Viral infection can regulate the cell cycle, thereby promoting viral replication. Hijacking and altering the cell cycle are important for the virus to establish and maintain a latent infection. Previously, Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV)-latently infected P8-Se301-C1 cells, which grew more slowly than Se301 cells and interfered with homologous SeMNNPV superinfection, were established. However, the effects of latent and superinfection with baculoviruses on cell cycle progression remain unknown. In this study, the cell cycle profiles of P8-Se301-C1 cells and SeMNPV or Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-infected P8-Se301-C1 cells were characterized by flow cytometry. The results showed that replication-related genes MCM4, PCNA, and BAF were down-regulated (p < 0.05) in P8-Se301-C1 cells, and the S phase of P8-Se301-C1 cells was longer than that of Se301 cells. P8-Se301-C1 cells infected with SeMNPV did not arrest in the G2/M phase or affect the expression of Cyclin B and cyclin-dependent kinase 1 (CDK1). Furthermore, when P8-Se301-C1 cells were infected with SeMNPV after synchronized treatment with hydroxyurea and nocodazole, light microscopy and qRT-PCR analysis showed that, compared with unsynchronized cells and S and G2/M phase cells, SeMNPV-infected P8-Se301-C1 cells in G1 phase induced G2/M phase arrest, and the amount of virus adsorption and intracellular viral DNA replication were significantly increased (p < 0.05). In addition, budded virus (BV) production and occlusion body (OB)-containing cells were both increased at 120 h post-infection (p < 0.05). The expression of Cyclin B and CDK1 was significantly down-regulated at 48 h post-infection (p < 0.05). Finally, the arrest of SeMNPV-infected G1 phase cells in the G2/M phase increased BV production (p < 0.05) and the number of OB-containing cells. In conclusion, G1 phase infection and G2/M arrest are favorable to SeMNPV proliferation in P8-Se301-C1 cells, thereby alleviating the homologous superinfection exclusion. The results contribute to a better understanding of the relationship between baculoviruses and insect cell cycle progression and regulation.

Viral infection can regulate the cell cycle, thereby promoting viral replication. Hijacking and altering the cell cycle are important for the virus to establish and maintain latent infection. Previously, Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) latently infected P8-Se301-C1 cells, which grew more slowly than Se301 cells and interfered with homologous SeMNNPV superinfection, were established. However, the effects of latent and superinfection with baculoviruses on cell cycle progression remain unknown. In this study, the cell cycle profiles of P8-Se301-C1 cells, and SeMNPV or Autographa californica mutiple nucleopolyhedrovirus (AcMNPV) infected P8-Se301-C1 cells were characterized by flow cytometry. The results showed that replication-related genes MCM4, PCNA, and BAF were down-regulated (p < 0.05) in P8-Se301-C1 cells and the S phase of P8-Se301-C1 cells was longer than that of Se301 cells. P8-Se301-C1 cells infected with SeMNPV did not arrest in G2/M phase and or affect the expression of Cyclin B and cyclin-dependent kinase 1 (CDK1). Furthermore, when P8-Se301-C1 cells were infected with SeMNPV after synchronized treatment with hydroxyurea and nocodazole, light microscopy and qRT-PCR analysis showed that compared with unsynchronized cells and S and G2/M phase cells, SeMNPV-infected P8-Se301-C1 cells in G1 phase induced G2/M phase arrest, and the amount of virus adsorption and intracellular viral DNA replication were significantly increased (p < 0.05). In addition, the BV production and the OB containing cells were both increased at 120 h post-infection (p < 0.05). The expression of Cyclin B and CDK1 was significantly down-regulated at 48 h post infection (p < 0.05). Finally, arrest of SeMNPV-infected G1 phase cells in the G2/M phase increased BV production (p < 0.05) and the number of OB containing cells. In conclusion, G1 phase infection and G2/M arrest are favorable to SeMNPV proliferation in P8-Se301-C1 cells, thereby alleviating the homologous superinfection exclusion. The results contribute to a better understanding of the relationship between baculoviruses and insect cell cycle progression regulation.

The genus Rumex, a member of the Polygonaceae family, includes approximately 200 edible or medicinal species around the world (Vasas et al. 2015). In China, the Rumex species R. longifolius is widely distributed in all regions, except for southern China. In May 2023, typical powdery mildew symptoms were found on R. longifolius on Longwen Hill (26°23'04'' N, 106°37'57'' E) of Guizhou Normal University, China. The incidence was ~90% among 100 R. longifolius plants observed. The mycelia initially showed a sporadic distribution on the leaf surface of R. longifolius. In the late stage of infection, the mycelia coalesced into extensive patches and covered nearly the entire leaf surface. White colonies occurred mainly on the leaf adaxial side, occasionally on the leaf abaxial side and petiole. Hyphae were flexuous to straight, branched, 3.5-6.5 μm wide. Hyphal appressoria were lobed to multilobed, in opposite pairs or solitary. Conidiophores were erect, straight to slightly flexuous, 60-180 µm long (n = 30). Foot cells were subcylindrical, followed by 2-3 cells. Conidia were ellipsoid to ovoid, 32-50 μm long × 14-25 µm wide (n = 50). Conidia formed singly, occasionally two in a chain. Based on these morphological characteristics, the powdery mildew fungus (named as GZRL-1) was provisionally identified as Erysiphe species (Braun and Cook 2012). To confirm the species, the ITS and LSU regions were amplified using primer pairs ITS1/ITS4 (White et al. 1990) and NL1/NL4 (Ziemiecki et al. 1990), respectively. The obtained 647-bp ITS sequence (GenBank accession no. PQ856357) shared 100% identity and 99.38% query coverage with previously reported ITS sequences (AF011308, LC328322, and LC328323) of E. polygoni (Saenz and Taylor 1999; Abasova et al. 2018). The obtained 618-bp LSU sequence (PQ857798) shared 100% identity and 100% query coverage with LSU sequences (LC328322 and LC328323) of E. polygoni (Abasova et al. 2018). Based on the combined ITS and LSU sequences of Erysiphe taxa (Takamatsu et al. 2015), phylogenetic tree was constructed using MEGA 12. The isolate GZRL-1 was grouped in the same clade as E. polygoni. To perform pathogenicity tests, leaves of three healthy potted R. longifolius plants were inoculated by gently pressing with diseased leaves. Three non-inoculated plants were used as controls. All plants were incubated in a greenhouse 25 ± 2°C, 80% relative humidity. After 15 days, the inoculated plants developed typical powdery mildew symptoms, whereas control plants remained symptomless. The fungus from inoculated R. longifolius plants was morphologically identical to that on originally diseased plants. Similar results were obtained from three independent pathogenicity tests. Based on the morphological and molecular characterization, the powdery mildew fungus was identified as E. polygoni. Although powdery mildew caused by E. polygoni has been reported on R. longifolius (synonym R. domesticus) in Finland, Romania, South Korea, and Sweden (Braun 1995; Cho and Shin 2004), this is the first report of E. polygoni on R. longifolius in China. This work expands the geographical range of E. polygoni on R. longifolius plants.