Evaluation of Ultra-Microscopic Changes and Proliferation of Apoptotic Glioblastoma Multiforme Cells Induced by Velogenic Strain of Newcastle Disease Virus AF2240

Aim: Newcastle disease virus (NDV) is a member of genus Avulavirus within the family Paramyxoviridae. Interestof using NDV as an anticancer agent has arisen from its ability to kill tumor cells with limited toxicity to normal cells.Methods: In this investigation, the proliferation of brain tumor cell line, glioblastoma multiform (DBTRG.05MG)induced by NDV strain AF2240 was evaluated in-vitro, by using MTT proliferation assay. Furthermore, Cytologicalobservations were studied using fluorescence microscopy and transmission electron microscopy, DNA laddering inagarose gel electrophoresis assay used to detect the mode of cell death and analysis of the cellular DNA content byflowcytometery. Results: MTT proliferation assay, Cytological observations using fluorescence microscopy andtransmission electron microscopy show the anti-proliferation effect and apoptogenic features of NDV on DBTRG.05MG.Furthermore, analysis of the cellular DNA content showed that there was a loss of treated cells in all cell cycle phases(G1, S and G2/M) accompanied with increasing in sub-G1 region (apoptosis peak). Conclusion: It could be concludedthat NDV strain AF2240 is a potent antitumor agent that induce apoptosis and its cytotoxicity increasing while increasingof time and virus titer.     

Validation of efficient high-throughput plasmid and siRNA transfection of human monocyte-derived dendritic cells without cell maturation

Transfection of primary immune cells is difficult to achieve at high efficiency and without cell activation and maturation. Dendritic cells (DCs) represent a key link between the innate and adaptive immune systems. Delineating the signaling pathways involved in the activation of human primary DCs and reverse engineering cellular inflammatory pathways have been challenging tasks. We optimized and validated an effective high-throughput transfection protocol, allowing us to transiently express DNA in naïve primary DCs, as well as investigate the effect of gene silencing by RNA interference. Using a high-throughput nucleofection system, monocyte-derived DCs were nucleoporated with a plasmid expressing green fluorescent protein (GFP), and transfection efficiency was determined by flow cytometry, based on GFP expression. To evaluate the effect of nucleoporation on DC maturation, the expression of cell surface markers CD86 and MHCII in GFP-positive cells was analyzed by flow cytometry. We established optimal assay conditions with a cell viability reaching 70%, a transfection efficiency of over 50%, and unchanged CD86 and MHCII expression. We examined the impact of small interfering RNA (siRNA)-mediated knockdown of RIG-I, a key viral recognition receptor, on the induction of the interferon (IFN) response in DCs infected with Newcastle disease virus. RIG-I protein was undetectable by Western blot in siRNA-treated cells. RIG-I knockdown caused a 75% reduction in the induction of IFNβ mRNA compared with the negative control siRNA. This protocol should be a valuable tool for probing the immune response pathways activated in human DCs.     

NDV7793体外激活的小鼠单核巨噬细胞(MΦ)对小鼠肝癌细胞的杀伤作用及其机制

 目的
初步研究NDV7793激活的小鼠单核巨噬细胞（MΦ）对小鼠肝癌Novikoff细胞的杀伤作用,并探讨其杀伤机制与TNF-
α和TRAIL的关系。 方法从腹腔分离6周龄BALB/C小鼠MΦ, 用NDV7793于体外刺激小鼠MΦ,以ELISA分别测定
NDV7793刺激小鼠MΦ后产生的TNF-α及TRAIL水平;NDV7793体外刺激MΦ后,与小鼠肝癌Novikoff细胞混合培养,
以LDH微量释放法测定小鼠MΦ对小鼠肝癌Novikoff细胞的杀伤效应。同时设立3组实验对照组：IFN-β阳性对照组
、紫外线灭活NDV（UV-NDV）对照组以及空白对照组。 结果与3个对照组相比,NDV7793在体外能提高MΦ分泌TNF-
α、TRAIL的水平;NDV体外刺激后的小鼠MΦ能杀伤小鼠肝癌Novikoff细胞。结论NDV7793在体外能激活小鼠MΦ,
小鼠MΦ被NDV7793刺激后,对小鼠肝癌Novikoff细胞的杀伤作用增强,NDV激活后的小鼠MΦ对小鼠肝癌Novikoff细
胞的杀伤机制可能与TNF-α和TRAIL有关。     

Virulence during Newcastle Disease Viruses Cross Species Adaptation

The hypothesis that host adaptation in virulent Newcastle disease viruses (NDV) has been accompanied by virulence modulation is reviewed here. Historical records, experimental data, and phylogenetic analyses from available GenBank sequences suggest that currently circulating NDVs emerged in the 1920–1940′s from low virulence viruses by mutation at the fusion protein cleavage site. These viruses later gave rise to multiple virulent genotypes by modulating virulence in opposite directions. Phylogenetic and pathotyping studies demonstrate that older virulent NDVs further evolved into chicken-adapted genotypes by increasing virulence (velogenic-viscerotropic pathotypes with intracerebral pathogenicity indexes [ICPIs] of 1.6 to 2), or into cormorant-adapted NDVs by moderating virulence (velogenic–neurotropic pathotypes with ICPIs of 1.4 to 1.6), or into pigeon-adapted viruses by further attenuating virulence (mesogenic pathotypes with ICPIs of 0.9 to 1.4). Pathogenesis and transmission experiments on adult chickens demonstrate that chicken-adapted velogenic-viscerotropic viruses are more capable of causing disease than older velogenic-neurotropic viruses. Currently circulating velogenic–viscerotropic viruses are also more capable of replicating and of being transmitted in naïve chickens than viruses from cormorants and pigeons. These evolutionary virulence changes are consistent with theories that predict that virulence may evolve in many directions in order to achieve maximum fitness, as determined by genetic and ecologic constraints.     

人用新城疫病毒活疫苗喷雾吸入剂的制备与质量检验

目的：探讨新城疫病毒活疫苗喷雾吸入剂的简便制备方法。方法：用SPF种蛋传种、扩增病毒,经超速离心等方法提纯后制成疫苗;以血凝试验等方法测定其滴度和活性;按生物制品的要求进行安全性试验。结果：本疫苗能保持较高的生物学活性,并能达到临床前安全性的内控质量要求。结论：用本法制备供肿瘤临床试验用的新城疫病毒活疫苗是可行的。     

NDV联合热固化瘤苗的抗肿瘤作用

为了研究新城疫病毒（ＮＤＶ） 联合热固化瘤苗抑制肿瘤生长和免疫调节的作用，检测了荷瘤小鼠的抑瘤率和细胞免疫。结果显示，实验组Ⅰ与Ⅱ抑瘤率分别为２４．８％ 、４１．１％ ，２ 组的平均瘤重明显小于对照组，实验组Ⅱ在不同时间段的ＮＫ 细胞活性高于对照组（ Ｐ＜０ ．０１） 。提示ＮＤＶ联合热瘤苗对荷瘤小鼠的抑瘤作用和增强ＮＫ 细胞活性均比单独应用ＮＤＶ效果好。     

Intranasal administration of a live-attenuated recombinant newcastle disease virus expressing the SARS-CoV-2 Spike protein induces high neutralizing antibody levels and protects from experimental challenge infection in hamsters

The emergence of SARS-CoV-2 in December 2019 resulted in the COVID-19 pandemic. Recurring disease outbreaks repeatedly overloaded the public health sector and severely affected the global economy. We developed a candidate COVID-19 vaccine based on a recombinant Newcastle disease virus (NDV) vaccine vector, encoding a pre-fusion stabilized full-length Spike protein obtained from the original SARS-CoV-2 Wuhan isolate. Vaccination of hamsters by intra-muscular injection or intra-nasal instillation induced high neutralizing antibody responses. Intranasal challenge infection with SARS-CoV-2 strain Lelystad demonstrated that both vaccination routes provided partial protection in the upper respiratory tract, and almost complete protection in the lower respiratory tract, as measured by suppressed viral loads and absence of histological lung lesions. Activity wheel measurements demonstrated that animals vaccinated by intranasal inoculation rapidly recovered to normal activity. NDV constructs encoding the spike of SARS-CoV-2 may be attractive candidates for development of intra-nasal COVID-19 booster vaccines.     

Characterization and genetic analysis of recent and emergent virulent newcastle disease viruses in Egypt

Extensive vaccination against Newcastle disease virus (NDV) induced more selective immune pressure from hosts that enhanced the evolutionary process of NDV. Herein, we characterized 13 recently isolated NDV isolates from vaccinated chicken flocks during 2014–2017. Sequence analysis of F gene showed the presence of ¹¹²RRQKRF¹¹⁷ velogenic cleavage motif in 11 isolates, whereas the other two isolates (Ck/ME3/Eg/16 and Ck/ME5/Eg/16) showed the monobasic motif ¹¹²GGRQGRL¹¹⁷. Interestingly, isolate Ck/ME5/Eg/16 showed 100% and 99.82% nucleotide identity with the LaSota F gene hypervariable region and full‐length gene, respectively. On the other hand, isolate Ck/ME3/Eg/16 revealed natural recombination with strains NDV/Ck/Egypt/3/2006 and NDV/Teal/VRLCU/Egypt/2015 that indicates re‐emergence of that old strain. Interestingly, all 13 isolates showed high intracerebral pathogenicity (ICPI) and mean death time (MDT) despite the presence of lentogenic motif in both Ck/ME5/Eg/16 and Ck/ME3/Eg/16 isolates. Comparative analysis of F antigenic epitopes in our isolates with other published sequences from Egypt revealed high sequence conservation; recent isolates had one fixed amino acid substitution (K78R) and a novel V168I substitution, whereas a D170N substitution was detected in older strains (NDV‐EG‐35‐2014 and NDV‐KFR‐B7‐2012). Taken together, our results support the first isolation of virulent NDV isolates with a lentogenic motif; isolate Ck/ME5/Eg/16 might be generated in nature from LaSota live vaccine, whereas isolate Ck/ME3/Eg/16 is emergent from NDV/Ck/Egypt/3/2006. We conclude that the current diagnostic evaluation of the virulence of NDV isolates by characteristic amino acid residues at the F protein cleavage site is insufficient. There is a need to link virologic and epidemiologic data together, and routine and emergency LaSota vaccination protocols should be carefully and optimally applied, with regards to the timing and presence of co‐infecting agents in the field.     

Synergy or interference of a H9N2 avian influenza virus with a velogenic Newcastle disease virus in chickens is dose dependent

Field observations indicate that the impact of velogenic Newcastle disease virus (vNDV) is more severe in countries with concomitant circulation of low pathogenicity avian influenza virus, as is the case in the Middle East, in particular in Israel, where H9N2 and NDV are endemic. In our study, we evaluated how the exposure of chickens to an H9N2 challenge either favours or interferes with a subsequent vNDV infection and its transmission to sentinels. For this purpose, single vNDV and sequential H9/NDV challenges were performed with increasing doses of vNDV (10¹–10⁶ EID₅₀). The H9N2 challenge made birds more susceptible to the vNDV, lowering the minimum dose required to cause an infection, exacerbating the clinical outcome, while delaying the onset of the disease and time of death. Interestingly, the presence and degree of these seemingly contrasting effects were dose-dependent and not mutually exclusive.