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Hematopoietic progenitor kinase 1 (HPK1) is a negative regulator of T-cell activation, and targeting HPK1 is considered a promising strategy for improving responses to antitumor immune therapies. The biggest challenge of HPK1 inhibitor design is to achieve a higher selectivity to GLK, an HPK1 homology protein as a positive regulator of T-cell activation. Herein, we report the design of a series of macrocycle-based HPK1 inhibitors via a conformational constraint strategy. The identified candidate compound

Tropomyosin receptor kinase (TRK) inhibition is an effective therapeutic approach for treatment of a variety of cancers. Despite the use of first-generation TRK inhibitor (TRKI) larotrectinib (1) resulting in significant therapeutic response in patients, acquired resistance develops invariably. The emergence of secondary mutations occurring at the solvent-front, xDFG, and gatekeeper regions of TRK represents a common mechanism for acquired resistance. However, xDFG mutations remain insensitive to second-generation macrocyclic TRKIs selitrectinib (3) and repotrectinib (4) designed to overcome the resistance mediated by solvent-front and gatekeeper mutations. Here, we report the structure-based drug design and discovery of a next-generation TRKI. The structure-activity relationship studies culminated in the identification of a promising drug candidate 8 that showed excellent in vitro potency on a panel of TRK mutants, especially TRKAG667C in the xDFG motif, and improved in vivo efficacy than 1 and 3 in TRK wild-type and mutant fusion-driven tumor xenograft models, respectively.

New 8-chloro-2-phenyl-2,7-naphthyridin-1(2H)-one building blocks bearing diverse substitutes on the 2-phenyl group were synthesized via an efficient diaryliodonium salt-based N-arylation strategy with the advantage of mild conditions, short reaction times, and high yields. A small combinatorial library of 8-amino substituted 2-phenyl-2,7-naphthyridin-1(2H)-one was further conveniently constructed based on the above chlorinated naphthyridinones and substituted aniline. Preliminary biochemical screening resulted in the discovery of the new 2,7-naphthyridone-based MET/AXL kinase inhibitors. More importantly, 17c (IC50,MET of 13.8 nM) or 17e (IC50,AXl of 17.2 nM) and 17i (IC50,AXl of 31.8 nM) can efficient selectively inhibit MET or AXL kinase, respectively, while commercial cabozantinib showed no selectivity. The further exploration of the 8-substituted 2-phenyl-2,7-naphthyridin-1(2H)-one combinatorial library would significantly accelerate the discovery of more potent and selective inhibitors against diverse kinases.

During a survey of entomopathogenetic fungi from Southwest China, a new species, Paracremonium lepidopterorum was isolated from a pupa. It differs from other Paracremonium species by its shorter cylindrical phialides and absence of slimy head of conidia and chlamydospores. Phylogenetic analyses based on the combined datasets of ITS+LSU+TUB confirmed that P. lepidopterorum is distinct from other species. The new species is formally described and illustrated, and compared to similar species.

Several secondary tropomyosin receptor kinase (TRK) mutations located in the solvent front, xDFG, and gatekeeper regions, are a common cause of clinical resistance. Mutations in the xDFG motif in particular limit sensitivity to second-generation TRK inhibitors, which represent an unmet clinical need. We designed a series of 3-pyrazolyl-substituted pyrazolo[1,5-a]pyrimidine derivatives toward these secondary mutations using ring-opening and scaffold-hopping strategies. Compound 5n was the most potent, with IC

Aberrant signaling from tropomyosin receptor kinases (TRKs) has been identified as the oncogenic driver in a large variety of cancers, suggesting that inhibition of TRK may be an attractive strategy of attack for anticancer therapeutics. Despite the encouraging therapeutic response to the first-generation TRK inhibitor Larotrectinib (1), the emergence of drug-resistant secondary mutations within its ATP-binding pocket cause relapse in cancer patients. A next-generation inhibitor that overcomes multiple Larotrectinib-resistant mutations is still a major “unmet clinical need”. In this study, the progressive exploration of the structure-activity relationship (SAR) through scaffold hopping, flexibilization, and fluoroalkoxy substitutions produced modifications on the reported compound 1 that led to the discovery of a superior derivative 7g. Compound 7g is a novel, orally available TRK inhibitor that showed excellent in vitro potency on a panel of Larotrectinib-resistant mutants both in biochemical and cellular assays. Compound 7g also exhibited improved in vivo antitumor efficacy in TRK wild type and mutant fusion-driven tumor xenograft models compared to the efficacy of Larotrectinib and Selitrectinib (3). These encouraging results suggest that compound 7g is a promising drug candidate to pursue for development of a TRK inhibitor to overcome clinically acquired resistance to Larotrectinib.

Coronavirus disease 2019, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is still a pandemic around the world. Currently, specific antiviral drugs to control the epidemic remain deficient. Understanding the details of SARS-CoV-2 structural biology is extremely important for development of antiviral agents that will enable regulation of its life cycle. This review focuses on the structural biology and medicinal chemistry of various key proteins (Spike, ACE2, TMPRSS2, RdRp and Mpro) in the life cycle of SARS-CoV-2, as well as their inhibitors/drug candidates. Representative broad-spectrum antiviral drugs, especially those against the homologous virus SARS-CoV, are summarized with the expectation they will drive the development of effective, broad-spectrum inhibitors against coronaviruses. We are hopeful that this review will be a useful aid for discovery of novel, potent anti-SARS-CoV-2 drugs with excellent therapeutic results in the near future.

New 8-chloro-2-phenyl-2,7-naphthyridin-1(2

As part of our effort to develop new molecular targeted antitumor drug, a novel 2,7-naphthyridone-based MET kinase inhibitor, 8-((4-((2-amino-3-chloropyridin-4-yl)oxy)- 3-fluorophenyl)amino)-2-(4-fluorophenyl)-2,7-naphthyridin-1(2H)-one (13f), was identified. Knowledge of the binding mode of BMS-777607 in MET led to the design of new inhibitors that utilize novel 2,7-naphthyridone scaffold to conformationally restrain the key pharmacophoric groups (block C). Detailed SAR studies resulted in the discovery of a new MET inhibitor 13f, displaying favorable in vitro potency and oral bioavailability. More importantly, 13f exhibited excellent in vivo efficacy (tumor growth inhibition/TGI of 114% and 95% in 50 mg/kg, respectively) both in the U-87 MG and HT-29 xenograft models. The favorable drug-likeness of 13f indicated that 2,7-naphthyridinone may be used a promising novel scaffold for antitumor drug development. The preclinical studies of 13f are under way.

As a privileged scaffold, the quinazoline ring is widely used in the development of EGFR inhibitors, while few quinazoline-based MET inhibitors are reported. In our ongoing efforts to develop new MET-targeted anticancer drug candidates, a series of quinazoline-based 1,6-naphthyridinone derivatives were designed, synthesized, and evaluated for their biological activities. The preliminary SARs studies indicate that the quinazoline scaffold was also acceptable for the block A of class II MET inhibitors. The further pharmacokinetic studies led to the identification of the most promising compound 22a with favorable in vitro potency (MET, IC50 = 9.0 nM), human microsomal metabolic stability (t1/2 = 621.2 min) and oral bioavailability (F = 42%). Moreover, 22a displayed good in vivo antitumor efficacy (IR of 81% in 75 mg/kg) in MET-positive human glioblastoma U-87 MG xenograft model. These positive results indicated that 22a is a potential new MET-targeted antitumor drug lead, which is worthy of further development.

A robust, practical, and scalable approach for the construction of 3-substituted 5-chloro-1,6-naphthyridin-4-one derivatives 13 via the addition of Grignard reagents to 4-amino-2-chloronicotinonitrile (15) was developed. Starting with various Grignard reagents, a wide range of 3-substituted 5-chloro-1,6-naphthyridin-4-one derivatives 13 were conveniently synthesized in moderate-to-good yields through addition&ndash, acidolysis&ndash, cyclocondensation. In addition, the robustness and applicability of this synthetic route was proven on a 100 g scale, which would enable convenient sample preparation in the preclinical development of 1,6-naphthyridin-4-one-based MET-targeting antitumor drug candidates.

A potent and novel MET inhibitor, 5-((4-((2-amino-3-chloropyridin-4-yl)oxy)-3-fluorophenyl)amino)-3-(4-fluorophenyl)-1,6-naphthyridin-4(1H)-ones (8), was designed and synthesized via a scaffold-hopping strategy of a 2,7-naphthyridinone MET kinase inhibitor 7. Lead compound 8 had good potency (IC50 of 9.8 nM), but unfavorable pharmacokinetic profiles (F = 12%, CL = 5.0 L/h/kg). Systematic structural optimization of compound 8 resulted in 9g (MET, IC50 = of 9.8 nM) with a comparable MET potency to that of compound 2 and a favorable pharmacokinetic profile (F = 63%, CL = 0.12 L/h/kg). Further study of the derivatization of N(1) amine group of 9g led to the discovery of 23a with good MET potency (IC50 of 7.1 nM), promising VEGFR-2 selectivity (3226-fold), and a markedly drug-likeness improvement (F = 57.7%, CL = 0.02 L/h/kg). The excellent VEGFR-2 selectivity and favorable drug-likeness of 23g suggest that the 1,6-naphthyridine moiety could be used as a new scaffold for kinase inhibitor discovery.

To investigate tetracycline resistance and resistant genotype in Riemerella anatipestifer, the tetracycline susceptibility of 212 R. anatipestifer isolates from China between 2011 and 2017 was tested. The results showed that 192 of 212 (90.6%) R. anatipestifer isolates exhibited resistance to tetracycline (the MICs ranged from 4 to 256 μg/ml). The results of PCR detection showed that, 170 of 212 (80.2%) R. anatipestifer isolates possessed the tet(X) gene. Other genes, including tet(A), tet(M), tet(Q), tet(O), tet(B), and tet(O/W/32/O), were found at frequencies of 20.8, 4.7, 1.4, 0.9, 0.9, and 0.5%, respectively. However, tet(C), tet(E), tet(G), tet(K), and tet(W) were not detected in any isolate. In these tet gene positive strains, 31 (14.6%), 2 (0.9%), 5 (2.4%), 1 (0.5%), 3 (1.4%) were detected containing tet(A)/tet(X), tet(M)/tet(O), tet(M)/tet(X), tet(O)/tet(X), and tet(Q)/tet(X) simultaneously, respectively. One isolates, R131, unexpectedly contained three tet genes, i.e., tet(M), tet(O), and tet(X). Sequence analysis of the tet gene ORFs cloned from R. anatipestifer isolates confirmed that tet(A), tet(B), tet(M), tet(O), tet(Q) and an unusual mosaic tet gene tet(O/W/32/O) were present in R. anatipestifer. The MIC results of R. anatipestifer ATCC 11845 transconjugants carrying tet(A), tet(B), tet(M), tet(O), tet(O/W/32/O), tet(Q), and tet(X) genes exhibited tetracycline resistance with MIC values ranging from 4 to 64 μg/ml. Additionally, the tet(X) gene could transfer into susceptible strain via natural transformation (transformation frequencies of ~10−6). In conclusion, the tet(A), tet(B), tet(M), tet(O), tet(O/W/32/O), tet(Q), and tet(X) genes were found and conferred tetracycline resistance in R. anatipestifer isolates. Moreover, the tet(X) is the main mechanism of tetracycline resistance in R. anatipestifer isolates. To our knowledge, this is the first report of tet(A), tet(B), tet(M), tet(O), tet(Q), and mosaic gene tet(O/W/32/O) in R. anatipestifer.

he DPV UL19 protein encoded by UL19 gene was identified in our laboratory. In this article, we report some information about the structures and functions of this protein by using bioinformatics software or website online. The prediction indicated that DPV UL19 protein had no signal peptide and no transmembrane region, and could be a protein outside the membrane. It might contain many potential epitopes and functional sites. According to the prediction of 3D structure, a high reliability model was found in this protein. These works would be useful for further structural and functional studies of the DPV UL19 protein but experiments are needed to check the predicted results.

A fused benzimidazole–imidazole scaffold was designed and synthesized using a facile procedure involving deprotection and cyclization reactions. The acidic conditions and high yields for the benzimidazole ring closure allow potential application of this efficient methodology for the design and synthesis of a range of bioactive compounds.

The origin and accession number of 25 R. anatipestifer genomes. Table S2. The CRISPR loci in R. anatipestifer genomes. Table S3. Cas9-related information for 39 Gram-negative type II system-containing bacteria. Figure S1. Multiple sequence alignment of Cas2 amino acid sequences. The asterisk represents the conserved base in all of the sequences for alignment. Figure S2. Multiple sequence alignment of Cas1 amino acid sequences. The asterisk represents the conserved base in all of the sequences for alignment. Figure S3. Multiple sequence alignment of Cas9 amino acid sequences. The asterisk represents the conserved base in all of the sequences for alignment. Based on previous report (Chylinski K, et al., [47]), we defined that the amino acid sequence located between 1 and 60 aa, 718â 775 aa and 910â 1131 aa are RuvC-like domains (black boxes). The amino acid sequence located between 776 and 909 aa is considered as HNH domain (red box). The yellow shadows indicate catalytic sites. Figure S4. Multiple sequence alignment for Cas9 from 40 Gram-negative type II system-containing bacteria. ClustalW (in MEGA software) was utilized to perform multiple sequence alignment for Cas9 from 40 Gram-negative type II system-containing bacteria. The default parameter values were chosen when computed. (DOCX 2633Â kb)

Aim Activation of peroxisome proliferator-activated receptor δ (PPARδ) subtypes increases expression of genes involved in fatty acid transport and oxidation and alters adiposity in animal models of obesity and type-2 diabetes. We aim to explore the effects of peroxisome proliferator-activated receptor δ (PPARδ) subtypes on serum lipid profiles in obese rhesus monkey, especially evaluate the efficacy of investigational new drug (HS00098). Methods: First, a prototype of obese rhesus monkey was established by continuously feeding test animals a high fat diet for 2 months. Fifteen obese rhesus monkeys were randomly divided into 3 groups, and the 2 test groups were treated with GW 501516 and HS00098. The test groups were administered doses of 0.3 mg/kg for 1 month, then with 1 mg/kg for 1 month, and finally with 3 mg/kg for 1 month. The control group received placebo treatment. In each experiment, the body weight of each animal was measured and recorded initially and prior to changing the dose of the drug each month. The total cholesterol, blood glucose, triglyceride, high density lipoprotein cholesterol, low-density lipoprotein cholesterol, serum Apo-A1, Apo-B100 and insulin were tested. Results: The average body weight gain of the GW501516 and HS00098 groups was significantly lower than that of the control group. The group receiving the HS00098 treatment had a higher signifycant increase in high density lipoprotein and apo-A1 (P < 0.05) than the control monkeys, while the total cholesterol, triglycerides, low density lipoproteins, apo-B100, and insulin (P < 0.05 or P < 0.01) were significantly decreased. Compared with GW50-1516, the effects of HS00098 on serum lipid profiles in diet-induced obese rhesus monkeys are more obvious. Conclusion: These results suggested that the investigational drug (HS00098) can effectively reduce body weight, blood lipid and blood sugar levels of diet-induced obese rhesus monkeys.

A convenient one-pot protocol is developed for the synthesis of the title compounds (V) and (VII) through Ugi reaction/deprotection/cyclization strategy followed by an intramolecular nucleophilic substitution reaction.

In this paper, we analyzed the UL43 gene of duck plague virus (DPV) of codon usage bias. The results may provide a basis for understanding the evolution and pathogenesis of DPV and for selecting appropriate host expression systems to improve the expression of target gene in vitro. In this study, the synonymous codon usage bias of UL43 gene in the 24 herpesviruses have been analyzed and the results showed obvious differences by the Codon Adaptation Index (CAI), effective number of codons (ENC) and the value of G + C content at the 3rd codon position (GC3s). In addition, the results revealed that the synonymous codons with A and T at the third codon positon have widely usage in the codon of UL43 gene of DPV. G + C compositional constraint is the main factor that determines the codon usage bias in UL43 gene. The phylogenetic analysis suggested that DPV was evolutionarily closer to fowl herpesviruses which further clustered into Alphaherpesvirinae. Furthermore the ORF of UL43 gene has sequential rare codons. There were 25 codons showing distinct usage differences between DPV with Escherichia coli, 24 codons showing distinct usage differences between DPV with yeast, and 32 between DPV and H. sapiens. Therefore the yeast and E. coli expression system may be suitable for the expression of DPV UL43 gene if some codons could be optimized.

Enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) is a molecular biological technology that can be used to study microbial community diversity and dynamics. In many reports, investigations of microbial diversity from environmental samples were based on the agarose gel electrophoresis (AGE) patterns of ERIC-PCR amplified products. This is not a sound practice, since bands with identical positions can contain different sequences; thus, this practice could possibly exaggerate the similarities or diversities among samples. To mitigate this issue, we employed a denaturing gradient gel electrophoresis (DGGE) strategy to explore DNA bands with the same size, between ERIC-PCR profiles of samples. DPS software was used with Shannon-Wiener diversity index (H') to analyze ERIC-PCR fingerprint profiles. H' revealed that the microbial community diversity at DGGE was higher than with AGE. The results of this study suggest that the ERIC-PCR assays with DGGE can provide a better assessment of electrophoresis pattern with regards to the structure of an intestinal microbial community.

This study aimed to examine the host cell apoptosis in the tissues of Peking ducks infected with duck swollen head hemorrhagic disease virus (DSHDV). The dynamic changes associated with apoptosis occurring in the internal tissues were evaluated at different time points postinoculation (PI) by performing hematoxylin and eosin (HE) staining, followed by light microscopy, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, and transmission electron microscopy (TEM). The results showed that DSHDV infection could induce apoptosis in host cells, including those of the bursa of Fabricius (BF), thymus, spleen, liver, intestinal tract, kidney, and esophagus. The apoptotic index (AI) values increased with time from 2 to 72 h PI, and the highest values were recorded at 72 h PI. Further, cell death due to classic necrosis was observed in the dying or deceased ducks after 72 h PI. In conclusion, host cell apoptosis can be induced by DSHDV and may play an important role in the pathogenesis of duck viral swollen head hemorrhagic disease (DVSHD).

This research was undertaken to understand the replication kinetics of Salmonella enteritidis (S. enteritidis) in the internal organs of ducklings after oral challenge over a 2 wk period. A serovar-specific real-time, fluorescence-based quantitative polymerase chain reaction (FQ-PCR) assay was used to detect genomic DNA of S. enteritidis in the blood and the internal organs at different time points respectively. The results showed that the spleen was positive at 12 h post inoculation (PI) and the blood was at 14 h PI. The organism was detected in the liver and heart at 16 h PI, the pancreas and kidney were positive at 20 h PI, and the final organ to show a positive results was the gallbladder at 22 h PI. The copy number of S. enteritidis DNA in each tissue reached a peak at 24 h-36 h PI, with the liver and spleen containing the highest concentration of S. enteritidis. The blood, heart, kidney, pancreas, and gallbladder had low concentrations. S. enteritidis populations began to decrease and were not detectable at 3 d PI, but were still present up to 2 wk for the spleen without causing apparent symptoms. To make the results meaningful, a side-by-side bacteriology method (IFA) was performed. The results of IFA were similar to the FQ-PCR assay. This research provided a significant data for understanding the life cycle of S. enteritidis in the internal organs, and may help to understand the pathogenesis of S.entertidis in the future.

Ducks were subcutaneously infected with a high-virulence strain of Salmonella enterica ssp. enterica serovar Enteritidis (Salmonella Enteritidis). The kinetics of the Salmonella Enteritidis genomic DNA loads, the immunohistochemical localization of the bacterial antigens, and the histopathological examination in various tissues were investigated. The results showed that the time course of the appearance of the Salmonella Enteritidis bacterial antigens and the lesions in various tissues was coincident with the bacterial load of the organism in various infected tissues. This suggests that Salmonella Enteritidis loads in systemic organs are closely correlated with the progression of the infection.

To explore the correlation between neuropsychological features and Chinese medical syndrome types in Wilson's disease (WD) patients, thus providing evidence for early intervention by syndrome typing.Totally 96 WD patients were assigned to Gan-Dan damp-heat syndrome (GDDHS, 31 cases), Gan-Shen yin deficiency syndrome (GSYDS, 47 cases), and qi-blood deficiency syndrome (QBDS, 18 cases) by syndrome typing. Three items of neuropsychological testing were performed in them, i.e., Raven's standard progressive matrices (R'SPM), Stroop color-word test (CWT), trail making test (TMT). The correlations between the integrals of Chinese medical syndrome types and results of the 3 tests were analyzed.(1) There was statistical difference in the total scores of R'SPM, the word interference time of CWT, and interference effects of TMT among the 3 syndrome types (P0.01, P0.05). There was statistical difference in the total scores of R'SPM and the word interference time of CWT in patients of QBDS, when compared with those of GDDHS and GSYDS (P0.05). There was statistical difference in interference effects of TMT in patients of GDDHS, when compared with those of QBDS and GSYDS (P0.05). (2) The integrals of the 3 syndrome types were negatively correlated with the total scores of R'SPM (P0.01). The integral of GDDHS was significantly positively correlated with the interference effects of TMT (P0.01). The integral of GSYDS was significantly positively correlated with TMT-B time consumption and interference effects of TMT (P0. 05). The integral of QBDS was significantly positively correlated with the word interference time of CWT (P0.05).There was correlation between neuropsychological changes of WD patients and Chinese medical syndrome types. The severity of asthenia syndrome was sequenced from high to low as QBDSGSYDSGDDHS. The severity of asthenia was higher than that of asthenia.

We determined the nucleotide sequence of a portion of the duck enteritis virus (DEV) strain CHv which was suspected to be the UL10 gene(GenBank accession no EU195100.) , encoding glycoprotein M(gM). The UL10 gene was cloned and sequenced, which was composed of 1230 nucleotides encoding 409 amino acids, with a predicted molecular mass of 45.752 kDa. We calculated the codon usage bias in the newly identified UL10 gene of Duck Enteritis Virus (DEV) and performed a correlation analysis of codon bias of DEV UL10 gene. There is a strong bias in the DEV UL10 gene towards the synonymous codons with A and T at the third codon position. Multiple sequence alignment suggested that the UL10 gene was highly conserved in Alphaherpesvirinae and similar to the other herpesviral UL10. Phylogenetic analysis showed that the gene had a close evolutionary relationship with the avian herperviruses, and DEV should be placed into a single cluster within the subfamily Alphaherpesvirinae. Furthermore, the UL10△ gene was cloned into a pET prokaryotic expression vector and transformed into Escherichia coli BL21 (DE3). A 6 kDa fusion protein was induced by the further culture at 30℃ after addition of 0.8mM IPTG. Our results may provide some insight for further research about the gene and also enrich the database of herpesvirus.

Based on the duck plague virus (DPV) UL35 gene sequence that our laboratory obtained (GenBank accession number EF643558), a pair of primers was designed using Oligo6.0 and primer5.0, then the UL35 gene was amplified from DPV CHv strain genomic DNA and cloned into the pMD18-T to construct a clone plasmid pMD18-T-UL35. After identification of the pMD18-T-UL35 by PCR amplification and restriction digestion, the fragment of the UL35 gene was subcloned into the prokaryotic expression vector pET-32a(+). The resultant recombinant plasmid pET-32a(+)-UL35 was then transformed into E. coli BL21 (DE3) strain and optimally-expressed under the induction of 1.0 mmol/L IPTG at 34 degrees C for 5 hours. SDS-PAGE analysis showed the recombinant protein (VP26) had a molecular weight of about 33KDa and accounted for 32.3% of total bacterial protein by gel scanning. The protein was then purified by Ni(2+)-affinity chromatography and used to immunize rabbit for producing the VP26 anti-serum and its antibody titer was up to 1:32 detected by agar diffusion reaction. After the IgG of the polyclonal antibodies was purified by High-Q anion-exchange chromatography, Western blot analysis indicated that the IgG had specific reaction with the VP26. Moreover, the subcellular localization detection was observed using immunofluorescence technique. The results showed that the specific fluorescences appeared relatively few in nucleus in 2 to 8 hours and increased gradually in 12 to 36 hours and eventually reached to the maximum, which aggregated in the spot region of the nucleus after the duck embryo fibroblast (DEF) were infected by DPV. However, there were only a small amount of specific fluorescences in the cytoplasm in 12 hours and increased with the extension of infection time in 24 to 48 hours. The specific fluorescences finally reached to the maximum in the cytoplasm in 72 hours. The results provided significant data for furthering the study on the function of DPV UL35 gene.

The characteristics changes of apoptosis of Duck Embryo Fibroblasts (DEF) cells induced by New type gosling viral enteritis virus, NGVEV) were observed by means of HE staining, electron microscopy and Annexin V-FITC/PI fluorescent staining. During 24-48 h post infection (pi), the difference of morphological change between infected DEF cells and the mock infected cells was invisible. At 72 h pi, the nuclear chromatin was getting condensed through HE staining; apoptotic morphological change such as abnormal shape of the nucleus, condensation of the cytoplasm and chromatin were observed under electron microscope; and the early apoptotic cells (Annexin V-FITC positive and PI negative) were detected under fluorescence microscope. At 96-120 h pi, by means of HE staining and electron microscopy, the advanced morphological change of apoptosis such as formation of different kinds of apoptotic bodies, and shrink of the DEF cells and nucleus were detected; under fluorescence microscope the different stages of the apoptotic DEF can be easily distinguished: early apoptotic cells (Annexin V-FITC postive and pi negative), advanced or late apoptotic cells (both Annexin V-FITC and PI positive), necrosis cells or dead cells (Annexin V-FITC negative and PI positive). This investigation shows that NGVEV might induce apoptosis and form characteristic apoptotic morphological changes in the DEF cells. NGVEV inducement of apoptosis may be an important mechanism of efficient dissemination of virus progeny.