Molecular characterization of Newcastle disease virus vaccines in Nigeria.

Background and Aim: Newcastle disease (ND) caused by ND virus (NDV) is a serious impediment to effective poultry production in developing countries such as Nigeria. Despite employing vaccination and other control measures to curtail this disease, its severe forms still persist. This study aimed to confirm the virus strains in the NDV vaccine brands commonly used in Nigeria. Materials and Methods: We employed reverse-transcription polymerase chain reaction (RT-PCR), sequencing, and sequence analysis to characterize NDV strains in four NDV vaccines commonly used in Nigeria. Fragments of 300 bp from NDV fusion genes from the vaccines were amplified. Polymerase chain reaction products were sequenced and analyzed using multiple sequence alignment and phylogenetic analyses to characterize the vaccine viruses as pathotypes. Results: All the vaccines gave positive results, confirming the presence of NDV. Multiple sequence alignment and phylogenetic analyses revealed that two of the vaccines had the lentogenic pathotype, while the other two had the mesogenic or velogenic pathotype. Conclusion: This study provides information to facilitate strategies for regular control of the quality of vaccines in Nigeria.

Diagnostic Performance of a Rapid Immunochromatographic Test Kit for Detecting Canine Parvovirus Infection.

In dogs, canine parvovirus (CPV) enteritis is associated with high morbidity and fatality rates requiring early diagnosis to facilitate treatment and reduce its spread. In recent times, various commercial immunochromatographic (IC) test kits are available for its rapid diagnosis, which require an assessment of their accuracy. Therefore, precision of a point-of-care IC combination test kit for canine coronavirus (CCoV)/CPV faecal antigen detection was evaluated in this study. Multicentred random faecal samples from 115 dogs with gastroenteritis were checked for the presence of CPV antigens using the SensPERT IC combination test kit and the result was compared with polymerase chain reaction (PCR) as a reference test. Parvovirus was detected in 105 (91.3%) and 108 (93.9%) faecal samples by the point-of-care test kit and PCR, respectively. The point-of-care IC test kit showed 95.4% relative sensitivity, 71.4% specificity, 98.1% positive predictive value, 50.0% negative predictive value, and 93.9% accuracy comparable to conventional PCR in the samples tested. This point-of-care test kit also demonstrated a fair positive likelihood ratio (3.34), a very low negative likelihood ratio (0.07) and a moderate agreement (Kappa = 0.6) compared with conventional PCR. This test kit has shown to be very useful in the screening of dogs for CPV infection, and is a reliable alternative for diagnosing CPV both in conventional laboratories and remote areas without laboratories. Negative results in the IC testing with high suspicion of CPV infection should be further confirmed using superior test such as PCR.

Molecular detection and characterization of Sarcoptes scabiei var canis using skin scrapings and skin biopsies.

Canine sarcopticosis is a highly infectious and debilitating parasitic skin disease of dogs. Its diagnosis stands challenging as the golden standard of diagnosis; skin scraping microscopy is characterized by several diagnostic variations. Study thus employed several alternate diagnostic approaches using Polymerase Chain Reaction (PCR) on skin scrapings and skin biopsies. Whole Sarcoptes scabiei var canis mites, thirty six "3 cm × 3cm" skin scrapings and 3 mm punch biopsies from six different lesioned sites per infested dog were all obtained from six severely sarcoptes ridden dogs. Samples were mechanically disrupted for DNA extraction and amplification. Positive samples were further commercially sequenced. Amongst the thirty six (36) skin biopsy and scraping samples processed, PCR detected the DNA of Sarcoptes scabiei var canis in thirty two (32) skin biopsy samples with a sensitivity of 88.88%. Twenty five (25) skin scraping samples were also positive for scabies with a sensitivity of 69.44%. The Phylogenetic analysis revealed a relationship between the Sarcoptes scabiei var canis mites from Nigeria and Sarcoptes scabiei of humans, raccoon dogs and rabbits in Pakistan, Japan and Egypt.  The diagnostic errors and false negatives accompanying the skin microscopy diagnostic technique can best be limited with the use of PCR diagnosis on skin scrapings and skin biopsies most especially. This highly sensitive diagnostic tool would certainly and effectively control the menace of sarcopticosis in dogs.

Molecular characterisation of canine parvoviruses from clinical samples and vaccines in Nigeria.

Canine parvovirus (CPV) the causative agent of canine parvovirus enteritis is an intractable pathogen of dogs characterised by mutations, evolutionary changes and eventual vaccine failure. The disease is a serious problem in dogs with limited studies conducted in Nigeria. Therefore, this study was designed to characterise the subtypes of CPV isolates in six commonly used vaccines and 157 clinical samples collected from seven states in Nigeria from June 2016 to March 2018. Faecal samples collected from the clinical cases were subjected to in-clinic immunoassay to detect viral antigens. Polymerase chain reaction (PCR) was used to amplify viral VP2 gene in the samples and commonly used vaccines in Nigeria. Thereafter, PCR products were sequenced and analysed. The result showed that 93.0% of the dogs tested positive for CPV in both assays; 72.8% were puppies less than six months old, with 58.3% of them vaccinated. Partial VP2 gene sequence and phylogenetic analysis of 11 random clinical samples showed that CPV-2c 7(63.6%) and CPV-2a 4(36.4%) were the predominant subtypes in Nigeria; with genetic signatures that are 98.7% to 99.9% closely related to Asian and European strains, respectively. No CPV-2b was detected. Amino acid mutation analysis divulged some imperative transmutation sites: D305Y, Y324I, Q370R, N375D, T440A, Y444S, I447M and Y451C in the isolates. The viruses in the vaccines were characterised as the wild-type CPV. The genetic variability, viral population heterogeneity and phylogenetic linkage with isolates from other countries probably suggest transboundary migrations and local differentiations are contributing to continuous CPV evolution and vaccine failure in Nigeria.

Pathology and molecular diagnosis of canine parvoviral enteritis in Nigeria: case report.

This report describes the clinical presentation, pathology and molecular diagnosis of canine parvovirus infection in male Boerboel and female Alsatian puppies. The history of the dogs was considered, examined clinically for vital parameters, haemogram changes and faeces screened for parasites and canine parvovirus faecal antigen. Tissue samples were taken at necropsy for confirmatory diagnosis using histopathology, immunohistochemistry, polymerase chain reaction (PCR) and sequence analysis. There was a severe regenerative anaemia, leucopenia and lymphopaenia. The positive antigen faecal test and pathological findings of haemorrhagic enteritis suggested canine parvoviral enteritis disease. Polymerase chain reaction and sequence analysis confirmed canine parvovirus-2a as the aetiology of the disease. Informed management is important to avoid complications resulting from secondary to severe dehydration, hypovolemia from marked gastrointestinal fluid and protein loss and sepsis from bacterial translocation and leukopenia.

Limited Intrahost Diversity and Background Evolution Accompany 40 Years of Canine Parvovirus Host Adaptation and Spread.

Canine parvovirus (CPV) is a highly successful pathogen that has sustained pandemic circulation in dogs for more than 40 years. Here, integrating full-genome and deep-sequencing analyses, structural information, and in vitro experimentation, we describe the macro- and microscale features that accompany CPV's evolutionary success. Despite 40 years of viral evolution, all CPV variants are more than ∼99% identical in nucleotide sequence, with only a limited number (<40) of substitutions becoming fixed or widespread during this time. Notably, most substitutions in the major capsid protein (VP2) gene are nonsynonymous, altering amino acid residues that fall within, or adjacent to, the overlapping receptor footprint or antigenic regions, suggesting that natural selection has channeled much of CPV evolution. Among the limited number of variable sites, CPV genomes exhibit complex patterns of variation that include parallel evolution, reversion, and recombination, compromising phylogenetic inference. At the intrahost level, deep sequencing of viral DNA in original clinical samples from dogs and other host species sampled between 1978 and 2018 revealed few subconsensus single nucleotide variants (SNVs) above ∼0.5%, and experimental passages demonstrate that substantial preexisting genetic variation is not necessarily required for rapid host receptor-driven adaptation. Together, these findings suggest that although CPV is capable of rapid host adaptation, a relatively low mutation rate, pleiotropy, and/or a lack of selective challenges since its initial emergence have inhibited the long-term accumulation of genetic diversity. Hence, continuously high levels of inter- and intrahost diversity are not necessarily required for virus host adaptation.IMPORTANCE Rapid mutation rates and correspondingly high levels of intra- and interhost diversity are often cited as key features of viruses with the capacity for emergence and sustained transmission in a new host species. However, most of this information comes from studies of RNA viruses, with relatively little known about evolutionary processes in viruses with single-stranded DNA (ssDNA) genomes. Here, we provide a unique model of virus evolution, integrating both long-term global-scale and short-term intrahost evolutionary processes of an ssDNA virus that emerged to cause a pandemic in a new host animal. Our analysis reveals that successful host jumping and sustained transmission does not necessarily depend on a high level of intrahost diversity nor result in the continued accumulation of high levels of long-term evolution change. These findings indicate that all aspects of the biology and ecology of a virus are relevant when considering their adaptability.

The aqueous tuber extract of Pueraria tuberosa (Willd.) D.C. caused cytotoxic effect on HT 29 cell lines with down regulation of nuclear factor-kappa B (NF-κB).

Background Pueraria tuberosa (Willd) D.C. (Fabaceae) tubers are already used in traditional medicine by Ayurvedic physicians for the management of fertility disorders, general weakness, and also as anti-ageing therapies. Other known pharmacological properties include: anti-hyperglycemics, hepatoprotective, anti-hyperlipidemic, diuretic, nutritive, and anti-fertility agents in male rats. Methods The anti-proliferative effect of the aqueous tuberous root extract of Pueraria tuberosa on vascular smooth muscle cells (VSMCs) and Human Colorectal Adenocarcinoma Cell lines (HT-29) was investigated using the Cell Titer 96 MTT Proliferation Assay where the viable cells were seeded at a density of 5 × 104 (100 µL/well). For VSMC, log concentrations of the extract at 200 and 800 µg/mL were added and incubated for 24 and 48 h time points. Incubation of the extract in the presence of vascular endothelial growth factor (VEGF) and ET-1 was also conducted at different times. Concentrations of the extract (200, 400 and 700 µg/mL) were also added and incubated with the HT 29 cell lines for 24, 48 and 72 h time points. The effect of the tuber aqueous extract of the plant on nuclear factor-κB (NF-κB) expression after 2 h was also carried out using immunoblotting technique. Results The result showed that after 24 h, the effect of the extract in the presence of the mitogens and on the VSMC was more of proliferation. However, at 48 h, the 200 µg/mL dose, both alone and in the presence of VEGF caused 11.1% and 25.9% decreases respectively, in cell proliferation. In the HT 29 cytotoxic study the 200 µg/mL concentration caused the greatest cytotoxic effect at 77.1% cell inhibition followed by 400 µg/mL concentration at 71.4% after 72 h. The immunoblotting assay showed a down regulation of NF-κB expressions with 0.7 µg/mL concentration showing the greatest effect. NF-κB, a pro-inflammatory agent is increasingly recognized as a crucial player in many steps of cancer initiation and progression. Conclusions It could therefore be concluded that the aqueous root extract of Pueraria tuberosa possesses cytotoxic effect and could serve as a lead compound for anticancer and anti-inflammatory agents.

Gonado-histopathological changes, intersex and endocrine disruptor responses in relation to contaminant burden in Tilapia species from Ogun River, Nigeria.

In the present study, we investigated the occurrence of intersex condition, histopathological changes in the gonad and endocrine disruptor biomarker responses in Tilapia species (Tilaipia guineensis, Sarotherodon galileaus and Oreochromis niloticus) along the Ogun River, Nigeria. The study sites covered a length of 320 km and a total of 1074 tilapias were collected from three sampling sites (Abeokuta, Isheri and Ikorodu) with different degrees of anthropogenic contamination. Samples were also collected from an upstream putative control site (Igboho) along the Ogun River. Hepatic transcript levels for vitellogenin (Vtg), zona radiata (Zrp) and aromatase (cyp19a1) were analyzed using real-time PCR. Gross gonadal morphology revealed a 24% prevalence of intersex showing visible testis and ovary in phenotypic females (25.4%) or males (74.6%). The intersex condition paralleled histopathological changes (ovotestis or testis-ova) in the gonads of female and male fish, respectively. Plasma concentrations of luteinizing hormone (LH), follicle stimulating hormone (FSH), 11-ketotestosterone (11-KT) and estradiol-17ß (E2) were measured using enzyme immunoassay, showing that male fish from downstream of the control site had significantly higher plasma E2, LH, and FSH concentrations compared to females. Similarly, Vtg, Zrp and cyp19a1 mRNA was significantly higher in males, compared to females. Analysis of contaminants showed the presence of 15 PCB congeners, lindane and dieldrin, and 4-iso-nonylphenol (4-iso-NP) and 4-tert-octylphenol (4-tert-OP) in fish muscle and sediment samples from Ogun River. Principal component analysis (PCA) revealed site and sex relationships between measured biological responses to groups of environmental contaminants, showing that the endocrine disruptive responses in fish were associated with biota and sediment contaminant burden. In addition, strong positive correlations were observed between male fish and Zrp, cyp19a1, E2, LH, FSH, PCBs, 4-iso-NP and 4-tert-OP, suggesting possible feminization effects of these contaminants on the male. In female fish, PCBs, 4-iso-NP and 4-tert-OP showed positive relationships with 11-KT and gonadosomatic index (GSI), suggesting masculinization effects by these contaminants. Overall, our findings demonstrate a causal relationship between endocrine disruption and contaminants burden in Tilapias species from Ogun River.

Metastatic prostate cancer cell-specific phage-like particles as a targeted gene-delivery system.

BACKGROUND: One of the cardinal requirements for effective therapeutic management of tumors is the selective delivery of cancer drugs to the right site by ligand-decorated nanomedicines. Screening of 2 × 109 clone landscape phage library provides a reliable avenue for generating protein ligands specific for tumor cells. It was shown that selective phage proteins derived from landscape phage libraries against breast and prostate cancer cells are able to navigate drug or siRNA loaded liposomes to corresponding cancer cells with minimal toxicity to non-neoplastic cells. In an alternative platform, glioma cell-specific phage proteins were used for assembling in vivo cancer-specific phage-like particles, named 'phagemid infective particles' as targeted gene-delivery vehicles. METHODS: To extend the panel of anticancer cell phages, we have screened a 2 × 109 clone landscape phage library f8/8 to select phage clones specific for metastatic prostate cancer cell PC-3M. The phage clones were characterized for their selective interaction with PC-3M cells using phage capture assay, immunofluorescence microscopy and electron microscopy. A prostate cancer selective phage was converted to phage-like particles harboring emerald green fluorescent protein. RESULTS: Phage clone EPTHSWAT (designated by the sequence of inserted peptide) was found to be most selective for PC-3M cells and was observed to internalize PC-3M cells as revealed by immunofluorescence microscopy and electron microscopy. Conversion of this phage to phage-like particles harboring emerald green fluorescent protein and the expression of emerald green fluorescent protein in the phage-like particles treated PC-3M cells showed potential of adoption of this phage-like particle in prostate cancer therapeutic gene delivery. CONCLUSION: Successful employment of phage-like particles expressing emerald green fluorescent protein genes targeted to prostate cancer cells PC-3M confirms a prospect of their use for targeted delivery of therapeutic genes to cancer cells.

Landscape phages and their fusion proteins targeted to breast cancer cells.

Breast cancer is a leading cause of death among women in the USA. The efficacy of existing anticancer therapeutics can be improved by targeting them through conjugation with ligands binding to cellular receptors. Recently, we developed a novel drug targeting strategy based on the use of pre-selected cancer-specific 'fusion pVIII proteins' (fpVIII), as targeting ligands. To study the efficiency of this approach in animal models, we developed a panel of breast cancer cell-binding phages as a source of targeted fpVIIIs. Two landscape phage peptide libraries (8-mer f8/8 and 9-mer f8/9) were screened to isolate 132 phage variants that recognize breast carcinoma cells MCF-7 and ZR-75-1 and internalize into the cells. When tested for their interaction with the breast cancer cells in comparison with liver cancer cells HepG2, human mammary cells MCF-10A cells and serum, 16 of the phage probes selectively interacted with the breast cancer cells whereas 32 bound both breast and liver cancer cells. The most prominent cancer-specific phage DMPGTVLP, demonstrating sub-nanomolar Kd in interaction with target cells, was used for affinity chromatography of cellular membrane molecules to reveal its potential binding receptor. The isolated protein was identified by direct sequencing as cellular surface nucleolin. This conclusion was confirmed by inhibition of the phage-cell interaction with nucleolin antibodies. Other prominent phage binders VPTDTDYS, VEEGGYIAA, and DWRGDSMDS demonstrate consensus motifs common to previously identified cancer-specific peptides. Isolated phage proteins exhibit inherent binding specificity towards cancer cells, demonstrating the functional activity of the selected fused peptides. The selected phages, their peptide inserts and intact fusion proteins can serve as promising ligands for the development of targeted nanomedicines and their study in model mice with xenograft of human cells MCF-7 and ZR-75-1.

Delivery of siRNA into breast cancer cells via phage fusion protein-targeted liposomes.

Efficacy of siRNAs as potential anticancer therapeutics can be increased by their targeted delivery into cancer cells via tumor-specific ligands. Phage display offers a unique approach to identify highly specific and selective ligands that can deliver nanocarriers to the site of disease. In this study, we proved a novel approach for intracellular delivery of siRNAs into breast cancer cells through their encapsulation into liposomes targeted to the tumor cells with preselected intact phage proteins. The targeted siRNA liposomes were obtained by a fusion of two parental liposomes containing spontaneously inserted siRNA and fusion phage proteins. The presence of pVIII coat protein fused to a MCF-7 cell-targeting peptide DMPGTVLP in the liposomes was confirmed by Western blotting. The novel phage-targeted siRNA-nanopharmaceuticals demonstrate significant down-regulation of PRDM14 gene expression and PRDM14 protein synthesis in the target MCF-7 cells. This approach offers the potential for development of new anticancer siRNA-based targeted nanomedicines. FROM THE CLINICAL EDITOR: In this study, the authors report a novel approach for targeted intracellular delivery of siRNAs into breast cancer cells through encapsulation into liposomes targeted to the tumor cells with preselected intact phage proteins.

Enhanced binding and killing of target tumor cells by drug-loaded liposomes modified with tumor-specific phage fusion coat protein.

AIM: To explore cancer cell-specific phage fusion pVIII coat protein, identified using phage display, for targeted delivery of drug-loaded liposomes to MCF-7 breast cancer cells. MATERIAL & METHODS: An 8-mer landscape library f8/8 and a biopanning protocol against MCF-7 cells were used to select a landscape phage protein bearing MCF-7-specific peptide. Size and morphology of doxorubicin-loaded liposomes modified with the tumor-specific phage fusion coat protein (phage-Doxil) were determined by dynamic light scattering and freeze-fraction electron microscopy. Topology of the phage protein in liposomes was examined by western blot. Association of phage-Doxil with MCF-7 cells was evaluated by fluorescence microscopy and fluorescence spectrometry. Selective targeting to MCF-7 was shown by FACS using a coculture model with target and nontarget cells. Phage-Doxil-induced tumor cell killing and apoptosis were confirmed by CellTiter-Blue Assay and caspase-3/CPP32 fluorometric assay. RESULTS: A chimeric phage fusion coat protein specific towards MCF-7 cells, identified from a phage landscape library, was directly incorporated into the liposomal bilayer of doxorubicin-loaded PEGylated liposomes (Doxil) without additional conjugation with lipophilic moieties. Western blotting confirmed the presence of both targeting peptide and pVIII coat protein in the phage-Doxil, which maintained the liposomal morphology and retained a substantial part of the incorporated drug after phage protein incorporation. The binding activity of the phage fusion pVIII coat protein was retained after incorporation into liposomes, and phage-Doxil strongly and specifically targeted MCF-7 cells, demonstrating significantly increased cytotoxicity towards target cells in vitro. CONCLUSION: We present a novel and straightforward method for making tumor-targeted nanomedicines by anchoring specific phage proteins (substitute antibodies) on their surface.