Green Monkey Research Articles

Long-Read RNA Sequencing Identifies Polyadenylation Elongation and Differential Transcript Usage of Host Transcripts During SARS-CoV-2 In Vitro Infection.

Better methods to interrogate host-pathogen interactions during Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infections are imperative to help understand and prevent this disease. Here we implemented RNA-sequencing (RNA-seq) using Oxford Nanopore Technologies (ONT) long-reads to measure differential host gene expression, transcript polyadenylation and isoform usage within various epithelial cell lines permissive and non-permissive for SARS-CoV-2 infection. SARS-CoV-2-infected and mock-infected Vero (African green monkey kidney epithelial cells), Calu-3 (human lung adenocarcinoma epithelial cells), Caco-2 (human colorectal adenocarcinoma epithelial cells) and A549 (human lung carcinoma epithelial cells) were analyzed over time (0, 2, 24, 48 hours). Differential polyadenylation was found to occur in both infected Calu-3 and Vero cells during a late time point (48 hpi), with Gene Ontology (GO) terms such as viral transcription and translation shown to be significantly enriched in Calu-3 data. Poly(A) tails showed increased lengths in the majority of the differentially polyadenylated transcripts in Calu-3 and Vero cell lines (up to ~101 nt in mean poly(A) length, padj = 0.029). Of these genes, ribosomal protein genes such as RPS4X and RPS6 also showed downregulation in expression levels, suggesting the importance of ribosomal protein genes during infection. Furthermore, differential transcript usage was identified in Caco-2, Calu-3 and Vero cells, including transcripts of genes such as GSDMB and KPNA2, which have previously been implicated in SARS-CoV-2 infections. Overall, these results highlight the potential role of differential polyadenylation and transcript usage in host immune response or viral manipulation of host mechanisms during infection, and therefore, showcase the value of long-read sequencing in identifying less-explored host responses to disease.

AK-2011 strain for the development of a vaccine against equine rhinopneumonitis.

Equine rhinopneumonitis is an acute, highly contagious disease found virtually worldwide. The purpose of the studies presented in this paper is to develop a technology for the manufacture of a cell-derived equine rhinopneumonitis vaccine, as well as to assess the safety and immunogenicity of the newly developed vaccine in laboratory animals model. The object of the studies was the AK-2011 strain isolated from the horses suffering from rhinopneumonitis during an outbreak of abortions. The viability of the AK-2011 strain was assessed using a continuous line of calf trachea cells, a continuous line of calf kidney cells, a continuous line of sheep kidney cells, a continuous line of bovine kidney cells, a continuous line of green monkey kidney cells, a continuous line of Syrian hamster kidney cells, a primary trypsinized culture of horse kidney cells grown in tubes and flasks and the AK-2011 laboratory strain of equine rhinopneumonitis virus with biological activity of 6.0lg TCID50/cm 3 . Sequencing and polymerase chain reaction analysis were performed. The virus isolated from the ORF68 gene in Kazakhstan appeared to be the most similar to the T-953 and 2222-03 strains isolated in the USA and Australia, respectively, in terms of phylogenetics. As to primary infections, cytopathic effects (CPEs) induced by the AK-2011 virus stain (dilution 101 ) in calf trachea and horse kidney cell cultures were stable from the first to tenth passages, with biological activity of 5.75-6.00 lg TCID50/cm 3 . CPEs caused by the virus were apparent on days 2-3, further developed intensively and extended to 60-80% of the cell monolayer on days 5-7. The vaccine results can be used to immunize horses on farms against rhinopneumonia, and horses should be immunized twice with an interval of 2-3 months.

Chemical Constituents and Antiproliferative Activity of Eleusine indica (L.) Gaertn.

Eleusine indica, locally known as ‘rumput sambau’ is widely used by traditional practitioners in many parts of the world to treat various ailments including sprained muscle, asthma, black jaundice, stomach pain, diarrhea, convulsions, and those related to infection such as malaria, influenza, dysentery as well as pneumonia. The plant has been reported to possess a wide array of biological activities, which were antioxidant, anti-inflammatory, antimicrobial, antidiabetic, antipyretic, antiplasmodial, antiviral, hepatoprotective, and urolithiasis. Despite all the reported traditional uses and biological activities, not many chemical constituents have been isolated from the plant. In this work, E. indica methanolic and hexane extracts were assessed for their anti-proliferative activities against human liver cancer (HepG2) and African green monkey kidney epithelial normal (Vero) cell lines through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Although both extracts were considered to be not active on both cell lines with an IC50 of 91.02 ± 5.74 and 85.30 ± 3.03 µg/mL, and CC50 of >1000 and 639.39 ± 13.97 µg/mL, they were nevertheless more selective towards HepG2 cancer cells compared to the Vero normal cells with selectivity indexes (SI) of 10.99 and 7.50, respectively. Chemical profiling of the extracts on ultra-high performance liquid chromatography (UHPLC) coupled with a diode array detector showed a mixture of different complexity and polarities of compounds. Further isolation and purification on the hexane extract afforded β-sitosterol and stigmasterol, while the methanolic extract yielded loliolide, a benzofuran type of compound. Loliolide has been previously reported to exhibit antitumor activity, which could correlate to the higher antiproliferative selectivity against HepG2 cancer cell line of the methanolic compared to the hexane extracts. This is the first report on the isolation of β-sitosterol, stigmasterol, and loliolide from E. indica.

High-Throughput Screening of a Marine Compound Library Identifies Anti-Cryptosporidium Activity of Leiodolide A.

Cryptosporidium sp. are apicomplexan parasites that cause significant morbidity and possible mortality in humans and valuable livestock. There are no drugs on the market that are effective in the population most severely affected by this parasite. This study is the first high-throughput screen for potent anti-Cryptosporidium natural products sourced from a unique marine compound library. The Harbor Branch Oceanographic Institute at Florida Atlantic University has a collection of diverse marine organisms some of which have been subjected to medium pressure liquid chromatography to create an enriched fraction library. Numerous active compounds have been discovered from this library, but it has not been tested against Cryptosporidium parvum. A high-throughput in vitro growth inhibition assay was used to test 3764 fractions in the library, leading to the identification of 23 fractions that potently inhibited the growth of Cryptosporidium parvum. Bioassay guided fractionation of active fractions from a deep-sea sponge, Leiodermatium sp., resulted in the purification of leiodolide A, the major active compound in the organism. Leiodolide A displayed specific anti-Cryptosporidium activity at a half maximal effective concentration of 103.5 nM with selectivity indexes (SI) of 45.1, 11.9, 19.6 and 14.3 for human ileocecal colorectal adenocarcinoma cells (HCT-8), human hepatocellular carcinoma cells (Hep G2), human neuroblastoma cells (SH-SY5Y) and green monkey kidney cells (Vero), respectively. The unique structure of leiodolide A provides a valuable drug scaffold on which to develop new anti-Cryptosporidium compounds and supports the importance of screening natural product libraries for new chemical scaffolds.

CHIKV strains Brazil (wt) and Ross (lab-adapted) differ with regard to cell host range and antiviral sensitivity and show CPE in human glioblastoma cell lines U138 and U251.

Chikungunya virus (CHIKV), a (re)emerging arbovirus, is the causative agent of chikungunya fever. To date, no approved vaccine or specific antiviral therapy are available. CHIKV has repeatedly been responsible for serious economic and public health impacts in countries where CHIKV epidemics occurred. Antiviral tests in vitro are generally performed in Vero-B4 cells, a well characterised cell line derived from the kidney of an African green monkey. In this work we characterised a CHIKV patient isolate from Brazil (CHIKVBrazil) with regard to cell affinity, infectivity, propagation and cell damage and compared it with a high-passage lab strain (CHIKVRoss). Infecting various cell lines (Vero-B4, A549, Huh-7, DBTRG, U251, and U138) with both virus strains, we found distinct differences between the two viruses. CHIKVBrazil does not cause cytopathic effects (CPE) in the human hepatocarcinoma cell line Huh-7. Neither CHIKVBrazil nor CHIKVRoss caused CPE on A549 human lung epithelial cells. The human astrocyte derived glioblastoma cell lines U138 and U251 were found to be effective models for lytic infection with both virus strains and we discuss their predictive potential for neurogenic CHIKV disease. We also detected significant differences in antiviral efficacies regarding the two CHIKV strains. Generally, the antivirals ribavirin, hydroxychloroquine (HCQ) and T-1105 seem to work better against CHIKVBrazil in glioblastoma cells than in Vero-B4. Finally, full genome analyses of the CHIKV isolates were done in order to determine their lineage and possibly explain differences in tissue range and antiviral compound efficacies.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11262-022-01892-x.

Development and optimization of a high-throughput screening assay for in vitro anti-SARS-CoV-2 activity: Evaluation of 5676 Phase 1 Passed Structures.

Although vaccines are currently used to control the coronavirus disease 2019 (COVID‐19) pandemic, treatment options are urgently needed for those who cannot be vaccinated and for future outbreaks involving new severe acute respiratory syndrome coronavirus virus 2 (SARS‐CoV‐2) strains or coronaviruses not covered by current vaccines. Thus far, few existing antivirals are known to be effective against SARS‐CoV‐2 and clinically successful against COVID‐19. As part of an immediate response to the COVID‐19 pandemic, a high‐throughput, high content imaging–based SARS‐CoV‐2 infection assay was developed in VeroE6 African green monkey kidney epithelial cells expressing a stable enhanced green fluorescent protein (VeroE6‐eGFP cells) and was used to screen a library of 5676 compounds that passed Phase 1 clinical trials. Eight drugs (nelfinavir, RG‐12915, itraconazole, chloroquine, hydroxychloroquine, sematilide, remdesivir, and doxorubicin) were identified as inhibitors of in vitro anti–SARS‐CoV‐2 activity in VeroE6‐eGFP and/or Caco‐2 cell lines. However, apart from remdesivir, toxicity and pharmacokinetic data did not support further clinical development of these compounds for COVID‐19 treatment.

Analysis of R848 as an Adjuvant to Improve Inactivated Influenza Vaccine Immunogenicity in Elderly Nonhuman Primates.

Elderly individuals are highly susceptible to developing severe outcomes as a result of influenza A virus (IAV) infection. This can be attributed to alterations that span the aged immune system, which also result in reduced responsiveness to the seasonal inactivated vaccine. Given the rapidly increasing number of individuals in this age group, it is imperative that we develop strategies that can better protect this population from IAV-associated disease. Based on our previous findings that the TLR7/8 agonist resiquimod (R848) could efficiently boost responses in the newborn, another population with decreased vaccine responsiveness, we evaluated this adjuvant in an elderly African green monkey (AGM) model. AGM aged 16–24 years old (equivalent to 64–96 in human years) were primed and boosted with inactivated A/PuertoRico/8/1934 (H1N1) (IPR8) alone or directly linked to R848 (IPR8-R848). We observed increases in the level of circulating virus-specific IgM antibody 10 days following primary vaccination in AGM that were vaccinated with IPR8-R848, but not IPR8 alone. In addition, there were significant increases in virus-specific IgG after boosting selectively in the IPR8-R848 vaccinated animals. These findings provide insights into the ability of R848 to modulate the aged immune system in the context of IAV vaccination.

Whole-Genome Sequencing of Vero E6 (VERO C1008) and Comparative Analysis of Four Vero Cell Sublines.

The Vero cell line is an immortalized cell line established from kidney epithelial cells of the African green monkey. A variety of Vero sublines have been developed and can be classified into four major cell lineages. In this study, we determined the whole-genome sequence of Vero E6 (VERO C1008), which is one of the most widely used cell lines for the proliferation and isolation of severe acute respiratory syndrome coronaviruses (SARS-CoVs), and performed comparative analysis among Vero JCRB0111, Vero CCL-81, Vero 76, and Vero E6. Analysis of the copy number changes and loss of heterozygosity revealed that these four sublines share a large deletion and loss of heterozygosity on chromosome 12, which harbors type I interferon and CDKN2 gene clusters. We identified a substantial number of genetic differences among the sublines including single nucleotide variants, indels, and copy number variations. The spectrum of single nucleotide variants indicated a close genetic relationship between Vero JCRB0111 and Vero CCL-81, and between Vero 76 and Vero E6, and a considerable genetic gap between the former two and the latter two lines. In contrast, we confirmed the pattern of genomic integration sites of simian endogenous retroviral sequences, which was consistent among the sublines. We identified subline-specific/enriched loss of function and missense variants, which potentially contribute to the differences in response to viral infection among the Vero sublines. In particular, we identified four genes (IL1RAP, TRIM25, RB1CC1, and ATG2A) that contained missense variants specific or enriched in Vero E6. In addition, we found that V739I variants of ACE2, which functions as the receptor for SARS-CoVs, were heterozygous in Vero JCRB0111, Vero CCL-81, and Vero 76; however, Vero E6 harbored only the allele with isoleucine, resulting from the loss of one of the X chromosomes.

MRNA expression analysis of the hippocampus in a vervet monkey model of fetal alcohol spectrum disorder

BackgroundFetal alcohol spectrum disorders (FASD) are common, yet preventable developmental disorders that stem from prenatal exposure to alcohol. This exposure leads to a wide array of behavioural and physical problems with a complex and poorly defined biological basis.Molecular investigations to date predominantly use rodent animal models, but because of genetic, developmental and social behavioral similarity, primate models are more relevant. We previously reported reduced cortical and hippocampal neuron levels in an Old World monkey (Chlorocebus sabaeus) model with ethanol exposure targeted to the period of rapid synaptogenesis and report here an initial molecular study of this model. The goal of this study was to evaluate mRNA expression of the hippocampus at two different behavioural stages (5 months, 2 years) corresponding to human infancy and early childhood.MethodsOffspring of alcohol-preferring or control dams drank a maximum of 3.5 g ethanol per kg body weight or calorically matched sucrose solution 4 days per week during the last 2 months of gestation. Total mRNA expression was measured with the Affymetrix GeneChip Rhesus Macaque Genome Array in a 2 × 2 study design that interrogated two independent variables, age at sacrifice, and alcohol consumption during gestation.Results and discussionStatistical analysis identified a preferential downregulation of expression when interrogating the factor ‘alcohol’ with a balanced effect of upregulation vs. downregulation for the independent variable ‘age’. Functional exploration of both independent variables shows that the alcohol consumption factor generates broad functional annotation clusters that likely implicate a role for epigenetics in the observed differential expression, while the variable age reliably produced functional annotation clusters predominantly related to development. Furthermore, our data reveals a novel connection between EFNB1 and the FASDs; this is highly plausible both due to the role of EFNB1 in neuronal development as well as its central role in craniofrontal nasal syndrome (CFNS). Fold changes for key genes were subsequently confirmed via qRT-PCR.ConclusionPrenatal alcohol exposure leads to global downregulation in mRNA expression. The cellular interference model of EFNB1 provides a potential clue regarding how genetically susceptible individuals may develop the phenotypic triad generally associated with classic fetal alcohol syndrome.

Identifying HSV-1 Inhibitors from Natural Compounds via Virtual Screening Targeting Surface Glycoprotein D.

Herpes simplex virus (HSV) infections are a worldwide health problem in need of new effective treatments. Of particular interest is the identification of antiviral agents that act via different mechanisms compared to current drugs, as these could interact synergistically with first-line antiherpetic agents to accelerate the resolution of HSV-1-associated lesions. For this study, we applied a structure-based molecular docking approach targeting the nectin-1 and herpesvirus entry mediator (HVEM) binding interfaces of the viral glycoprotein D (gD). More than 527,000 natural compounds were virtually screened using Autodock Vina and then filtered for favorable ADMET profiles. Eight top hits were evaluated experimentally in African green monkey kidney cell line (VERO) cells, which yielded two compounds with potential antiherpetic activity. One active compound (1-(1-benzofuran-2-yl)-2-[(5Z)-2H,6H,7H,8H-[1,3] dioxolo[4,5-g]isoquinoline-5-ylidene]ethenone) showed weak but significant antiviral activity. Although less potent than antiherpetic agents, such as acyclovir, it acted at the viral inactivation stage in a dose-dependent manner, suggesting a novel mode of action. These results highlight the feasibility of in silico approaches for identifying new antiviral compounds, which may be further optimized by medicinal chemistry approaches.

In-vitro Assessment of Antiviral Activity of the Herbo Mineral Capsule, Fema SakthiTM, against Human Coronavirus (HCoV) on VERO Cells

Introduction: With the spread of COVID-19 pandemic, healthcare workers and patients look for alternate medicines including Siddha, Ayurveda, Unani and other forms of traditional medicines as we still do not have promising antiviral drugs for COVID-19. Objective: To evaluate the in-vitro antiviral activity of Fema Sakthi™ (FS) by eliciting the inhibition of cytopathic effect of Human Coronavirus (HCoV) on African Green Monkey kidney cells (VERO cells). Materials and Method:The cytopathic effect (CPE) was performed on Vero cells with Human Coronavirus 229E, a type of Coronavirus associated with respiratory infections. The Median Tissue Culture Infectious Dose (TCID50) was evaluated using Reed-Muench method. 100 TCID50 of HCoV 229E viral suspensions were added to VERO cell culture to induce the cytopathic effect. Uninfected and untreated cells were used as control and five concentrations (62.5, 125, 250, 500 & 1000 μg/mL) of Fema Sakthi™ (FS) were used to study the anti-viral activity. After incubation for 72 hours, the cell viability was observed under the inverted microscope after staining with 0.1% crystal violet. Results: Fema Sakthi™ (FS) was found to exhibit inhibition of cytopathic effect at lower concentrations (62.5, 125 and 250 μg/mL) but at higher concentrations (500 and 1000 μg/mL), the formulation itself was cytotoxic to the cells. Conclusion: This preliminary study showed that FS has antiviral activity at lower concentrations 62.5, 125 and 250 μg/mL on the VERO cells. However, further specific studies have to be carried out to confirm the anti-viral activity and clinical efficacy using other preclinical and clinical models of Human Coronavirus (HCoV) including COVID-19.

A Comprehensive Atlas of Immunological Differences Between Humans, Mice, and Non-Human Primates.

Animal models are an integral part of the drug development and evaluation process. However, they are unsurprisingly imperfect reflections of humans, and the extent and nature of many immunological differences are unknown. With the rise of targeted and biological therapeutics, it is increasingly important that we understand the molecular differences in the immunological behavior of humans and model organisms. However, very few antibodies are raised against non-human primate antigens, and databases of cross-reactivity between species are incomplete. Thus, we screened 332 antibodies in five immune cell populations in blood from humans and four non-human primate species generating a comprehensive cross-reactivity catalog that includes cell type-specificity. We used this catalog to create large mass cytometry universal cross-species phenotyping and signaling panels for humans, along with three of the model organisms most similar to humans: rhesus and cynomolgus macaques and African green monkeys; and one of the mammalian models most widely used in drug development: C57BL/6 mice. As a proof-of-principle, we measured immune cell signaling responses across all five species to an array of 15 stimuli using mass cytometry. We found numerous instances of different cellular phenotypes and immune signaling events occurring within and between species, and detailed three examples (double-positive T cell frequency and signaling; granulocyte response to Bacillus anthracis antigen; and B cell subsets). We also explore the correlation of herpes simian B virus serostatus on the immune profile. Antibody panels and the full dataset generated are available online as a resource to enable future studies comparing immune responses across species during the evaluation of therapeutics.

Novel antiviral activity of PAD inhibitors against human beta-coronaviruses HCoV-OC43 and SARS-CoV-2

The current SARS-CoV-2 pandemic, along with the likelihood that new coronavirus strains will appear in the nearby future, highlights the urgent need to develop new effective antiviral agents. In this scenario, emerging host-targeting antivirals (HTAs), which act on host-cell factors essential for viral replication, are a promising class of antiviral compounds. Here we show that a new class of HTAs targeting peptidylarginine deiminases (PADs), a family of calcium-dependent enzymes catalyzing protein citrullination, is endowed with a potent inhibitory activity against human beta-coronaviruses (HCoVs). Specifically, we show that infection of human fetal lung fibroblasts with HCoV-OC43 leads to enhanced protein citrullination through transcriptional activation of PAD4, and that inhibition of PAD4-mediated citrullination with either of the two pan-PAD inhibitors Cl-A and BB-Cl or the PAD4-specific inhibitor GSK199 curbs HCoV-OC43 replication. Furthermore, we show that either Cl-A or BB-Cl treatment of African green monkey kidney Vero-E6 cells, a widely used cell system to study beta-CoV replication, potently suppresses HCoV-OC43 and SARS-CoV-2 replication. Overall, our results demonstrate the potential efficacy of PAD inhibitors, in suppressing HCoV infection, which may provide the rationale for the repurposing of this class of inhibitors for the treatment of COVID-19 patients.

Introduced Monkey Populations of Florida - History, Status, and Potential Impacts

At least nine populations of non-human primates have established in Florida since the 1930s. These include three species: rhesus macaques (Macaca mulatta), vervet monkeys (Chlorocebus sabaeus), and squirrel monkeys (Saimiri sp.). Invasion success has varied among the three species. As of 2021, a single, growing population of rhesus macaques was present in central Florida; a single, stable population of vervet monkeys was present in south Florida; and only two squirrel monkeys remained of a previous population on a private property in south Florida. Demonstrated and potential impacts of the three species also varied. Continued monitoring and research of introduced primate populations in Florida is critical to better understand impacts and management needs.

Validation of Excitation-Scan Hyperspectral Multi-faceted Mirror Array Prototype System Advancements to Hyperspectral Imaging Applications.

Hyperspectral imaging technologies (HSI) have undergone rapid development since their beginning stages. While original applications were in remote sensing, other uses include agriculture, food safety and medicine. HSI has shown great utility in fluorescence microscopy for detecting signatures from many fluorescent molecules; however, acquisitions speeds have been slow due to light losses associated with spectral filtering. Therefore, we designed a novel light emitting diode (LED)-based rapid excitation scanning hyperspectral imaging platform allowing users to obtain simultaneous measurements of fluorescent labels without compromising acquisition speeds. Previously, we reported our results of the optical ray trace simulations and the geometrical capability of designing a multifaceted mirror imaging system as an initial approach to combine light at many wavelengths. The design utilized LEDs and a multifaceted mirror array to combine light sources into a liquid light guide. The computational model was constructed using Monte Carlo optical ray software (TracePro, Lambda Research Corp.). Recent prototype validation results show that when compared to a commercial emission scanning spectral confocal microscope (Zeiss-LSM-980), the novel LED-based excitation scanning HSI prototype successfully detected and separated six fluorescent labels from a custom 6-label African green monkey kidney epithelial cells. We report on the prototype's ability to overcome limitations of acquisition speeds, sensitivity, and specificity present in conventional systems. Future work will evaluate prototype's light losses to determine latent design modifications needed to demonstrate the system's feasibility as a promising solution for overcoming HSI acquisition speeds. This work was supported by NSF award MRI1725937.

Intrathecal amyloid-beta oligomer administration increases tau phosphorylation in the medial temporal lobe in the African green monkey: A nonhuman primate model of Alzheimer's disease.

An obstacle to developing new treatment strategies for Alzheimer's disease (AD) has been the inadequate translation of findings in current AD transgenic rodent models to the prediction of clinical outcomes. By contrast, nonhuman primates (NHPs) share a close neurobiology with humans in virtually all aspects relevant to developing a translational AD model. The present investigation used African green monkeys (AGMs) to refine an inducible NHP model of AD based on the administration of amyloid-beta oligomers (AβOs), a key upstream initiator of AD pathology. AβOs or vehicle were repeatedly delivered over 4 weeks to age-matched young adult AGMs by intracerebroventricular (ICV) or intrathecal (IT) injections. Induction of AD-like pathology was assessed in subregions of the medial temporal lobe (MTL) by quantitative immunohistochemistry (IHC) using the AT8 antibody to detect hyperphosphorylated tau. Hippocampal volume was measured by magnetic resonance imaging (MRI) scans prior to, and after, intrathecal injections. IT administration of AβOs in young adult AGMs revealed an elevation of tau phosphorylation in the MTL cortical memory circuit compared with controls. The largest increases were detected in the entorhinal cortex that persisted for at least 12 weeks after dosing. MRI scans showed a reduction in hippocampal volume following AβO injections. Repeated IT delivery of AβOs in young adult AGMs led to an accelerated AD-like neuropathology in MTL, similar to human AD, supporting the value of this translational model to de-risk the clinical trial of diagnostic and therapeutic strategies.

Effect of Pien Tze Huang against EV71

This study aims to investigate the inhibitory effect of Pien Tze Huang(PZH) on enterovirus 71(EV71). To be speci-fic, chemiluminescence method was adopted to evaluate the toxicity of PZH to African green monkey kidney(Vero) cells and human rhabdomyosarcoma(RD) cells, and cytopathic effect(CPE) method to assess the inhibition on EV71-GFP reporter virus and EV71 C4 wild-type virus. The results showed that PZH had low cytotoxicity to Vero cells and RD cells, with the half-maximal cytotoxic concentration(CC_(50)) of about 0.691 3-0.879 2 mg·mL~(-1) for the two. In addition, PZH can effectively inhibit the replication of EV71 within the non-cytotoxic concentration range, and dose-dependently alleviate the cytopathic changes caused by virus infection, with the half-maximal effective concentration(EC_(50)) of 0.009 2-0.106 3 mg·mL~(-1). On the basis of the above results, the green fluorescent protein(GFP), indirect immunofluorescence assay(IFA), and median tissue culture infective dose(TCID_(50)) were employed to assess and verify the anti-EV71-GFP and anti-EV71 C4 activity of PZH. The results demonstrated that PZH can dose-dependently lower the expression of GFP by EV71-GFP and structural protein VP-1 by EV71 C4 and decrease the production of progeny infectious viruses. The EC_(50) of PZH for EV71-GFP and EV71 C4 was about 0.006 0-0.006 2 mg·mL~(-1) and 0.006 6-0.025 6 mg·mL~(-1), respectively. This study suggested that PZH may exert antiviral activity by acting on EV71 and interfering with the expression of VP-1. At the moment, there is still a lack of specific anti-EV71 drugs. This study proposed a new idea for the symptomatic treatment of EV71 infections such as hand-foot-mouth disease and verified an effective drug for the treatment of EV71 infections.

560G>A (rs4986782) (R187Q) Single Nucleotide Polymorphism in Arylamine N-Acetyltransferase 1 Increases Affinity for the Aromatic Amine Carcinogens 4-Aminobiphenyl and N-Hydroxy-4-Aminobiphenyl: Implications for Cancer Risk Assessment.

Human arylamine N-acetyltransferase 1 (NAT1) catalyzes the N-acetylation of arylamine carcinogens such as 4-aminobiphenyl (ABP), and following N-hydroxylation, the O-acetylation of N-hydroxy-arylamine carcinogens such as N-hydroxy-ABP (N-OH-ABP). Genetic polymorphisms in NAT1 are linked to cancer susceptibility following exposures. The effects of individual single nucleotide polymorphisms (SNPs) in the NAT1 coding exon on Michaelis-Menten kinetic constants was assessed for ABP N-acetyltransferase and N-OH-ABP O-acetyltransferase activity following transfection of human NAT1 into COS-1 cells (SV40-transformed African green monkey kidney cells). NAT1 coding region SNPs 97C > T (rs56318881) (R33stop), 190C > T (rs56379106) (R64W), 559C > T (rs5030839) (R187stop) and 752A > T (rs56172717) (D251V) reduced ABP N- acetyltransferase and N-OH-ABP O-acetyltransferase activity below detection. 21T > G (rs4986992) (synonymous), 402T > C (rs146727732) (synonymous), 445G > A (rs4987076) (V149I), 613A > G (rs72554609) (M205V) and 640T > G (rs4986783) (S241A) did not significantly affect Vmax for ABP N-acetyltransferase or N-OH-ABP O-acetyltransferase. 781G > A (rs72554610) (E261K), and 787A > G (rs72554611) (I263V) slightly reduced ABP N-acetyltransferase and N-OH-ABP O-acetyltransferase activities whereas 560G > A (rs4986782) (R187Q) substantially and significantly reduced them. 560G > A (rs4986782) (R187Q) significantly reduced the apparent Km for ABP and N-OH-ABP a finding that was not observed with any of the other NAT1 SNPs tested. These findings suggest that the role of the 560G > A (rs4986782) (R187Q) SNP cancer risk assessment may be modified by exposure level to aromatic amine carcinogens such as ABP.

Detailed analysis of the pathologic hallmarks of Nipah virus (Malaysia) disease in the African green monkey infected by the intratracheal route.

Disease associated with Nipah virus infection causes a devastating and often fatal spectrum of syndromes predominated by both respiratory and neurologic conditions. Additionally, neurologic sequelae may manifest months to years later after virus exposure or apparent recovery. In the two decades since this disease emerged, much work has been completed in an attempt to understand the pathogenesis and facilitate development of medical countermeasures. Here we provide detailed organ system-specific pathologic findings following exposure of four African green monkeys to 2.41×105 pfu of the Malaysian strain of Nipah virus. Our results further substantiate the African green monkey as a model of human Nipah virus disease, by demonstrating both the respiratory and neurologic components of disease. Additionally, we demonstrate that a chronic phase of disease exists in this model, that may provide an important opportunity to study the enigmatic late onset and relapse encephalitis as it is described in human disease.

Chronic Collection of Cerebrospinal Fluid from Rhesus Macaques (Macaca mulatta) with Cisterna Magna Ports: Update on Refinements.

More than 20 y ago, we developed an animal model for chronic and continuous collection of cerebrospinal fluid (CSF) from conscious rhesus macaques. Since our previous publication in 2003, we have successfully implanted 168 rhesus macaques using this approach. Our experience enables us to provide up-to-date information regarding the model, including refine- ments to our implant design, reductions in maintenance, and new procedures for dealing with contamination. The results of our experiences have reduced the number of surgeries required and helped to increase the longevity of the implant, with some functioning for more than 18 y. Building on our success in rhesus macaques, we attempted to develop similar animal models in the African green monkeys and dogs but have been unable to develop reliable chronic models for CSF collection in these species.