Sulfated Polyglycerol-Modified Hydrogels for Binding HSV-1 and RSV.

Heparan sulfate (HS) is a highly sulfated polysaccharide on the surface of mammalian cells and in the extracellular matrix and has been found to be important for virus binding and infection. In this work, we designed synthetic hydrogels with viral binding and deactivation activities through the postfunctionalization of an HS-mimicking polyelectrolyte and alkyl chains. Three polyglycerol-based hydrogels were prepared as substrates and postfunctionalized by sulfated linear polyglycerol (lPGS) via thiol-ene click reaction. The viral binding properties were studied using herpes simplex virus type 1 (HSV-1) and respiratory syncytial virus (RSV). The effect of hydrogel types and molecular weight (Mw) of conjugated lPGS on viral binding properties was also assessed, and promising binding activities were observed in all lPGS-functionalized samples. Further coupling of 11 carbons long alkyl chains to the hydrogel revealed virucidal properties caused by destruction of the viral envelope, as shown by atomic force microscopy (AFM) imaging.

Advanced fluorescence microscopy in respiratory virus cell biology.

Respiratory viruses are a major public health burden across all age groups around the globe, and are associated with high morbidity and mortality rates. They can be transmitted by multiple routes, including physical contact or droplets and aerosols, resulting in efficient spreading within the human population. Investigations of the cell biology of virus replication are thus of utmost importance to gain a better understanding of virus-induced pathogenicity and the development of antiviral countermeasures. Light and fluorescence microscopy techniques have revolutionized investigations of the cell biology of virus infection by allowing the study of the localization and dynamics of viral or cellular components directly in infected cells. Advanced microscopy including high- and super-resolution microscopy techniques available today can visualize biological processes at the single-virus and even single-molecule level, thus opening a unique view on virus infection. We will highlight how fluorescence microscopy has supported investigations on virus cell biology by focusing on three major respiratory viruses: respiratory syncytial virus (RSV), Influenza A virus (IAV) and SARS-CoV-2. We will review our current knowledge of virus replication and highlight how fluorescence microscopy has helped to improve our state of understanding. We will start by introducing major imaging and labeling modalities and conclude the chapter with a perspective discussion on remaining challenges and potential opportunities.

Heat shock protein 19.9 (Hsp19.9) from Bombyx mori is involved in host protection against viral infection.

Adverse environmental conditions cause serious economic losses in sericulture; Bombyx mori nucleopolyhedrovirus (BmNPV) is the primary biotic stress and high temperature is the major abiotic stress in this industry. B. mori heat shock protein 19.9 (Bmhsp19.9) overexpression was previously demonstrated to protect transgenic silkworm H19.9 against extreme temperature. This study analyzed the role of Bmhsp19.9 in H19.9A and H19.9B silkworm lines and BmE cells infected with BmNPV at regular and high temperatures. qPCR results showed that Bmhsp19.9 expression was upregulated in BmE cells and silkworm after BmNPV challenge. Bmhsp19.9 overexpression significantly inhibited BmNPV proliferation in BmE cells. The viral DNA content was significantly decreased in transgenic H19.9 silkworm compared to the control. These results suggested that Bmhsp19.9 was involved in antiviral immunity against BmNPV. Furthermore, Bmhsp19.9 overexpression protected BmE cells against BmNPV under high temperature shock. This indicates that Bmhsp19.9 is a promising candidate for improving silkworm resistance to biotic and abiotic stresses, thereby reducing sericulture losses.

Identification of the Vo domain of V-ATPase in Bombyx mori silkworm.

Vacuolar H+-ATPase (V-ATPase) is very important for eukaryotes and consists of a conserved V1 domain and slightly variable Vo domain. However, the Vo domain has not been systematically identified in the silkworm Bombyx mori. In this study, 11 Vo domain subunit members were identified throughout the genome of B. mori, including four isoforms of subunit a (BmVoa1-4), two isoforms of subunit e (BmVoe1-2), one each of subunit c″ (BmVob), subunit c (BmVoc), and subunit d (BmVod), and two accessory subunits (BmVoap1 and BmVoap2). Further analysis revealed BmVoa3 and BmVoa4 were located on the same chromosome and had similar molecular weights and isoelectric points, but separated to different small branches on the phylogenetic tree. Reverse transcription polymerase chain reaction results indicated that most Vo domain subunits were expressed during all silkworm developmental stages. Quantitative polymerase chain rection (qPCR) showed BmVoa1 was hemocyte-specific and BmVoe1 was testis-specific. BmVoa2 was not expressed in the midgut, while the other members were specifically or highly expressed in the midgut and Malpighian tubules. Further qPCR analysis indicated BmVoa4 in the midgut and BmVoa3 in BmE cells were significantly induced by B. mori nucleopolyhedrovirus (BmNPV), suggesting that these two genes may be involved in BmNPV infection.

Potential of transferring transgenic DNA from silkworm to chicken.

Safety assessment must be conducted before the commercial release of transgenic silkworms. This study was conducted to assess the potential of transferring transgenic DNA from silkworms to other organisms. One hundred hatched male chickens were evenly assigned into 4 groups (T1-4). Groups T1-3 were fed transgenic silkworms P3+5UI with enhanced green fluorescent protein DNA (EGFP) inserted, A4SOR with superoxide reductase DNA (SOR) inserted, and normal silkworm, respectively. Each chicken was fed one silkworm larva every day for 3 weeks. T4 was the normal feeding control. Twenty chickens were randomly selected from each treatment for sacrifice at 22 days of age. The serum was collected individually for biochemical examination, revealing no difference in the analyzed serum parameters between T4 and T1-3. DNA from the duodenum, jejunum, ileum, liver, kidney, and jejunal digesta was extracted for PCR analysis of EGFP, SOR, silkworm housekeeping gene TIF-4A, and chicken ovalbumin gene. No transgenic DNA or TIF-4A was detected in the digesta and tissues of chickens. The same results were observed in chicken upon increasing the amount and frequency of feeding transgenic silkworms, suggesting that the transgenic DNA from silkworms was degraded in the digestive tract and not transferred into the tissues of chicken. This study revealed that transferr recombinant DNA from transgenic silkworm to another organism is unlikely.

Toxicological evaluation of transgenic silkworms.

Safety of transgenic silkworms must be evaluated before their commercial application. We assessed subacute toxicity using a 28-day feeding study in rats. Eighty rats were evenly allocated into four groups, with each group containing 10 male and 10 female rats. Rats of three groups were fed dried transgenic silkworm H19.9A pupae with overexpressed endogenous Bmhsp19.9, transgenic silkworm A4SOR pupae with overexpressed exogenous SOR, or normal silkworm pupae at a dose of 3.0 g/kg/day, respectively. The fourth group served as a normal feeding control. The body weight, feed consumption, hematology response variables, serum biochemical parameters, organ weights, gross necropsy, and histopathologic of animals were evaluated. No mortality, adverse effects, or major differences in the evaluated parameters were observed in the groups fed transgenic pupae in comparison with the control, suggesting that transgenic silkworms are toxicologically equivalent to normal silkworms and are safe for consumption in rats.

Overexpression of Bmhsp19.9 protects BmE cells and transgenic silkworm against extreme temperatures.

Temperature stress disrupts protein homeostasis in biological cells. High temperature causes death of the silkworm Bombyx mori, while low temperature leads to slow growth of silkworm in sericulture. Small heat shock proteins (sHsps) play a crucial role in maintaining protein homeostasis and protecting cells against various harmful conditions. In this study, the expression patterns of Bmhsp19.9 were analyzed by reverse transcription (RT)-PCR and quantitative PCR. The promoters hsp19.9P1 and hsp19.9P2 of Bmhsp19.9 were cloned, quantitative PCR and western blotting confirmed that the longer promoter of hsp19.9P1 showed higher activity compared to hsp19.9P2. Overexpression of Bmhsp19.9 increased the viability of BmE cells at high temperatures. The transgenic vector pb-H19.9 was constructed, in which Bmhsp19.9 was overexpressed under the control of hsp19.9P1. Two transgenic lines, H19.9A and H19.9B, were generated via microinjection of P50 silkworm embryos. Quantitative PCR and western blotting revealed that the content of Bmhsp19.9 in transgenic silkworms was higher than that in the control. After exposure to 35 °C for 49 h, the survival rate was increased by approximately 40% in transgenic silkworms compared to non-transgenic P50. When P50 and H19.9A were reared at 13 °C, growth was faster and the survival rate and surviving larval weight were significantly higher in H19.9A compared to P50. These results suggest that overexpression of Bmhsp19.9 increased the resistance of transgenic silkworms to extreme temperatures, which may be useful for sericulture in regions and seasons with no optimal average temperatures for silkworm.

Spry is downregulated by multiple viruses to elevate ERK signaling and ensure viral reproduction in silkworm.

Viral diseases of silkworm are mainly caused by Bombyx mori nucleopolyhedrovirus (BmNPV), B. mori cytoplasmic polyhedrosis virus (BmCPV) and B. mori bidensovirus (BmBDV). The virus alters host cellular pathways to facilitate its proliferation. It is still unclear whether the three silkworm viruses regulate a certain host pathway. Spry is a negative regulator upstream of ERK. In this study, we found that BmSpry was decreased and p-ERK was increased in silkworm after infection with each virus. A transgenic RNAi vector of BmSpry was constructed and used for embryo microinjection to generate the transgenic line Spry-I. The expression of BmSpry was significantly reduced in Spry-I compared to that in non-transgenic silkworm. The viral content and mortality in Spry-I were significantly higher than those in non-transgenic larvae after infection with the three viruses. p-ERK was increased in Spry-I compared to that in non-transgenic control after virus infection. These results suggest that BmSpry is downregulated by multiple different classes of viruses to elevate p-ERK and ensure viral reproduction in the silkworm.

Screening of PI3K-Akt-targeting Drugs for Silkworm against Bombyx mori Nucleopolyhedrovirus.

Bombyx mori nucleopolyhedrovirus (BmNPV) is the most prevalent threat to silkworms. Hence, there is a need for antiviral agents in sericulture. The PI3K-Akt pathway is essential for the efficient replication of the baculovirus. In an attempt to screen antiviral drugs against BmNPV, we summarized the commercial compounds targeting PI3K-Akt and selected the following seven oral drugs for further analyses: afuresertib, AZD8835, AMG319, HS173, AS605240, GDC0941, and BEZ235. Cell viability assay revealed that the cytotoxicity of these drugs at 10 µM concentration was not strong. Viral fluorescence observation and qPCR analysis showed that these candidate drugs significantly inhibited BmNPV in BmE cells. Only AMG319 and AZD8835 inhibited viral proliferation in silkworm larvae. The mortality of AZD8835-treated silkworms was lower than that of the control silkworms. Western blotting showed that AMG319 and AZD8835 decreased p-Akt expression after BmNPV infection. These results suggest that AZD8835 has application potential in sericulture.

Distinct Functions of Bombyx mori Peptidoglycan Recognition Protein 2 in Immune Responses to Bacteria and Viruses.

Peptidoglycan recognition protein (PGRP) is an important pattern recognition receptor in innate immunity that is vital for bacterial recognition and defense in insects. Few studies report the role of PGRP in viral infection. Here we cloned two forms of PGRP from the model lepidopteran Bombyx mori: BmPGRP2-1 is a transmembrane protein, whereas BmPGRP2-2 is an intracellular protein. BmPGRP2-1 bound to diaminopimelic acid (DAP)-type peptidoglycan (PGN) to activate the canonical immune deficiency (Imd) pathway. BmPGRP2-2 knockdown reduced B. mori nucleopolyhedrovirus (BmNPV) multiplication and mortality in cell lines and in silkworm larvae, while its overexpression increased viral replication. Transcriptome and quantitative PCR (qPCR) results confirmed that BmPGRP2 negatively regulated phosphatase and tensin homolog (PTEN). BmPGRP2-2 expression was induced by BmNPV, and the protein suppressed PTEN-phosphoinositide 3-kinase (PI3K)/Akt signaling to inhibit cell apoptosis, suggesting that BmNPV modulates BmPGRP2-2-PTEN-PI3K/Akt signaling to evade host antiviral defense. These results demonstrate that the two forms of BmPGRP2 have different functions in host responses to bacteria and viruses.

Phosphoenolpyruvate carboxykinase is involved in antiviral immunity against Bombyx mori nucleopolyhedrovirus.

Phosphoenolpyruvate carboxykinase (PEPCK) has cytoplasmic isoform (PEPCK-C) and a mitochondrial isoform (PEPCK-M). PEPCK-C plays an important role in gluconeogenesis, but the function of PEPCK-M is largely unknown. In this study, we cloned two isoforms of PEPCK (BmPEPCK-1 and BmPEPCK-2; both of PEPCK-M) from the lepidopteran model Bombyx mori. BmPEPCK-1 and BmPEPCK-2 were adjacently located in the silkworm genome, and both contained 13 exons. The main difference in the sequences was the 13th exon and 3'UTR. The expression of BmPEPCK-1 was higher than that of BmPEPCK-2, the overexpression of which did not affect BmNPV proliferation. The expression levels of BmPEPCK-2 and ATG6/7/8/13 decreased after BmNPV infection. Overexpression of BmPEPCK-2 increased the expression of ATG6/7/8 and significantly decreased viral fluorescence and content, suggesting that BmPEPCK-2 suppressed the multiplication of BmNPV by increasing ATGs expression. These results revealed that PEPCK-M has an important function in antiviral immunity.

Expression, purification, and characterization of a novel acid phosphatase that displays protein tyrosine phosphatases activity from Metarhizium anisopliae strain CQMa102.

The protein tyrosine phosphatase (PTPase) plays an important role in insect immune system. Our group has purified a type of acid phosphatase that could specifically dephosphorylate trans-Golgi p230 in vitro. In order to study this phosphatase further, we have identified and cloned the phosphatase gene from a locust specific Metarhizium anisopliae Strain CQMa102. The CQMa102 phosphatase was expressed in Pichia pastoris to verify its protease activity. The molecular weight (MW) and the isoelectric point (pI) of the phosphatase were about 85 kDa and 6.15, respectively. Substrate specificity evaluation showed that the purified enzyme exhibited high activity on O-phospho-L-tyrosine. At its optimal pH of 6.5 and optimum temperature of 70 °C, the protein showed the highest activity respectively. It can be activated by Ca2+, Mg2+, Mn2+, Ba2+, Co2+ and phosphate analogs, but inhibited by Zn2+, Cu2+, fluoride, dithiothreitol, ß-mercaptoethanol and N-ethylmaleimide.

Nitrogen Removal Characteristics of Pseudomonas putida Y-9 Capable of Heterotrophic Nitrification and Aerobic Denitrification at Low Temperature.

The cold-adapted bacterium Pseudomonas putida Y-9 was investigated and exhibited excellent capability for nitrogen removal at 15°C. The strain capable of heterotrophic nitrification and aerobic denitrification could efficiently remove ammonium, nitrate, and nitrite at an average removal rate of 2.85 mg, 1.60 mg, and 1.83 mg NL-1 h-1, respectively. Strain Y-9 performed nitrification in preference to denitrification when ammonium and nitrate or ammonium and nitrite coexisted in the solution. Meantime, the presence of nitrate had no effect on the ammonium removal rate of strain Y-9, and yet the presence of high concentration of nitrite would inhibit the cell growth and decrease the nitrification rate. The experimental results indicate that P. putida Y-9 has potential application for the treatment of wastewater containing high concentrations of ammonium along with its oxidation products at low temperature.