The global landscapes of lysine crotonylation in pseudorabies virus infection.

Lysine crotonylation is a common occurrence in eukaryotic cells, regulating various physiological functions, including chromatin remodeling, cellular growth, and development. However, its involvement in viral infections has rarely been documented. In this study, we reveal that pseudorabies virus (PRV) infection significantly alters the global lysine crotonylation levels in porcine kidney PK-15 cells. Specifically, we identified a few viral proteins, including UL54, gM, gD, UL19, UL37, and UL46, which undergo crotonylation modification. Our observations indicate that at 20 h post-infection (hpi), 551 crotonylation sites were reduced across 345 proteins, while 47 new sites emerged in 37 proteins compared to the control group. By 40 hpi, 263 sites had decreased in 190 proteins, while 389 new sites appeared in 240 proteins. Deeper analysis revealed that the proteins with altered crotonylation levels were primarily involved in binding, catalytic activity, biosynthetic processes, ribosome activity, and metabolic processes. Additionally, our findings underscored the significance of ribosomes and the endoplasmic reticulum (ER), which were enriched with proteins exhibiting altered crotonylation. Overall, our study for the first time offers new insights into the relationship between crotonylation and herpes virus infection, paving the way for future investigations into the role of crotonylation in viral infections.

Erratum: MiR-375 Has Contrasting Effects on Newcastle Disease Virus Growth Depending on the Target Gene : Erratum.

[This corrects the article DOI: 10.7150/ijbs.25106.].

Microbiome analysis reveals the effects of black soldier fly oil on gut microbiota in pigeon.

The gut microbiota plays a vital roles in poultry physiology, immunity and metabolism. Black soldier fly oil is known to have a positive effect on the gut microbiota. However, the specific effect of black soldier fly oil on the composition and structure of the gut microbiota of the pigeon is unknown. In this experiment, 16S rDNA high-throughput sequencing was performed to study the effect of different doses of black soldier fly oil on the changes of pigeon intestinal microbes. Results indicated that the different doses of black soldier fly oil had no effect on the gut microbial diversity of the pigeon. Although the dominant phyla (Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria) and genus (uncultured_bacterium_f_Lachnospiraceae and Desulfovibrio) in control group and experimental group with different doses were the same, the abundances of some beneficial bacteria (Megasphaera, Intestinimonas, Prevotella_9, Lachnospiraceae_UCG-001, Faecalibacterium, Coprococcus_2, Parabacteroides, Megasphaera, Leuconostoc, Prevotellaceae_UCG-001, Lactococcus, Ruminococcaceae_UCG-014, and Coprococcus_2) increased significantly as the concentration of black soldier fly oil increased. Taken together, this study indicated that black soldier fly oil supplementation could improve gut microbial composition and structure by increasing the proportions of beneficial bacteria. Notably, this is the first report on the effects of black soldier fly oil on the gut microbiota of pigeon, which contribute to understanding the positive effects of black soldier fly oil from the gut microbial perspective.

Newcastle disease virus selectively infects dividing cells and promotes viral proliferation.

Newcastle disease virus (NDV) can select cells to infect, but the mechanism of its cell selectivity has not been comprehensively investigated. Here, we use HeLa cells to establish that NDV can selectively infect cells at the single-cell level. We labeled proliferating cells with 5'-bromo-2-deoxyuridine (BrdU) and examined the colocalization of BrdU with NDV in cells to clarify the relationships between NDV infection and cell proliferation. Receptors at the plasma membrane mediate NDV entry into host cells. We labeled sialic acid receptor isoforms, compared their densities between different cell types and measured the sialic acid receptor densities in different cell phases. Our results suggest that NDV displays host tropism to HeLa cells compared to BHK cells and that the differences in the receptor isoform expression patterns between cell types contribute to the selection of HeLa by NDV. At the single-cell level, the dynamics of receptor expression changes during different cell phases contributing to the selection of cells in S/G2 phase for NDV infection. Furthermore, cell proliferation benefits viral replication, and enhanced virus replication leads to increased damage to cells. The elucidation of the mechanisms underlying host cell selection by NDV may help in the screening and characterizing of additional candidate oncolytic virus strains.

Truncated chicken MDA5 enhances the immune response to inactivated NDV vaccine.

Melanoma Differentiation-Associated protein 5 (MDA5) is a cytoplasmic sensor for viral invasion and plays an important role in regulation of the immune response against Newcastle disease virus (NDV) in chickens. MDA5 was used as an adjuvant to enhance the humoral immune response against influenza virus. In the current study, truncated chicken MDA5 [1-483 aa, chMDA5(483aa)] expressed by recombinant adenovirus was administered to specific-pathogen-free (SPF) chickens to improve the immune response induced by inactivated NDV vaccine. A total of 156 SPF chickens were divided into six groups, and after two rounds of immunization, the humoral immune response, cell-mediated immune (CMI) response and the protective efficacy of the vaccines against NDV challenge were evaluated. The results showed that co-administration of chMDA5(483aa) expressed by adenovirus increased the NDV-specific antibody response by 1.7 times and chickens received chMDA5(483aa) also gained a higher level of CMI response. Consistently, the protective efficacy of the inactivated NDV vaccine against virulent NDV (vNDV) challenge was improved by co-administrate with chMDA5(483aa), as indicated by the reduced morbidity and pathological lesions, lower levels of viral load in organs and reduced virus shedding. Our study demonstrated that chMDA5(433aa) expressed by adenovirus could enhance the immune efficacy of inactivated NDV vaccine in chickens and could be a potential adjuvant candidate in developing chicken NDV vaccines.

MiR-375 Has Contrasting Effects on Newcastle Disease Virus Growth Depending on the Target Gene.

MicroRNAs regulate post-transcriptional gene expression via either translational repression or mRNA degradation. They have important roles in both viral infection and host anti-infection processes. We discovered that the miR-375 is significantly upregulated in Newcastle disease virus (NDV)-infected chicken embryonic visceral tissues using a small RNA sequencing approach. Further research revealed that the overexpression of miR-375 markedly decreases the replication of the velogenic NDV F48E9 and the lentogenic NDV La Sota by targeting the M gene of NDV in DF-1 cells. Interestingly, miR-375 has another target, ELAVL4, which regulates chicken fibrocyte cell cycle progression and decreases NDV proliferation. In addition, miR-375 can influence bystander cells by its secretion in culture medium. Our results indicated that miR-375 is an inhibitor of NDV, but can also enhance NDV growth by reducing the expression of its target ELAVL4. These results emphasize the complex roles of microRNAs in the regulation of viral infections.

Newcastle Disease Virus V Protein Inhibits Cell Apoptosis and Promotes Viral Replication by Targeting CacyBP/SIP.

Newcastle disease virus (NDV) has been classified by the World Organization for Animal Health (OIE) as a notable disease-causing virus, and this virus has the ability to infect a wide range of birds. V protein is a non-structural protein of NDV. V protein has been reported to inhibit cell apoptosis (Park et al., 2003a) and promote viral replication (Huang et al., 2003), however, the mechanisms of action of V protein have not been elucidated. In the present study, a yeast two-hybrid screen was performed, and V protein was found to interact with the CacyBP/SIP protein. The results of co-immunoprecipitation and immuno-colocalization assays confirmed the interaction between V protein and CacyBP/SIP. The results of quantitative-PCR and viral plaque assays showed that overexpression of CacyBP/SIP inhibited viral replication in DF-1 cells. Overexpression of CacyBP/SIP in DF-1 cells induced caspase3-dependent apoptosis. The effect of knocking down CacyBP/SIP by siRNA was the opposite of that observed upon overexpression. Moreover, it is known that NDV induces cell apoptosis via multiple caspase-dependent pathways. Furthermore, V protein inhibited cell apoptosis and downregulated CacyBP/SIP expression in DF-1 cells. Taken together, the findings of the current study indicate that V protein interacts with CacyBP/SIP, thereby regulating cell apoptosis and viral replication.

Coadministration of Recombinant Adenovirus Expressing GM-CSF with Inactivated H5N1 Avian Influenza Vaccine Increased the Immune Responses and Protective Efficacy Against a Wild Bird Source of H5N1 Challenge.

Wild birds play a key role in the spread of avian influenza virus (AIV). There is a continual urgent requirement for AIV vaccines to address the ongoing genetic changes of AIV. In the current study, we trialed a novel AIV vaccine against the wild bird source of H5N1 type AIV with recombinant adenovirus expressing granulocyte monocyte colony-stimulating factor (GM-CSF) as an adjuvant. A total of 150-day-old commercial chicks, with AIV-maternal-derived antibody, were divided into 6 groups. The primary vaccination was performed at day 14 followed by a subsequent boosting and intramuscular challenge on day 28 and 42, respectively. Recombinant GM-CSF (rGM-CSF) expressed by adenovirus, named as rAd-GM-CSF, raised the hemagglutination inhibition (HI) titers (log2) against AIV from 7.0 (vaccinate with inactivated vaccine alone) to 8.4 after booster immunization. Moreover, the rGM-CSF addition markedly increased the expression of interferon-γ, interleukin-4, and major histocompatibility complex-II in the lungs, compared with those immunized with inactivated vaccine alone on day 29, that is, 18 h post booster immunization. Following challenge, chicks inoculated with the inactivated AIV vaccine and rAd-GM-CSF together exhibited mild clinical signs and 62% survivals compared to 33% in the group immunized with inactivated AIV vaccine alone. Higher level of HI titers, immune related molecule expressions, and protection ratio demonstrates a good potential of rGM-CSF in improving humoral and cell mediated immune responses of inactivated AIV vaccines.

Phylogenetic and pathogenic characterization of a pigeon paramyxovirus type 1 isolate reveals cross-species transmission and potential outbreak risks in the northwest region of China.

Pigeon paramyxovirus type-1 (PPMV-1) is enzootic in pigeons, causing severe economic loss in the poultry industry in many countries. However, the exact epidemic process of PPMV-1 transmission is still unclear. In this study, we analyzed the complete genome of the PPMV-1/SX-01/15 isolate. Sequence results show that the virus genome contains 15,192 nucleotides, with the gene order 3'-NP-P-M-F-HN-L-5'. Phylogenetic analysis revealed that this genome belongs to subgenotype VIc in class II. The mean death time (MDT) and intracerebral pathogenicity index (ICPI) were 62.4 h and 1.13, respectively, indicating that this isolate is a mesogenic PPMV-1 strain. To our knowledge, this is the first report of a subgenotype VIc mesogenic PPMV-1 strain circulating in commercial pigeon flocks in the northwest region of China. In a comparative infection experiment, the morbidity and mortality rates were 100% and 80%, respectively, in 4-week-old pigeons, whereas they were 50% and 30%, respectively, in 5-week-old chickens. Furthermore, this virus caused severe neurological symptoms in a 4-week-old pigeon and mild neurological symptoms in a 5-week-old chicken. A histopathological examination of the brain showed a classical nonsuppurative encephalitis lesion. The pattern of viral shedding, and viral load, and virus distribution differed between infected chickens and pigeons. Genomic characteristics suggest that there was cross-species transmission of PPMV-1 subgenotype VIc in this region at least from the years 2006 to 2015.

Re-evaluation the immune efficacy of Newcastle disease virus vaccine in commercial laying chickens.

Newcastle disease virus (NDV) infection causes serious problems in laying chickens, like reducing egg production, increasing rate of abnormal eggs in spite of strict vaccination in layer farms program. A new evaluation system is needed to show complete protection of the immunization in laying chickens based on the egg-laying performance, rather than clinical signs of the disease. In this study, laying chickens with different anti-NDV HI (hemagglutination-inhibition) antibody titer after vaccination were divided into different groups. These chickens were then challenged with field isolated highly virulent NDV strains. Results showed that the chickens in low HI titers group (5log2 to 8log2) and medium HI titers group (9log2 to 11log2) had atypical symptoms, produced abnormal eggs, and shed virus. Whereas, with HI titers≥12log2, the chickens were completely protected, and did not show symptoms, or produce abnormal eggs or shed virus. Morbidity, positive viral shedding rate and abnormal egg-rate decreased with increase in pre-challenge HI antibody titer. Our result suggested that 12log2 is the threshold of the HI antibody in providing complete protection to laying chickens under field condition, and protective efficacy is correlated with HI antibody titer. This study provides a valuable reference for the vaccination and control of ND in poultry.

Sonodynamically-induced apoptosis, necrosis, and active oxygen generation by mono-l-aspartyl chlorin e6.

In this study, we investigated the induction of apoptosis by ultrasound in the presence of a photochemically active chlorin, mono-l-aspartyl chlorin e6 (NPe6). HL-60 cells were exposed to ultrasound for up to 3 min in the presence and absence of NPe6, and the induction of apoptosis was examined by analyzing cell morphology, DNA fragmentation, and caspase-3 activity. Cells treated with 80 microM NPe6 and ultrasound clearly showed membrane blebbing and cell shrinkage, whereas significant morphologic changes were not observed in cells exposed to either ultrasound alone, at the same intensity, or NPe6 alone. Also, DNA ladder formation and caspase-3 activation were observed in cells treated with both ultrasound and NPe6 but not in cells treated with ultrasound or NPe6 alone. In addition, NPe6 substantially enhanced nitroxide generation by ultrasound in the same acoustical arrangement. Sonodynamically-induced apoptosis, caspase-3 activation, and nitroxide generation were significantly suppressed by histidine. These results suggest that the combination of ultrasound and NPe6 sonochemically induces apoptosis as well as necrosis in HL-60 cells. They further suggest that some ultrasonically-generated active species, deactivatable by histidine, are the major mediators to induce the observed apoptosis.

Sonodynamically induced apoptosis with porfimer sodium in HL-60 cells.

Sonodynamically induced apoptosis with porfimer sodium in HL-60 cells was investigated. HL-60 cells were exposed to ultrasound for up to 3 min in the presence and absence of porfimer sodium. After the exposure, sonodynamically induced apoptosis was assessed according to morphologic changes, DNA fragmentation and caspase-3 activation. The cells treated with 50 mug/ml porfimer sodium and ultrasound clearly showed membrane blebbing and cell shrinkage, whereas no significant morphologic change was observed in the cells exposed to either ultrasound alone or porfimer sodium alone. DNA ladder formation was observed in the cells treated with ultrasound in the presence of porfimer sodium. Activation of caspase-3 was also observed after the treatment with ultrasound and porfimer sodium. Both sonodynamically induced apoptosis and caspase-3 activation were significantly suppressed by histidine. These results indicate that combination treatment with ultrasound and porfimer sodium induced apoptosis in HL-60 cells. Significant reduction by histidine in both sonodynamically induced apoptosis and caspase-3 activation suggests that some ultrasonically generated active species, deactivatable by histidine, are the major mediators to induce the observed apoptosis.