SARS-CoV and SARS-CoV -2 cross-reactive antibodies in domestic animals and wildlife in Nigeria suggest circulation of sarbecoviruses.

Anthropogenic exposure of domestic animals, as well as wildlife, can result in zoonotic transmission events with known and unknown pathogens including sarbecoviruses. During the COVID-19 pandemic, SARS-CoV-2 infections in animals, most likely resulting from spill-over from humans, have been documented worldwide. However, only limited information is available for Africa. The anthropozoonotic transmission from humans to animals, followed by further inter- and intraspecies propagation may contribute to viral evolution, and thereby subsequently alter the epidemiological patterns of transmission. To shed light on the possible role of domestic animals and wildlife in the ecology and epidemiology of sarbecoviruses in Nigeria, and to analyze the possible circulation of other, undiscovered, but potentially zoonotic sarbecoviruses in animals, we tested 504 serum samples from dogs, rabbits, bats, and pangolins collected between December 2020 and April 2022. The samples were analyzed using an indirect multi-species enzyme-linked immunosorbent assay (ELISA) based on the receptor binding domain (RBD) of SARS-CoV and SARS-CoV -2, respectively. ELISA reactive sera were further analyzed by highly specific virus neutralization test and indirect immunofluorescence assay for confirmation of the presence of antibodies. In this study, we found SARS-CoV reactive antibodies in 16 (11.5%) dogs, 7 (2.97%) rabbits, 2 (7.7%) pangolins and SARS-CoV-2 reactive antibodies in 20 (13.4%) dogs, 6 (2.5%) rabbits and 2 (7.7%) pangolins, respectively. Interestingly, 2 (2.3%) bat samples were positive only for SARS-CoV RBD reactive antibodies. These serological findings of SARS-CoV and/or SARS-CoV-2 infections in both domestic animals and wildlife indicates exposure to sarbecoviruses and requires further One Health-oriented research on the potential reservoir role that different species might play in the ecology and epidemiology of coronaviruses at the human-animal interface.

An international, interlaboratory ring trial confirms the feasibility of an extraction-less "direct" RT-qPCR method for reliable detection of SARS-CoV-2 RNA in clinical samples.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is used worldwide to test and trace the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). "Extraction-less" or "direct" real time-reverse transcription polymerase chain reaction (RT-PCR) is a transparent and accessible qualitative method for SARS-CoV-2 detection from nasopharyngeal or oral pharyngeal samples with the potential to generate actionable data more quickly, at a lower cost, and with fewer experimental resources than full RT-qPCR. This study engaged 10 global testing sites, including laboratories currently experiencing testing limitations due to reagent or equipment shortages, in an international interlaboratory ring trial. Participating laboratories were provided a common protocol, common reagents, aliquots of identical pooled clinical samples, and purified nucleic acids and used their existing in-house equipment. We observed 100% concordance across laboratories in the correct identification of all positive and negative samples, with highly similar cycle threshold values. The test also performed well when applied to locally collected patient nasopharyngeal samples, provided the viral transport media did not contain charcoal or guanidine, both of which appeared to potently inhibit the RT-PCR reaction. Our results suggest that direct RT-PCR assay methods can be clearly translated across sites utilizing readily available equipment and expertise and are thus a feasible option for more efficient COVID-19 coronavirus disease testing as demanded by the continuing pandemic.

An international, interlaboratory ring trial confirms the feasibility of an open-source, extraction-less "direct" RT-qPCR method for reliable detection of SARS-CoV-2 RNA in clinical samples.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is used worldwide to test and trace the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). "Extraction-less" or "direct" real time-reverse transcription polymerase chain reaction (RT-PCR) is an open-access qualitative method for SARS-CoV-2 detection from nasopharyngeal or oral pharyngeal samples with the potential to generate actionable data more quickly, at a lower cost, and with fewer experimental resources than full RT-qPCR. This study engaged 10 global testing sites, including laboratories currently experiencing testing limitations due to reagent or equipment shortages, in an international interlaboratory ring trial. Participating laboratories were provided a common protocol, common reagents, aliquots of identical pooled clinical samples, and purified nucleic acids and used their existing in-house equipment. We observed 100% concordance across laboratories in the correct identification of all positive and negative samples, with highly similar cycle threshold values. The test also performed well when applied to locally collected patient nasopharyngeal samples, provided the viral transport media did not contain charcoal or guanidine, both of which appeared to potently inhibit the RT-PCR reaction. Our results suggest that open-access, direct RT-PCR assays are a feasible option for more efficient COVID-19 coronavirus disease testing as demanded by the continuing pandemic.

Fatal multiple outbreaks of equine influenza H3N8 in Nigeria, 2019: The first introduction of Florida clade 1 to West Africa.

In December 2018, suspected outbreaks of equine influenza (EI) were observed in donkeys in Sokoto State, in the extreme northwest of Nigeria bordering the Republic of the Niger. Equine influenza virus (EIV) subtype H3N8 was the etiologic agent identified in the outbreaks using real-time RT-qPCR and sequencing of both the partial haemagglutinin (HA) gene and the complete genome. Since then the H3N8 virus spread to 7 of the 19 northern states of Nigeria, where it affected both donkeys and horses. Phylogenetic analysis of the partial and complete HA gene revealed the closest nucleotide similarity (99.7%) with EIVs belonging to the Florida clade 1 (Fc-1) of the American lineage isolated in 2018 from Argentina and Chile. In total, 80 amino acid substitutions were observed in the viral proteins when compared to the OIE-recommended Fc-1 vaccine strains. The HA and neuraminidase proteins respectively had 13 and 16 amino acid substitutions. This study represents the first reported outbreak of EI caused by an Fc-1 virus in Nigeria and in the West Africa sub-region. Based on this report, extensive disease surveillance in equids is required to establish the circulating lineages and design an effective control strategy to protect the considerable population of horses and donkeys in the country.

The potential of putative zinc-binding motifs of haemagglutinin (HA) protein for categorization and prediction of pathogenicity of H5 subtypes of avian influenza virus.

In the present study, we used the potential of bioinformatics and computational analysis to predict the existence and biological relevance of zinc finger (ZF) motifs in heamagglutinin (HA) protein of Avian Influenza (AI) virus. Sequence data of Avian Influenza (AI) viruses were retrieved from accessible databases (GenBank, GISAID, IRD) and analyzed for the existence, as well as functional prediction of the putative zinc finger or ''zinc-binding'' motif(s) of HA protein. It is hypothesized that the ZF motif(s) in HA of AI virus can be used as a ''novel'' biomarker for categorization of the virus and/or its virulence. As a model for analysis, we used the H5 subtypes of highly pathogenic, non-pathogenic and low pathogenic avian influenza (HPAI, NPAI and LPAI) viruses of H5N1 and H5N2 of avian and human origins. Interestingly, our method of characterization using the zinc-finger agrees with the existing classification in distinguishing between highly pathogenic and non-pathogenic or low pathogenic subtypes. The new method also clearly distinguished between low and non-pathogenic strains of H5N2 and H5N1 which are indistinguishable by the existing method that utilizes the sequence of the polybasic amino acids of the proteolytic cleavage site for pathogenicity. It is hypothesized that zinc through the activities of zinc-binding proteins modulates the virulence property of the viral subtypes. Our observation further revealed that only the HA protein among the eight encoded proteins of influenza viruses contain high numbers of Cys-His residues. It is expected that the information gathered from the analysis of the data will be useful to generate more research hypotheses/designs that will give further insight towards the identification and control of avian influenza virus through the molecular manipulation of zinc finger motifs present in viral HA protein.

Occurrence of infectious bronchitis in layer birds in Plateau state, north central Nigeria.

A flock of 54 wk-old layer birds exhibiting signs of respiratory distress, greenish diarrhea, and drop in egg production was investigated. A marked drop in egg production (55%) was recorded with eggs appearing white and soft-shelled. Mortality was in the range of 1%-2% with post-mortem lesions revealing cloudy air sacs, frothy, and congested lungs. Viral RNA was extracted from pooled tissue samples (trachea, lungs, spleen, and liver) and tested for Avian influenza virus (AIV), Newcastle disease virus (NDV), and infectious bronchitis virus (IBV) by reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, virus isolation was attempted in 9-11 day-old embryonating chicken eggs (ECE). In order to determine the prevalence of IBV serotype(s) in the flock, serum samples were screened by hemagglutination-inhibition (HI) test using IBV antigens and antisera (Arkansas, Connecticut, and Massachusetts). Neither AIV nor NDV but IBV was detected in the tissue samples by RT-PCR. In addition, virus isolate obtained after four serial passages in ECE produced dwarfed, stunted, and hemorrhagic embryos, and the isolate was confirmed by RT-PCR to be IBV. The serum samples were 100% seropositive for three serotypes with HI titres ranging from 5 to 12 Log2. In this study, IBV was confirmed as the causative agent of the observed respiratory distress and drop in egg production. Also, the evidence of co-circulation of multiple IBV serotypes was established, this to the best of our knowledge is the first of such report in Nigeria. We recommend extensive molecular and sero-epidemiology of circulating IBV genotypes and serotypes in Nigeria with the aim of developing better control strategies, including vaccination.

Genomic comparison of Newcastle disease viruses isolated in Nigeria between 2002 and 2015 reveals circulation of highly diverse genotypes and spillover into wild birds.

Newcastle disease virus (NDV) has a wide avian host range and a high degree of genetic variability, and virulent strains cause Newcastle disease (ND), a worldwide concern for poultry health. Although NDV has been studied in Nigeria, genetic information about the viruses involved in the endemicity of the disease and the transmission that likely occurs at the poultry-wildlife interface is still largely incomplete. Next-generation and Sanger sequencing was performed to provide complete (n = 73) and partial genomic sequence data (n = 38) for NDV isolates collected from domestic and wild birds in Nigeria during 2002-2015, including the first complete genome sequences of genotype IV and subgenotype VIh from the African continent. Phylogenetic analysis revealed that viruses of seven different genotypes circulated in that period, demonstrating high genetic diversity of NDV for a single country. In addition, a high degree of similarity between NDV isolates from domestic and wild birds was observed, suggesting that spillovers had occurred, including to three species that had not previously been shown to be susceptible to NDV infection. Furthermore, the first spillover of a mesogenic Komarov vaccine virus is documented, suggesting a previous spillover and evolution of this virus. The similarities between viruses from poultry and multiple bird species and the lack of evidence for host adaptation in codon usage suggest that transmission of NDV between poultry and non-poultry birds occurred recently. This is especially significant when considering that some viruses were isolated from species of conservation concern. The high diversity of NDV observed in both domestic and wild birds in Nigeria emphasizes the need for active surveillance and epidemiology of NDV in all bird species.

Advancements in Nucleic Acid Based Therapeutics against Respiratory Viral Infections.

Several viruses cause pulmonary infections due to their shared tropism with cells of the respiratory tract. These respiratory problems due to viral infection become a public health concern due to rapid transmission through air/aerosols or via direct-indirect contact with infected persons. In addition, the cross-species transmission causes alterations to viral genetic makeup thereby increasing the risk of emergence of pathogens with new and more potent infectivity. With the introduction of effective nucleic acid-based technologies, post translational gene silencing (PTGS) is being increasingly used to silence viral gene targets and has shown promising approach towards management of many viral infections. Since several host factors are also utilized by these viruses during various stages of infection, silencing these host factors can also serve as promising therapeutic tool. Several nucleic acid-based technologies such as short interfering RNAs (siRNA), antisense oligonucleotides, aptamers, deoxyribozymes (DNAzymes), and ribozymes have been studied and used against management of respiratory viruses. These therapeutic nucleic acids can be efficiently delivered through the airways. Studies have also shown efficacy of gene therapy in clinical trials against respiratory syncytial virus (RSV) as well as models of respiratory diseases including severe acute respiratory syndrome (SARS), measles and influenza. In this review, we have summarized some of the recent advancements made in the area of nucleic acid based therapeutics and highlighted the emerging roles of nucleic acids in the management of some of the severe respiratory viral infections. We have also focused on the methods of their delivery and associated challenges.

Draft Genome Sequences of Five Novel Ochrobactrum spp. Isolated from Different Avian Hosts in Nigeria.

Here, we present the draft genome sequences of five multidrug-resistant novel Ochrobactrum species strains isolated from a pigeon, a duck, and chickens from Nigeria in 2009.

Highly pathogenic avian influenza (H5N1) in Nigeria in 2015: evidence of widespread circulation of WA2 clade 2.3.2.1c.

Genetic analysis of the complete haemagglutinin (HA) gene of fourteen Nigerian avian influenza isolates showed multiple basic amino acids at the cleavage site (321PQRERRRK del R*GLF333), characteristic of highly pathogenic avian influenza (HPAI). Substitution of Gln to Lys at position 322 (H5-specific numbering) was identified in one isolate. In some isolates, amino acid substitutions were observed across the HA gene, however the receptor binding, antigenic and glycosylation sites were conserved in all. Phylogenetic analysis revealed two clusters of the HPAI H5N1 clade 2.3.2.1c. Cluster I has close genetic relatedness (97.8-99.8%) with viruses circulating in some West Africa countries. Cluster II shared close identity (98.9-100.0%) with isolates from Europe, Côte d'Ivoire and Niger and viruses from this cluster were detected in five of the eleven states investigated in Nigeria. In view of the continuous HPAI outbreaks being recorded in Nigerian poultry and the zoonotic potential of the virus, extensive and continued characterization of HPAI isolates is advocated.

Serological evidence of equine influenza virus in horse stables in Kaduna, Nigeria.

Equine influenza virus (EIV) is a major cause of acute respiratory diseases in horses in most parts of the world that results in severe economic losses. Information on the epidemiology of EIV in tropical Africa is scanty. An enzyme-linked immunosorbent assay (ELISA) was used to detect the presence of influenza A virus nucleoprotein (NP) in 284 horse sera in Kaduna State, Northern Nigeria. The ELISA-positive sera were further examined for hemagglutination inhibition (HI) antibodies to two strains each of H3N8 and H7N3 subtypes of influenza A virus. The results showed that antibodies against influenza A virus nucleoprotein were detected in 60.9% (173 of 284) of horses examined by NP-ELISA. Equine H3 and H7 subtypes were detected in 60% (21 of 35) and 20% (7 of 35) of horse sera respectively across the stables. Adequate quarantine of all imported horses, a national equine influenza surveillance plan and an appropriate EIV control program in Nigeria are recommended to safeguard the large horse population.