Newcastle Disease Virus Manipulates Mitochondrial MTHFD2-Mediated Nucleotide Metabolism for Virus Replication.

Viruses require host cell metabolic reprogramming to satisfy their replication demands; however, the mechanism by which the Newcastle disease virus (NDV) remodels nucleotide metabolism to support self-replication remains unknown. In this study, we demonstrate that NDV relies on the oxidative pentose phosphate pathway (oxPPP) and the folate-mediated one-carbon metabolic pathway to support replication. In concert with [1,2-13C2] glucose metabolic flow, NDV used oxPPP to promote pentose phosphate synthesis and to increase antioxidant NADPH production. Metabolic flux experiments using [2,3,3-2H] serine revealed that NDV increased one-carbon (1C) unit synthesis flux through the mitochondrial 1C pathway. Interestingly, methylenetetrahydrofolate dehydrogenase (MTHFD2) was upregulated as a compensatory mechanism for insufficient serine availability. Unexpectedly, direct knockdown of enzymes in the one-carbon metabolic pathway, except for cytosolic MTHFD1, significantly inhibited NDV replication. Specific complementation rescue experiments on small interfering RNA (siRNA)-mediated knockdown further revealed that only a knockdown of MTHFD2 strongly restrained NDV replication and was rescued by formate and extracellular nucleotides. These findings indicated that NDV replication relies on MTHFD2 to maintain nucleotide availability. Notably, nuclear MTHFD2 expression was increased during NDV infection and could represent a pathway by which NDV steals nucleotides from the nucleus. Collectively, these data reveal that NDV replication is regulated by the c-Myc-mediated 1C metabolic pathway and that the mechanism of nucleotide synthesis for viral replication is regulated by MTHFD2. IMPORTANCE Newcastle disease virus (NDV) is a dominant vector for vaccine and gene therapy that accommodates foreign genes well but can only infect mammalian cells that have undergone cancerous transformation. Understanding the remodeling of nucleotide metabolic pathways in host cells by NDV proliferation provides a new perspective for the precise use of NDV as a vector or in antiviral research. In this study, we demonstrated that NDV replication is strictly dependent on pathways involved in redox homeostasis in the nucleotide synthesis pathway, including the oxPPP and the mitochondrial one-carbon pathway. Further investigation revealed the potential involvement of NDV replication-dependent nucleotide availability in promoting MTHFD2 nuclear localization. Our findings highlight the differential dependence of NDV on enzymes for one-carbon metabolism, and the unique mechanism of action of MTHFD2 in viral replication, thereby providing a novel target for antiviral or oncolytic virus therapy.

Dietary inclusion of mushroom (Flammulina velutipes) stem waste on growth performance and immune responses in growing layer hens.

BACKGROUND: Medicinal mushrooms contain biologically active substances that can be used as an immune-modulating agent in poultry. The present study aimed to investigate the effects of Flammulina velutipes mushroom waste (FVW) on performance, immune response and serum immunity in growing layer hens. RESULTS: No significant differences (P > 0.05) were observed with respect to average daily feed intake, body weight gain and feed conversion ratio among the experimental groups during the entire study period (1-70 days). Antibody titers against Newcastle disease and infectious bronchitis were higher (P < 0.05) in the FVW fed groups than in the control and antibiotic groups. On day 28, serum immunoglobulin (Ig)A and IgG were higher (P < 0.05) in the 6% FVW group than in the control and antibiotic fed groups. On day 70, serum IgA was higher (P < 0.05) in FVW fed groups than in the control group; IgG was higher (P < 0.05) in the FVW groups than in the control and antibiotic groups. However, IgM was higher (P < 0.05) in both the 4% and 6% FVW groups than in the control and antibiotic groups for both experimental periods. Serum cytokine interleukin (IL)-2 and tumor necrosis factor-α concentrations were significantly higher (P < 0.05) in both the 4% and 6% FVW grousp than in the control and antibiotic groups; IL-4 was significantly higher (P < 0.05) in the FVW groups than in the control group; and IL-6 was significantly higher (P < 0.05) in the 6% FVW group than in the control and antibiotic groups. CONCLUSION: FVW at the 6% level can be used as a potential phytogenic feed stuff in growing layer hen rations with respect to improving the immune response without affecting normal weight gain. © 2018 Society of Chemical Industry.

Genomic and biological characterization of Newcastle disease viruses isolated from migratory mallards (Anas platyrhynchos).

Given the global evolutionary dynamics of Newcastle disease viruses (NDVs), it is imperative to continue extensive surveillance, routine monitoring and characterization of isolates originating from natural reservoirs (waterfowls). In this report, we isolated and characterized two virulent NDV strains from clinically healthy mallard (Anas platyrhynchos). Both isolates had a genome of 15,192 nucleotides encoding six genes in an order of 3´-NP-P-M-F-HN-L-5´. The biological characteristics (mean death time: 49.5-50 hr, EID50108.5 ml-1) and presence of a typical cleavage site in the fusion (F) protein (112R-R-Q-K-R↓F117) confirmed the velogenic nature of these isolates. Phylogenetic analysis classified both isolates as members of genotype VII within class-II. Furthermore, based upon the hypervariable region of the F gene (375 nt), isolates showed clustering within sub-genotype VIIi. Similarity index and parallel comparison revealed a higher nucleotide divergence from commonly used vaccine strains; LaSota (21%) and Mukteswar (17%). A comparative residues analysis with representative strains of different genotypes, including vaccine strains, revealed a number of substitutions at important structural and functional domains within the F and hemagglutinin-neuraminidase (HN) proteins. Together, the results highlight consistent evolution among circulating NDVs supporting extensive surveillance of the virus in waterfowl to better elucidate epidemiology, evolutionary relationships and their impacts on commercial and backyard poultry.

Newcastle disease virus-like particles induce dendritic cell maturation and enhance viral-specific immune response.

Circulating of genotype VII Newcastle disease virus (NDV) is a great threat to the poultry industry worldwide. Virus-like particles (VLPs) are increasingly being considered as potential viral vaccines due to their safety and efficacy. In this study, we analyzed in vitro the stimulatory effects of VLPs containing the matrix and hemagglutinin-neuraminidase of genotype VII NDV on dendritic cells (DCs) and evaluated their immunogenicity in mice. The results showed that immature bone marrow-derived dendritic cells (BMDCs) responded to stimulation with VLPs by up-regulating expressions of MHC II, CD40, CD80, and CD86 molecules and by increasing the cytokine secretions of TNF-α, IFN-γ, IL-6, and IL-12p70. Besides, VLPs enhanced the immunostimulatory capacity of DCs to stimulate autologous T cell proliferation. Furthermore, VLPs can induce efficient humoral and cellular immune responses, and recruit mature DCs to the spleen in C57BL/6 mice, as shown by an obvious increase in double-positive proliferation of splenic CD11c+CD86+ cells. These data indicate that NDV VLPs can be a valuable candidate for NDV vaccine development.

Perturbations in the antioxidant metabolism during Newcastle disease virus (NDV) infection in chicken : protective role of vitamin E.

The aim of the present study was to investigate the effect of vitamin E on pro/anti-oxidant status in the liver, brain and heart of Newcastle disease virus (NDV) infected chickens. Activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferase (GST) and the levels of reduced glutathione and malonaldehyde were estimated in selected tissues of uninfected, NDV-infected and NDV + vit. E-treated chickens. A significant increase in MDA levels in brain and liver (p < 0.05) was observed in NDV-infected chickens when compared to controls. The activities of SOD, CAT, GPx, GR, GST and levels of GSH were significantly (p < 0.05) decreased in brain and liver of NDV-infected chickens over controls. On the other hand, a significant decreased MDA levels and enhanced antioxidant enzyme activity levels were observed in NDV + vit. E-treated animals compared to NDV-infected chickens. Histopathological studies revealed that liver of NDV infected chicken shows focal coagulation and infiltration of hepatocytes, whereas neuronal necrosis and degeneration of Purkinje cells were observed in brain and moderate infiltration of inflammatory cells was observed in heart. However such histological alterations were not observed in NDV + vit. E-treated animals. The results of the present study, thus demonstrated that antioxidant defense mechanism is impaired after the induction of NDV, suggesting its critical role in cellular injury in brain and liver. Further, the results also suggest that vitamin E treatment will ameliorate the antioxidant status in the infected animals. The findings could be beneficial to understand the role of oxidative stress in the pathogenesis of NDV and therapeutic interventions of antioxidants.

Combined exposure of Japanese quails to cyanotoxins, Newcastle virus and lead: oxidative stress responses.

Wild birds are continually exposed to many anthropogenic and natural stressors in their habitats. Over the last decades, mass mortalities of wild birds constitute a serious problem and may possibly have more causations such as natural toxins including cyanotoxins, parasitic diseases, industrial chemicals and other anthropogenic contaminants. This study brings new knowledge on the effects of controlled exposure to multiple stressors in birds. The aim was to test the hypothesis that influence of cyanobacterial biomass, lead and antigenic load may combine to enhance the effects on birds, including modulation of antioxidative and detoxification responses. Eight treatment groups of model species Japanese quail (Coturnix coturnix japonica) were exposed to various combinations of these stressors. The parameters of detoxification and oxidative stress were studied in liver and heart after 30 days of exposure. The antioxidative enzymatic defense in birds seems to be activated quite efficiently, which was documented by the elevated levels and activities of antioxidative and detoxification compounds and by the low incidence of damage to lipid membranes. The greatest modulations of glutathione level and activities of glutathione-S-transferase, glutathione peroxidase, glutathione reductase, superoxide dismutase, catalase and lipid peroxidation were shown mostly in the groups with combined multiple exposures. The results indicate that the antioxidative system plays an important role in the protective response of the tissues to applied stressors and that its greater induction helps to protect the birds from more serious damage. Most significant changes of these "defense" parameters in case of multiple stressors suggest activation of this universal mechanism in situation with complex exposure and its crucial role in protection of the bird health in the environment.

Newcastle disease virus-induced cytopathic effect in infected cells is caused by apoptosis.

The velogenic Newcastle disease virus (NDV) causes highly infectious and economically significant Newcastle disease (ND) in birds of various species. In cell culture NDV induces cytopathic effect (CPE) characterized by rounding, vacuolation, syncytia formation and cell death. Aside from cell to cell fusion caused by the F and HN glycoprotein of the virus molecular events leading to cell death are not known. In the current study, NDV-infected Vero cells, at 48 h p.i., showed nuclear condensation, cytoplasm blebbing, DNA fragmentation, and phosphatidylserine translocation to the cell surface. In addition, virus-infected cells demonstrated decreased DNA content and an increased Bax to Bcl-2 ratio, p53 level and caspase 3, 8, 9 expression compared to mock-infected cells. Based on these results, it was concluded that CPE in NDV-infected cells was caused by to the induction of apoptosis with the involvement of p53 and the Bax, dependent apoptotic pathways.

Partial characterisation of five cloned viruses differing in pathogenicity, obtained from a single isolate of pigeon paramyxovirus type 1 (PPMV-1) following passage in fowls' eggs.

Viruses with intracerebral pathogenicity indices (ICPIs) of 0.025, 0.55, 1.013 and 1.3. were cloned from a PPMV-1 isolate with an ICPI of 0.32 by passage in embryonated fowls' eggs. Deduced amino acid sequences of the haemagglutinin-neuraminidase (HN) and precursor fusion proteins (F0) showed them to have only a single amino acid difference: those with an ICPI value <0.7 had proline at amino acid position 453 of the F0 protein, and those with an ICPI value >0.7 contained a serine. The virus with an ICPI of 0.025 was further passaged, and the ICPI of non-cloned virus increased to 0.76/0.79, which was then reduced to 0.49 on cloning. The proline at residue 453 was retained, but there were two nucleotide changes in the virus of ICPI 0.49, T --> C at position 1769 in the untranslated region of the fusion gene and G --> A at position 437 of the HN gene, resulting in the amino acid change G --> R at position 116 in the HN protein.

Differentially regulated interferon response determines the outcome of Newcastle disease virus infection in normal and tumor cell lines.

Newcastle disease virus (NDV) is a negative-strand RNA virus with oncolytic activity against human tumors. Its effectiveness against tumors and safety in normal tissue have been demonstrated in several clinical studies. Here we show that the spread of NDV infection is drastically different in normal cell lines than in tumor cell lines and that the two cell types respond differently to beta interferon (IFN-beta) treatment. NDV rapidly replicated and killed HT-1080 human fibrosarcoma cells but spread poorly in CCD-1122Sk human skin fibroblast cells. Pretreatment with endogenous or exogenous IFN-beta completely inhibited NDV replication in normal cells but had little or no effect in tumor cells. Thus, the outcome of NDV infection appeared to depend on the response of uninfected cells to IFN-beta. To investigate their differences in IFN responsiveness, we analyzed and compared the expression and activation of components of the IFN signal transduction pathway in these two types of cells. The levels of phosphorylated STAT1 and STAT2 and that of the ISGF3 complex were markedly reduced in IFN-beta-treated tumor cells. Moreover, cDNA microarray analysis revealed significantly fewer IFN-regulated genes in the HT-1080 cells than in the CDD-1122Sk cells. This finding suggests that tumor cells demonstrate a less-than-optimum antiviral response because of a lesion in their IFN signal transduction pathway. The rapid spread of NDV in HT-1080 cells appears to be caused by their deficient expression of anti-NDV proteins upon exposure to IFN-beta.

Effect of fusion protein cleavage site mutations on virulence of Newcastle disease virus: non-virulent cleavage site mutants revert to virulence after one passage in chicken brain.

Virulence of Newcastle disease virus (NDV) is mainly determined by the amino acid sequence of the fusion (F0) protein cleavage site. Full-length NDV cDNA clone pNDFL was used to generate infectious NDV with defined mutations in the F0 cleavage site (RRQRR downward arrow L, GRQGR downward arrow F, RRQGR downward arrow F, RGQRR downward arrow F and RKQKR downward arrow F). All the mutants were viable and the mutations were maintained after virus propagation in embryonated eggs. The mutants showed single-cell infections on chicken embryo fibroblasts, which suggested that they were non-virulent. However, virulence tests in 1-day-old chickens resulted in an intracerebral pathogenicity index (ICPI) between 0 and 1.3. Moreover, virulent virus was isolated from chickens that had died in the virulence tests. Subsequent sequence analysis showed that the mutants RRQRR downward arrow L, RRQGR downward arrow F, RGQRR downward arrow F and RKQKR downward arrow F gave rise to the appearance of revertants containing the virulent cleavage site RRQ(K/R)R downward arrow F and an ICPI of 1.4 or higher. This indicated that reversion to virulence was caused by alteration of the amino acid sequence of the F0 cleavage site from a non-virulent to a virulent type. Furthermore, the ICPI of the revertants was higher than that of cDNA-derived strain NDFLtag, which has the same cleavage site, RRQRR downward arrow F (ICPI=1.3). NDFLtag(Pass), which was isolated from dead chickens after intracerebral inoculation of NDFLtag, also showed an increase in the ICPI from 1.3 to 1.5. This study proves that reversion to virulence occurs within non-virulent NDV populations and that the virulence may increase after one passage in chicken brain.

Effect of adrenal blocking chemicals on viral and respiratory infections of chickens.

In a series of experiments chickens were treated with chemicals which block the production of corticosterone by the adrenal cortex prior to being challenged with respiratory disease (and other) agents in order to determine if the course of the diseases could be altered. Some chickens received a single intramuscular injection (14 mg/kg) of 1,1-dichloro-2,2-bis/p-chlorophenyl/ethane (ABC) dissolved in corn oil (20 mg/mL) at least 12 h before challenge. Other chickens received feed containing 500 mg/kg of metyrapone for at least 12 h before and during the challenge infection. Treated chickens were more resistant than the untreated controls to Newcastle disease virus, Mycoplasma gallisepticum, combined M. gallisepticum-Newcastle disease virus infection, and avian adenovirus group II infection. The feeding of erythromycin (1 g/kg of feed), one day before and during the challenge, reduced the severity of M. gallisepticum infection. The effects of feeding both metyrapone and erythromycin resulted in a further increase in resistance. Chickens which had been treated with ABC had less severe lesions and greater postchallenge weight gain than the controls in response to a secondary Escherichia coli infection.

Effects of Newcastle disease virus infection on the binding, phagocytic, and bactericidal activities of respiratory macrophages of the turkey.

Effects of Newcastle disease virus (NDV) infection on the binding, phagocytic, and bactericidal activities of turkey respiratory macrophages were studied. Respiratory macrophages of the turkey demonstrated the presence of immunoglobulin (Ig) G and complement receptors but lacked IgM receptors. Respiratory macrophages from NDV-infected turkeys showed little or no depression of binding of sheep erythrocyte-IgG complexes and sheep erythrocyte-IgM-complement complexes to their appropriate membrane receptors. In contrast, respiratory macrophages from NDV-infected turkeys showed significant (P less than or equal to 0.05) depression of phagocytosis of similar complexes. Bacterial killing by respiratory macrophages from NDV-infected turkeys was significantly (P less than or equal to 0.05) inhibited.

Electrocardiographic and respiratory responses to viscerotropic and neurotropic strains of Newcastle disease virus measured by radio telemetry.

Two groups each of White Rock chickens from 6 to 23 weeks old were monitored by radio telemetry to determine their electrocardiogram and respiration response after they were infected with either a velogenic viscerotropic isolate of Newcastle disease virus (VVND) or the neurotropic GB strain of Newcastle disease virus (NGB). Significant changes were found in the heart rate, R wave amplitude, ST segment elevation, T wave amplitude, RS complex interval, ST segment duration T wave interval, TP segment duration, PR segment duration and TP interval of the birds infected with VVND, but no significant ECG changes were found in the birds infected with NGB. There were no significant changes in respiration rate in any birds.