In vitro antiviral activity of peptide-rich extracts from seven Nigerian plants against three non-polio enterovirus species C serotypes.

BACKGROUND: As frequent viral outbreaks continue to pose threat to public health, the unavailability of antiviral drugs and challenges associated with vaccine development underscore the need for antiviral drugs discovery in emergent moments (endemic or pandemic). Plants in response to microbial and pest attacks are able to produce defence molecules such as antimicrobial peptides as components of their innate immunity, which can be explored for viral therapeutics. METHODS: In this study, partially purified peptide-rich fraction (P-PPf) were obtained from aqueous extracts of seven plants by reverse-phase solid-phase extraction and cysteine-rich peptides detected by a modified TLC method. The peptide-enriched fractions and the aqueous (crude polar) were screened for antiviral effect against three non-polio enterovirus species C members using cytopathic effect reduction assay. RESULTS: In this study, peptide fraction obtained from Euphorbia hirta leaf showed most potent antiviral effect against Coxsackievirus A13, Coxsackievirus A20, and Enterovirus C99 (EV-C99) with IC50 < 2.0 µg/mL and selective index ≥ 81. EV-C99 was susceptible to all partially purified peptide fractions except Allamanda blanchetii leaf. CONCLUSION: These findings establish the antiviral potentials of plants antimicrobial peptides and provides evidence for the anti-infective use of E. hirta in ethnomedicine. This study provides basis for further scientific investigation geared towards the isolation, characterization and mechanistic pharmacological study of the detected cysteine-rich peptides.

Dynamics of Evolution of Poliovirus Neutralizing Antigenic Sites and Other Capsid Functional Domains during a Large and Prolonged Outbreak.

We followed the dynamics of capsid amino acid replacement among 403 Nigerian outbreak isolates of type 2 circulating vaccine-derived poliovirus (cVDPV2) from 2005 through 2011. Four different functional domains were analyzed: (i) neutralizing antigenic (NAg) sites, (ii) residues binding the poliovirus receptor (PVR), (iii) VP1 residues 1 to 32, and (iv) the capsid structural core. Amino acid replacements mapped to 37 of 43 positions across all 4 NAg sites; the most variable and polymorphic residues were in NAg sites 2 and 3b. The most divergent of the 120 NAg variants had no more than 5 replacements in all NAg sites and were still neutralized at titers similar to those of Sabin 2. PVR-binding residues were less variable (25 different variants; 0 to 2 replacements per isolate; 30/44 invariant positions), with the most variable residues also forming parts of NAg sites 2 and 3a. Residues 1 to 32 of VP1 were highly variable (133 different variants; 0 to 6 replacements per isolate; 5/32 invariant positions), with residues 1 to 18 predicted to form a well-conserved amphipathic helix. Replacement events were dated by mapping them onto the branches of time-scaled phylogenies. Rates of amino acid replacement varied widely across positions and followed no simple substitution model. Replacements in the structural core were the most conservative and were fixed at an overall rate ∼20-fold lower than the rates for the NAg sites and VP1 1 to 32 and ∼5-fold lower than the rate for the PVR-binding sites. Only VP1 143-Ile, a non-NAg site surface residue and known attenuation site, appeared to be under strong negative selection.IMPORTANCE The high rate of poliovirus evolution is offset by strong selection against amino acid replacement at most positions of the capsid. Consequently, poliovirus vaccines developed from strains isolated decades ago have been used worldwide to bring wild polioviruses almost to extinction. The apparent antigenic stability of poliovirus obscures a dynamic of continuous change within the neutralizing antigenic (NAg) sites. During 7 years of a large outbreak in Nigeria, the circulating type 2 vaccine-derived polioviruses generated 120 different NAg site variants via multiple independent pathways. Nonetheless, overall antigenic evolution was constrained, as no isolate had fixed more than 5 amino acid differences from the Sabin 2 NAg sites, and the most divergent isolates were efficiently neutralized by human immune sera. Evolution elsewhere in the capsid was also constrained. Amino acids binding the poliovirus receptor were strongly conserved, and extensive variation in the VP1 amino terminus still conserved a predicted amphipathic helix.

In vitro antiviral activity of twenty-seven medicinal plant extracts from Southwest Nigeria against three serotypes of echoviruses.

BACKGROUND: Echoviruses, a serotype of enteroviruses, infect millions of people globally and there is no specific drug treatment or vaccine available for its management. The screening of medicinal plants used locally for the treatment of infectious diseases, can provide a reliable option in the discovery of potent therapeutic compounds. This study was carried out to investigate the antiviral activities of 27 medicinal plant extracts, belonging to 26 different plant species, selected from Nigerian ethnobotany, against echovirus 7, 13 and 19 serotypes (E7, E13 and E19, respectively). METHODS: The plants were macerated in methanol and the cytotoxicities of the crude extracts were evaluated on the rhabdomyosarcoma cell line using the MTT assay. The antiviral activity of the plant extracts and fractions against echoviruses (E7, E13, and E19) was determined using the neutralisation assay, an assay that measures the inhibition of cytopathic effect on cell culture. RESULTS: The crude extract of Macaranga barteri leaves had the highest cytotoxicity with CC50 value of 0.27 µg/mL. This was followed by Crinum jagus (9.88 µg/mL) and Terminalia ivorensis (12.14 µg/mL). The antiviral screening showed that ten out of the 27 crude plant extracts tested were active on E7 and E19, inhibiting the cytopathic effect of the virus in tissue culture. None of the extracts inhibited the cytopathic effect caused by E13 serotype. Amongst the active plant extracts, the methanol extract of M. barteri leaves had the highest antiviral activity on both E7 and E9 with IC50 values of 0.028 and 0.0017 ng/mL, respectively, followed by the Ageratum conyzoides extract (0.208 µg/mL, E7; 0.006 µg/mL, E19) and Mondia whitei extract (0.038 µg/mL, E7; 0.005 µg/mL, E19). Amongst the fractions of M. barteri, the DCM fraction was most the active and selective on E7 (IC50 = 0.0075 ng/mL; SI = 19,896.54) and E19 (IC50 = 0.0175 ng/mL; SI = 8581.24). CONCLUSION: Our research has demonstrated that Macaranga barteri extracts has potent antiviral activity against echoviruses E7 and E19, and our findings suggest that this extract may have potential as a therapeutic agent in the treatment of enteroviral infections.

In vitro cytotoxic activity of medicinal plants from Nigeria ethnomedicine on Rhabdomyosarcoma cancer cell line and HPLC analysis of active extracts.

BACKGROUND: Cancer is a leading cause of death world-wide, with approximately 17.5 million new cases and 8.7 million cancer related deaths in 2015. The problems of poor selectivity and severe side effects of the available anticancer drugs, have demanded the need for the development of safer and more effective chemotherapeutic agents. The present study was aimed at determining the cytotoxicities of 31 medicinal plants extracts, used in Nigerian ethnomedicine for the treatment of cancer. METHODS: The plant extracts were screened for cytotoxicity, using the brine shrimp lethality assay (BSLA) and MTT cytotoxicity assay. Rhabdomyosarcoma (RD) cell line, normal Vero cell line and the normal prostate (PNT2) cell line were used for the MTT assay, while Artemia salina nauplii was used for the BSLA. The phytochemical composition of the active plant extracts was determined by high performance liquid chromatography (HPLC) analysis. RESULTS: The extract of Eluesine indica (L.) Gaertn (Poaceae), with a LC50 value of 76.3 µg/mL, had the highest cytotoxicity on the brine shrimp larvae compared to cyclophosphamide (LC50 = 101.3 µg/mL). Two plants extracts, Macaranga barteri Mull. Arg. (Euphorbiaceae) and Calliandra portoricensis (Jacq.) Benth (Leguminosae) exhibited significant cytotoxic activity against the RD cell line and had comparable lethal activity on the brine shrimps. Further cytotoxic investigation showed that the dichloromethane fraction of Macaranga barteri (DMB) and the ethyl acetate fraction of Calliandra portoricensis (ECP), exhibited approximately 6-fold and 4-fold activity, respectively, compared to cyclophosphamide on RD cell line. Determination of selective index (SI) using Vero and PNT2 cell line indicated that DMB and ECP displayed a high degree of selectivity against the cancer cell under investigation. HPLC analysis showed that 3,5dicaffeoylquinic acid, acteoside, kampferol-7-O-glucoside and bastadin 11 were the major components of DMB while the major components of ECP were neurolenin B, nigrosporolide and trans-geranic acid. CONCLUSION: The results demonstrate the cytotoxicity of Macaranga barteri and Calliandra portoricensis extracts, which are used in Nigerian folklore for cancer treatment.

Environmental Isolation of Circulating Vaccine-Derived Poliovirus After Interruption of Wild Poliovirus Transmission - Nigeria, 2016.

In September 2015, more than 1 year after reporting its last wild poliovirus (WPV) case in July 2014 (1), Nigeria was removed from the list of countries with endemic poliovirus transmission,* leaving Afghanistan and Pakistan as the only remaining countries with endemic WPV. However, on April 29, 2016, a laboratory-confirmed, circulating vaccine-derived poliovirus type 2 (cVDPV2) isolate was reported from an environmental sample collected in March from a sewage effluent site in Maiduguri Municipal Council, Borno State, a security-compromised area in northeastern Nigeria. VDPVs are genetic variants of the vaccine viruses with the potential to cause paralysis and can circulate in areas with low population immunity. The Nigeria National Polio Emergency Operations Center initiated emergency response activities, including administration of at least 2 doses of oral poliovirus vaccine (OPV) to all children aged <5 years through mass campaigns; retroactive searches for missed cases of acute flaccid paralysis (AFP), and enhanced environmental surveillance. Approximately 1 million children were vaccinated in the first OPV round. Thirteen previously unreported AFP cases were identified. Enhanced environmental surveillance has not resulted in detection of additional VDPV isolates. The detection of persistent circulation of VDPV2 in Borno State highlights the low population immunity, surveillance limitations, and risk for international spread of cVDPVs associated with insurgency-related insecurity. Increasing vaccination coverage with additional targeted supplemental immunization activities and reestablishment of effective routine immunization activities in newly secured and difficult-to-reach areas in Borno is urgently needed.

Polio virus neutralizing antibody dynamics among children in a north-central and South-Western Nigeria state.

Northern Nigeria accounts for the highest number of confirmed wild polio viruses globally. Transmission to neighboring countries is worrisome after the country failed to meet several deadlines for polio eradication. Most studies on neutralizing antibody have focused on the Northeastern and Northwestern regions. This study measured polio virus neutralizing antibody levels among children in North-central and South-western Nigeria. Children between the ages of 10 months and 13 yr were randomly selected from Abanishe-lolu Hospital Ilorin (North-central) and Oni Memorial and Adeoyo Hospitals in Ibadan (South-west) Nigeria. The alpha neutralization method was employed. Herd immunity was 1.4% in Ilorin, 36.6% in Oni Memorial Hospital, and 49.5% in Adeoyo Hospital. Out of 299 children studied, 49 (16.4%) children had no protection against all poliovirus serotypes while 100 (33.4%) were fully protected against all three serotypes. Five (7.9%) children in Ilorin and 5 (3.4%) children in Oni Memorial Hospital Ibadan had no detectable neutralizing antibody. A significant difference was observed (p=0.000) when the GMT against poliovirus 1, 2, and 3 was compared. A significant proportion of children were not fully protected. Sero-surveillance is recommended for effective monitoring of vaccination efforts to guide health policy formulators.

Environmental surveillance for polioviruses in the Global Polio Eradication Initiative.

This article summarizes the status of environmental surveillance (ES) used by the Global Polio Eradication Initiative, provides the rationale for ES, gives examples of ES methods and findings, and summarizes how these data are used to achieve poliovirus eradication. ES complements clinical acute flaccid paralysis (AFP) surveillance for possible polio cases. ES detects poliovirus circulation in environmental sewage and is used to monitor transmission in communities. If detected, the genetic sequences of polioviruses isolated from ES are compared with those of isolates from clinical cases to evaluate the relationships among viruses. To evaluate poliovirus transmission, ES programs must be developed in a manner that is sensitive, with sufficiently frequent sampling, appropriate isolation methods, and specifically targeted sampling sites in locations at highest risk for poliovirus transmission. After poliovirus ceased to be detected in human cases, ES documented the absence of endemic WPV transmission and detected imported WPV. ES provides valuable information, particularly in high-density populations where AFP surveillance is of poor quality, persistent virus circulation is suspected, or frequent virus reintroduction is perceived. Given the benefits of ES, GPEI plans to continue and expand ES as part of its strategic plan and as a supplement to AFP surveillance.

Multiple independent emergences of type 2 vaccine-derived polioviruses during a large outbreak in northern Nigeria.

Since 2005, a large poliomyelitis outbreak associated with type 2 circulating vaccine-derived poliovirus (cVDPV2) has occurred in northern Nigeria, where immunization coverage with trivalent oral poliovirus vaccine (tOPV) has been low. Phylogenetic analysis of P1/capsid region sequences of isolates from each of the 403 cases reported in 2005 to 2011 resolved the outbreak into 23 independent type 2 vaccine-derived poliovirus (VDPV2) emergences, at least 7 of which established circulating lineage groups. Virus from one emergence (lineage group 2005-8; 361 isolates) was estimated to have circulated for over 6 years. The population of the major cVDPV2 lineage group expanded rapidly in early 2009, fell sharply after two tOPV rounds in mid-2009, and gradually expanded again through 2011. The two major determinants of attenuation of the Sabin 2 oral poliovirus vaccine strain (A481 in the 5'-untranslated region [5'-UTR] and VP1-Ile143) had been replaced in all VDPV2 isolates; most A481 5'-UTR replacements occurred by recombination with other enteroviruses. cVDPV2 isolates representing different lineage groups had biological properties indistinguishable from those of wild polioviruses, including efficient growth in neuron-derived HEK293 cells, the capacity to cause paralytic disease in both humans and PVR-Tg21 transgenic mice, loss of the temperature-sensitive phenotype, and the capacity for sustained person-to-person transmission. We estimate from the poliomyelitis case count and the paralytic case-to-infection ratio for type 2 wild poliovirus infections that ∼700,000 cVDPV2 infections have occurred during the outbreak. The detection of multiple concurrent cVDPV2 outbreaks in northern Nigeria highlights the risks of cVDPV emergence accompanying tOPV use at low rates of coverage in developing countries.

Whole genome sequencing unravels cryptic circulation of divergent dengue virus lineages in the rainforest region of Nigeria.

Dengue is often misclassified and underreported in Africa due to inaccurate differential diagnoses of nonspecific febrile illnesses such as malaria, sparsity of diagnostic testing and poor clinical and genomic surveillance. There are limited reports on the seroprevalence and genetic diversity of dengue virus (DENV) in humans and vectors in Nigeria. In this study, we investigated the epidemiology and genetic diversity of dengue in the rainforest region of Nigeria. We screened 515 febrile patients who tested negative for malaria and typhoid fever in three hospitals in Oyo and Ekiti States in southern Nigeria with a combination of anti-dengue IgG/IgM/NS1 rapid test kits and metagenomic sequencing. We found that approximately 28% of screened patients had previous DENV exposure, with the highest prevalence in persons over sixty. Approximately 8% of the patients showed evidence of recent or current infection, and 2.7% had acute infection. Following sequencing of sixty samples, we assembled twenty DENV-1 genomes (3 complete and 17 partial). We found that all assembled genomes belonged to DENV-1 genotype III. Our phylogenetic analyses showed evidence of prolonged cryptic circulation of divergent DENV lineages in Oyo state. We were unable to resolve the source of DENV in Nigeria owing to limited sequencing data from the region. However, our sequences clustered closely with sequences in Tanzania and sequences reported in Chinese with travel history to Tanzania in 2019. This may reflect the wider unsampled bidirectional transmission of DENV-1 in Africa, which strongly emphasizes the importance of genomic surveillance in monitoring ongoing DENV transmission in Africa.

Metagenomic Detection and Genetic Characterization of Human Sapoviruses among Children with Acute Flaccid Paralysis in Nigeria.

Using a metagenomic sequencing approach on stool samples from children with Acute Flaccid Paralysis (AFP), we describe the genetic diversity of Sapoviruses (SaVs) in children in Nigeria. We identified six complete genome sequences and two partial genome sequences. Several SaV genogroups and genotypes were detected, including GII (GII.4 and GII.8), GIV (GIV.1), and GI (GI.2 and GI.7). To our knowledge, this is the first description of SaV infections and complete genomes from Nigeria. Pairwise identity and phylogenetic analysis showed that the Nigerian SaVs were related to previously documented gastroenteritis outbreaks with associated strains from China and Japan. Minor variations in the functional motifs of the nonstructural proteins NS3 and NS5 were seen in the Nigerian strains. To adequately understand the effect of such amino acid changes, a better understanding of the biological function of these proteins is vital. The identification of distinct SaVs reinforces the need for robust surveillance in acute gastroenteritis (AGE) and non-AGE cohorts to better understand SaVs genotype diversity, evolution, and its role in disease burden in Nigeria. Future studies in different populations are, therefore, recommended.

Human rotavirus VP4 and VP7 genetic diversity and detection of GII norovirus in Ibadan as Nigeria introduces rotavirus vaccine.

OBJECTIVE: This cross-sectional study investigated the circulating strains of rotavirus and screened for noravirus in Ibadan, Nigeria as the country introduces the rotavirus vaccine into its national immunization program. METHODS: Sixty-five stool samples were collected from children younger than 5 years with clinically diagnosed diarrhea and screened for the presence of rotavirus and norovirus using RT-PCR. Rotavirus-positive samples were further analyzed to determine the G and P genotypes using semi-nested multiplex PCR. RESULTS: The rates of rotavirus and norovirus positivity were 30.8% and 10.8%, respectively, whereas the rate of rotavirus and norovirus mixed infection was 4.6%. G1 was the predominant VP7 genotype, followed by G2, G9, and G1G2G9, whereas the predominant VP4 genotype was P[4], followed by P[6], P[8], and P[9]. The mixed P types P[4]P[8] and P[4]P[6] were also detected. G1P[4] was the most common VP4 and VP7 combination, followed by G2P[4], G1[P6], G1P[8], G2P[6], G2P[9], G9P[6], G2G9P[4], G2P[4]P[6], G1P[4]P[8], G2G9P[8], G1G2G9P[8], and G1[non-typable] P[non-typable], which were detected in at least 5% of the samples. Four samples had a combination of non-typable G and P types. CONCLUSIONS: It is essential to monitor the circulation of virus strains prior to and during the implementation of the immunization program.

In vitro anti-enteroviral activity of stilbenoids isolated from the leaves of Macaranga barteri.

The anti-enteroviral activity of three stilbenoids isolated from the leaves of Macaranga barteri was investigated using the cytopathic effect reduction assay. The stilbenes were inactive against echovirus E13 but showed activity against echoviruses E7 and E19. In particular, vedelianin (2), schweinfurthin G (3) and mappain (1) elicited antiviral activity on E19 with IC50 values of 0.0036 nM, 0.018 nM and 0.24 µM, respectively. Vedelianin (2) showed the best selectivity profile amongst the isolated compounds with selectivity index values of 31 and 216 against E7 and E19, respectively. It is possible these compounds may be responsible for the traditional use of Macaranga barteri in the treatment of viral infections.

FIRST MOLECULAR DETECTION AND VP7 (G) GENOTYPING OF GROUP A ROTAVIRUS BY SEMI-NESTED RT-PCR FROM SEWAGE IN NIGERIA.

Rotavirus is the leading cause of viral gastroenteritis worldwide, and sewage is a major source of the virus dissemination in the environment. Our aim was to detect and genotype rotaviruses from sewages in Nigeria. One hundred and ninety sewage samples were collected between June 2014 and January 2015. The two phase concentration method using PEG 6000 and dextran was used to concentrate sewage samples following WHO protocols. Molecular detection was performed by RT-PCR, and VP7 genotyping by semi-nested multiplex PCR. A total of 14.2% (n = 27) samples tested positive. Monthly distribution showed that June to September had a lower rate (3.7% to 7.4%), while October to January recorded 11% to 26%. Genotype G1 predominated followed by G8, G9, G4 and lastly G2, 7.4% (n = 2) of isolates were nontypeable. This is the first report of rotavirus detection in sewages from Nigeria. Genotype G1 remains the most prevalent genotype. This observation calls for an effort by the governmental authorities to implement a molecular surveillance, both clinical and environmental, in order to provide vital information for the control and the vaccine efficacy not only in Nigeria, but globally.

Kinetics of measles antibody by hemagglutination inhibition assay in children in south-west and north-central Nigeria.

OBJECTIVES: We investigated the antibody level of children against wild measles virus in view of recurrent measles epidemics, in order to provide information on immunization status for health policies and for the global measles mortality reduction initiative. METHODS: Two hundred and seventy-three children between the ages of 10 months and 13 years were recruited for this study from three hospital facilities in south-west and north-central Nigeria. Serum samples were collected from February to July 2009, and laboratory examination commenced in August of the same year. Measles hemagglutinin (HA) antigen was prepared by culturing the measles vaccine virus strain (Edmonston-Zagreb) in a vero/hSLAM cell line. Serum samples were treated to get rid of potentiating factors, non-specific inhibitors, and agglutinins before the HA/hemagglutination inhibition (HI) procedure. RESULTS: Out of the 175 children vaccinated in Ibadan, 60 (34.3%) had an antibody level not sufficient to protect against measles infection. Likewise, 12 (25.0%) vaccinated children from Ilorin had an antibody level not sufficient to protect against measles infection. There was no significant difference in the level of protection between the children in Ibadan and Ilorin (p>0.05). The geometric mean titer (GMT) was 53.83 for males and 48.64 for females. There was no significant difference between the GMTs of females and males in both locations (p>0.05). Also, no significant difference was observed in the GMTs of children in both locations (p>0.05). CONCLUSIONS: Of the vaccinated children, 157 (57.5%) developed protective measles virus HI antibody, which is not enough to maintain protective herd immunity. Hence there is a need for catch-up and follow-up vaccination programs, especially in rural areas and places with difficult terrains, in order to reduce measles mortality.

Phytochemicals from Kigelia pinnata leaves show antioxidant and anticancer potential on human cancer cell line.

Studies suggest that the traditional applications of Kigelia pinnata leaves have beneficial effects against oxidative stress-mediated diseases and cancers. The pulverized dried leaves of K. pinnata were extracted with hexane, ethyl acetate, and methanol sequentially, and the crude extracts were fractionated by silica gel column chromatography with solvent gradient of increasing polarity. 3-hydro-4,8-phytene, trans-phytol, (9Z,12Z)-methyl octadeca-9,12-dienoate, and two oil fractions were obtained. The chemical compositions of chromatographic fractions were determined using gas chromatography-mass spectroscopy. The structure elucidations of the isolated compounds were based on FTIR, MS, and NMR spectral data analyses. These along with the crude extracts were examined for their antioxidant activities using ferric reducing antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, and 2,2-azinobis(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS) assays. Total phenolic contents were also determined. The crude extracts and purified compounds were evaluated on the rhabdomyosarcoma human cancer cell for their cytotoxicity using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assays. The methanol extract was richer in phenolics and was most potent as antioxidant and cytotoxic agent among all the substances tested. Among the fractions and pure compounds, the two oil fractions showed more cytotoxicity potency, with IC50s of 143.4±0.5 and 147.9±1.3 ng/mL, which is more significant than the reference standard, cyclophosphamide (165.6±1.0 ng/mL). 3-hydro-4,8-phytene showed lower antioxidant and cytotoxicity potential (IC50=1818±5.2 µg/mL and 171.7±0.8 ng/mL, respectively). Trans-phytol did not show a high cytotoxic power (IC50=769.8±4.3 ng/mL). The comparatively high cytotoxicity index of (9Z, 12Z)-methyl octadeca-9,12-dienoate (IC50=153.3±0.1 ng/mL) indicated that it may be one of the principal cytotoxic agent in the ethyl acetate extract. These results suggest that the leaves of K. pinnata possess tumor cytotoxic potential and could be part of a drug combination for future cancer chemotherapy.