramma increta granulovirus is a representative of a prospective new species of the genus Betabaculovirus. Phylogenetic analysis based on the baculovirus core genes showed that PsinGV is closely related to Choristoneura fumiferana granulovirus, Clostera anastomosis granulovirus-B , and Erinnyis ello granulovirus. These four species therefore share a common ancestor residing in the Betabaculovirus genus. The genome of the PsinGV isolate contained two p10 copies: p10 and p10-2. Phylogenetic reconstruction of P10 suggests a transfer event of the p10-2 gene from an alphabaculovirus to the aforementioned common ancestor. Analysis of genomic diversity showed that 203 intrahost variants, including 182 single nucleotide variants and 21 short insertions/deletions, are present within the PsinGV isolate. Meanwhile, allele frequency indicated that the isolate contains three major genotypes, implying the archived isolate consists of several P. increta carcasses killed by PsinGV with different genotypes. [ABSTRACT FROM AUTHOR]

• Amino acids N318, V319, C320, R321, and I323 of P48 are critical for the association with Ac93. • Amino acids N318, V319, C320, R321, and I323 of P48 are required for the nuclear egress of nucleocapsids and efficient formation of intranuclear microvesicles.. • The association between P48 and Ac93 may be important for both BV and ODV morphogenesis. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) undergoes a biphasic life cycle with the production of two physically and functionally distinct virions: budded virions (BVs) and occlusion-derived virions (ODVs). Nuclear egress of nucleocapsids and intranuclear microvesicle formation are critical for the morphogenesis of BVs and ODVs, respectively, but the mechanisms and details of these two processes remain unknown. Our previous studies have shown that AcMNPV p48 (ac103) gene is essential for the nuclear egress of nucleocapsids and efficient formation of intranuclear microvesicles, and protein P48 associates with Ac93, which is also involved in the above processes in virion morphogenesis. In this study, we present evidence that alanine substitution for residues N318, V319, C320, R321, and I323 of P48 disrupted the association with Ac93. Moreover, mutation of these residues blocked the nuclear egress of nucleocapsids and efficient formation of intranuclear microvesicles, and subsequent BV formation, as well as ODV envelopment and embedding of ODVs into polyhedra. These results suggested that the association between P48 and Ac93 may be important for both BV and ODV morphogenesis. [ABSTRACT FROM AUTHOR]

Abstract: Many viruses activate the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, thereby modulating diverse downstream signaling pathways associated with antiapoptosis, proliferation, cell cycling, protein synthesis and glucose metabolism, in order to augment their replication. To date, the role of the PI3K-Akt pathway in Baculovirus replication has not been defined. In the present study, we demonstrate that infection of Sf9 cells with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) elevated cellular Akt phosphorylation at 1 h post-infection. The maximum Akt phosphorylation occurred at 6 h post-infection and remained unchanged until 18 h post-infection. The PI3K-specific inhibitor, LY294002, suppressed Akt phosphorylation in a dose-dependent manner, suggesting that AcMNPV-induced Akt phosphorylation is PI3K-dependent. The inhibition of PI3K-Akt activation by LY294002 significantly reduced the viral yield, including a reduction in budded viruses and occlusion bodies. The virus production was reduced only when the inhibitor was added within 24 h of infection, implying that activation of PI3K occurred early in infection. Correspondingly, both viral DNA replication and late (VP39) and very late (POLH) viral protein expression were impaired by LY294002 treatment; LY294002 had no effect on immediate-early (IE1) and early-late (GP64) protein expression. These results demonstrate that the PI3K-Akt pathway is required for efficient Baculovirus replication. [Copyright &y& Elsevier]

Abstract: Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf53 (ac53) is a highly conserved gene existing in all sequenced Lepidoptera and Hymenoptera baculoviruses, but its function remains unknown. To investigate its role in the baculovirus life cycle, an ac53 deletion virus (vAcac53KO-PH-GFP) was generated through homologous recombination in Escherichia coli. Fluorescence and light microscopy and titration analysis revealed that vAcac53KO-PH-GFP could not produce infectious budded virus in infected Sf9 cells. Real-time PCR demonstrated that the ac53 deletion did not affect the levels of viral DNA replication. Electron microscopy showed that many lucent tubular shells devoid of the nucleoprotein core are present in the virogenic stroma and ring zone, indicating that the ac53 knockout affected nucleocapsid assembly. With a recombinant virus expressing an Ac53-GFP fusion protein, we observed that Ac53 was distributed within the cytoplasm and nucleus at 24 h post-infection, but afterwards accumulated predominantly near the nucleus–cytoplasm boundary. These data demonstrate that ac53 is involved in nucleocapsid assembly and is an essential gene for virus production. [Copyright &y& Elsevier]

Abstract: orf18 (ac18) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved gene in lepidopteran nucleopolyhedroviruses, but its function remains unknown. In this study, an ac18 knockout AcMNPV bacmid was generated to determine the role of ac18 in baculovirus life cycle. After transfection of Sf-9 cells, the ac18-null mutant showed similar infection pattern to the parent virus and the ac18 repair virus with respect to the production of infectious budded virus, occlusion bodies, or the formation of nucleocapsids as visualized by electron microscopy. The deletion mutant did not reduce AcMNPV infectivity for Trichoplusia ni in LD50 bioassay; however, it did take 24 h longer for deleted mutant to kill T. ni larvae than wild-type virus in LT50 bioassay. Our results demonstrate that ac18 is not essential for viral propagation both in vitro and in vivo, but it may play a role in efficient virus infection in T. ni larvae. [Copyright &y& Elsevier]

• SfMEF interacts with Ac34. • Knocking down Sfmef resulted in failure of nuclear import of Ac34. • Mutations in the potential zinc finger motif of Ac34 disrupted the interaction between Ac34 and SfMEF. • The loss of interaction between Ac34 and SfMEF resulted in decreased budded virion production. Autographa californica multiple nucleopolyhedrovirus orf34 (ac34) is one of the unique genes of alphabaculoviruses. For successful alphabaculovirus replication, viral proteins must be transported to the nucleus. Our previous study showed that the nuclear localization of Ac34 was required for optimal production of budded virions. To investigate the mechanism of Ac34 nuclear import, mass spectrometric analysis was performed to identify potential proteins that may be involved in the nuclear import of Ac34. The result indicated that Spodoptera frugiperda mRNA export factor (SfMEF) may interact with Ac34 during baculovirus infection. Co-immunoprecipitation assays confirmed that Ac34 could interact with SfMEF in the absence of other baculovirus proteins. The deletion of ac34 did not affect the subcellular localization of SfMEF; however, knocking down Sfmef prevented the nuclear import of Ac34 in virus-infected cells. The mutations of C116 or C119 in a potential CCCH zinc finger motif (C116-X 2 -C119-X 8 -C128-X 2 -H131) of Ac34 led to an exclusive cytoplasmic distribution of Ac34, in consistent with our previous finding of mutations of C128 or H131 in this motif. Co-immunoprecipitation analysis showed that the above mutations in the potential zinc finger motif disrupted the interaction between Ac34 and SfMEF, and the loss of the interaction resulted in decreased BV production. Our findings demonstrated that SfMEF interacts with and mediates the nuclear import of Ac34, which is a new nucleocytoplasmic transport pathway used by alphabaculovirus to achieve successful viral replication within the nucleus of the infected cells. [ABSTRACT FROM AUTHOR]

• pkip is required for hyperexpression of viral very late genes. • Knockdown of pkip specifically down-regulates transcription of viral very late genes. • PKIP associates with P6.9 during virus infection. • The deletion of pkip resulted in decreased hyperphosphorylation of P6.9. A previous study showed that a mutation in Autographa californica multiple nucleopolyhedrovirus pkip (ac24) led to severe defects in progeny budded virion production and very late gene transcription at non-permissive temperature. To dissect the underlying mechanism, our early study showed that PKIP is associated with nucleocapsid of budded virion and involved in nucleocapsid assembly. However, how pkip affects very late gene transcription has not been determined. In the present study, double-stranded RNA was used to silence pkip expression during virus infection, resulting in the significant reduction of occlusion body production and polyhedrin expression. To find out whether PKIP regulates polyhedrin expression by affecting the transcription of other viral genes for very late gene expression, a comparative transcriptome analysis of viral genes was performed by RNA sequencing and the result showed that silencing pkip specifically down-regulated transcription of very late genes, while the transcription patterns of the viral genes associated with very late gene transcription were not affected. Since PKIP was reported to interact with and stimulate the activity of virus-encoded protein kinase PK1 and PK1 was involved in the hyperphosphorylation of viral basic protein P6.9, which was required for the maximal hyperexpression of very late genes, we sought to determine the association between PKIP and P6.9. Further experiments showed that PKIP interacted with P6.9 during virus infection, and the deletion of pkip resulted in decreased hyperphosphorylation of P6.9. Taken together, our results indicated that PKIP is involved in hyperphosphorylation of P6.9, which in return maybe required for hyperexpression of very late genes. [ABSTRACT FROM AUTHOR]