Nanopore MinION Sequencing Generates a White Spot Syndrome Virus Genome from a Pooled Cloacal Swab Sample of Domestic Chickens in South Africa.

White spot syndrome virus is a highly contagious pathogen affecting shrimp farming worldwide. The host range of this virus is primarily limited to crustaceans, such as shrimps, crabs, prawns, crayfish, and lobsters; however, several species of non-crustaceans, including aquatic insects, piscivorous birds, and molluscs may serve as the vectors for ecological dissemination. The present study was aimed at studying the faecal virome of domestic chickens (Gallus gallus domesticus) in Makhanda, Eastern Cape, South Africa. The cloacal swab specimens (n = 35) were collected from domestic chickens in December 2022. The cloacal swab specimens were pooled-each pool containing five cloacal swabs-for metagenomic analysis using a sequence-independent single-primer amplification protocol, followed by Nanopore MinION sequencing. While the metagenomic sequencing generated several contigs aligning with reference genomes of animal viruses, one striking observation was the presence of a White spot syndrome virus genome in one pool of cloacal swab specimens. The generated White spot syndrome virus genome was 273,795 bp in size with 88.5% genome coverage and shared 99.94% nucleotide sequence identity with a reference genome reported in China during 2018 (GenBank accession: NC_003225.3). The Neighbour-Joining tree grouped South African White spot syndrome virus genome with other White spot syndrome virus genomes reported from South East Asia. To our knowledge, this is the first report of a White spot syndrome virus genome generated from domestic chickens. The significance of White spot syndrome virus infection in domestic chickens is yet to be determined.

Overview of Diagnostic Methods, Disease Prevalence and Transmission of Mpox (Formerly Monkeypox) in Humans and Animal Reservoirs.

Mpox-formerly monkeypox-is a re-emerging zoonotic virus disease, with large numbers of human cases reported during multi-country outbreaks in 2022. The close similarities in clinical symptoms that Mpox shares with many orthopoxvirus (OPXV) diseases make its diagnosis challenging, requiring laboratory testing for confirmation. This review focuses on the diagnostic methods used for Mpox detection in naturally infected humans and animal reservoirs, disease prevalence and transmission, clinical symptoms and signs, and currently known host ranges. Using specific search terms, up to 2 September 2022, we identified 104 relevant original research articles and case reports from NCBI-PubMed and Google Scholar databases for inclusion in the study. Our analyses observed that molecular identification techniques are overwhelmingly being used in current diagnoses, especially real-time PCR (3982/7059 cases; n = 41 studies) and conventional PCR (430/1830 cases; n = 30 studies) approaches being most-frequently-used to diagnose Mpox cases in humans. Additionally, detection of Mpox genomes, using qPCR and/or conventional PCR coupled to genome sequencing methods, offered both reliable detection and epidemiological analyses of evolving Mpox strains; identified the emergence and transmission of a novel clade 'hMPXV-1A' lineage B.1 during 2022 outbreaks globally. While a few current serologic assays, such as ELISA, reported on the detection of OPXV- and Mpox-specific IgG (891/2801 cases; n = 17 studies) and IgM antibodies (241/2688 cases; n = 11 studies), hemagglutination inhibition (HI) detected Mpox antibodies in human samples (88/430 cases; n = 6 studies), most other serologic and immunographic assays used were OPXV-specific. Interestingly, virus isolation (228/1259 cases; n = 24 studies), electron microscopy (216/1226 cases; n = 18 studies), and immunohistochemistry (28/40; n = 7 studies) remain useful methods of Mpox detection in humans in select instances using clinical and tissue samples. In animals, OPXV- and Mpox-DNA and antibodies were detected in various species of nonhuman primates, rodents, shrews, opossums, a dog, and a pig. With evolving transmission dynamics of Mpox, information on reliable and rapid detection methods and clinical symptoms of disease is critical for disease management.

Characterization of a Near Full-Length Hepatitis E Virus Genome of Subtype 3c Generated from Naturally Infected South African Backyard Pigs.

Eight genotypes of the hepatitis E virus (Orthohepevirus A; HEV) designated HEV-1 to HEV-8 have been reported from various mammalian hosts. Notably, domestic pigs and wild boars are the natural reservoirs of HEV-3 and HEV-4 genotypes with zoonotic propensity. Since HEV infection in domestic pigs is usually subclinical, it may remain undetected, facilitating zoonotic spillover of HEV to the exposed human populations. A previous study from our group in 2021, using deep sequencing of a pooled saliva sample, generated various swine enteric virus genomes, including a near full-length swine HEV genome (7040 nt; 97.7% genome coverage) from five-month-old grower pigs at a backyard pig farm in the uMgungundlovu District, KwaZulu-Natal, South Africa. In the present study, we describe the further characterization, including genotyping and subtyping of the swine HEV isolate using phylogenetics and 'HEVnet Typing Tool'. Our analyses confirmed that the South African swine HEV genome characterized in this study belonged to HEV genotype 3 subtype 3c (HEV-3c). While HEV-3c infections in domestic pigs have been previously reported from Brazil, Germany, Italy, and the Netherlands, they only generated partial genome sequences of open reading frame 1 (ORF1) and/or ORF2. To our knowledge, this is the first near full-length swine HEV-3c genome generated from naturally infected domestic pigs (Sus scrofa domesticus) in South Africa. However, due to the gap in the information on the HEV-3c genome sequences in various geographical locations worldwide, including South Africa, the epidemiology of the South African swine HEV genome characterized in this study remains inconclusive. Molecular and genomic surveillance of HEV in domestic pig populations in South Africa would be useful to determine their prevalence, circulating subtypes, and zoonosis risk.

Metagenomic Analysis of RNA Fraction Reveals the Diversity of Swine Oral Virome on South African Backyard Swine Farms in the uMgungundlovu District of KwaZulu-Natal Province.

Numerous RNA viruses have been reported in backyard swine populations in various countries. In the absence of active disease surveillance, a persistent knowledge gap exists on the diversity of RNA viruses in South African backyard swine populations. This is the first study investigating the diversity of oral RNA virome of the backyard swine in South Africa. We used three samples of backyard swine oral secretion (saliva) collected from three distantly located backyard swine farms (BSFs) in the uMgungundlovu District, KwaZulu-Natal, South Africa. Total viral RNA was extracted and used for the library preparation for deep sequencing using the Illumina HiSeq X instrument. The FASTQ files containing paired-end reads were analyzed using Genome Detective v 1.135. The assembled nucleotide sequences were analyzed using the PhyML phylogenetic tree. The genome sequence analysis identified a high diversity of swine enteric viruses in the saliva samples obtained from BSF2 and BSF3, while only a few viruses were identified in the saliva obtained from BSF1. The swine enteric viruses belonged to various animal virus families; however, two fungal viruses, four plant viruses, and five unclassified RNA viruses were also identified. Specifically, viruses of the family Astroviridae, according to the number of reads, were the most prevalent. Of note, the genome sequences of Rotavirus A (RVA) and Rotavirus C (RVC) at BSF2 and RVC and Hepatitis E virus (HEV) at BSF3 were also obtained. The occurrence of various swine enteric viruses in swine saliva suggests a high risk of diarrhoeic diseases in the backyard swine. Of note, zoonotic viruses in swine saliva, such as RVA, RVC, and HEV, indicate a risk of zoonotic spillover to the exposed human populations. We recommend the implementation of biosecurity to ensure sustainable backyard swine farming while safeguarding public health.

An overview of influenza A virus genes, protein functions, and replication cycle highlighting important updates.

The recent research findings on influenza A virus (IAV) genome biology prompted us to present a comprehensive overview of IAV genes, protein functions, and replication cycle. The eight gene segments of the IAV genome encode 17 proteins, each having unique functions contributing to virus fitness in the host. The polymerase genes are essential determinants of IAV pathogenicity and virulence; however, other viral components also play crucial roles in the IAV replication, transmission, and adaptation. Specific adaptive mutations within polymerase (PB2, PB1, and PA) and glycoprotein-hemagglutinin (HA) and neuraminidase (NA) genes, may facilitate interspecies transmission and adaptation of IAV. The HA-NA interplay is essential for establishing the IAV infection; the low pH triggers the inactivation of HA-receptor binding, leading to significantly lower NA activities, indicating that the enzymatic function of NA is dependent on HA binding. While the HA and NA glycoproteins are required to initiate infection, M1, M2, NS1, and NEP proteins are essential for cytoplasmic trafficking of viral ribonucleoproteins (vRNPs) and the assembly of the IAV virions. The mechanisms that enable IAV to exploit the host cell resources to advance the infection are discussed. A comprehensive understanding of IAV genome biology is essential for developing antivirals to combat the IAV disease burden.

A systematic review of influenza A virus prevalence and transmission dynamics in backyard swine populations globally.

BACKGROUND: Backyard swine farming is critical to generating subsistence and food security in rural and peri-urban households in several developing countries. The objective of this systematic review was to analyze the molecular and serological prevalence of influenza A virus (IAV) in backyard swine populations globally. RESULTS: We identified 34 full-text research articles in NCBI-PubMed and Google Scholar databases that have reported IAV sero- and/or virological prevalence in backyard swine up to 11 July 2021. The highest number of studies were reported from Asia (n = 11) followed by North America (n = 10), South America (n = 6), Africa (n = 6), and Europe (n = 1). While the maximum number of studies (44.12%) reported human-to-swine transmission of IAV, swine-to-human (5.88%), poultry-to-swine (5.88%), and wild birds-to-swine (2.94%) transmissions were also reported. An overall higher IAV seroprevalence (18.28%) in backyard swine was detected compared to the virological prevalence (1.32%). The human-origin pandemic A(H1N1)pdm09 virus clade 1A.3.3.2 was the more frequently detected IAV subtype in virological studies (27.27%) than serological studies (18.92%). In addition, the avian-origin highly pathogenic H5N1 and H5N8 viruses were also detected, which further substantiated the evidence of avian-swine interactions in the backyards. CONCLUSION: Human-swine and avian-swine interactions in backyards may transmit IAV between species. Monitoring the circulation and evolution of IAV in backyard swine would help stakeholders make informed decisions to ensure sustainable backyard swine farming and public safety.

Review of genome sequencing technologies in molecular characterization of influenza A viruses in swine.

The rapidly evolving antigenic diversity of influenza A virus (IAV) genomes in swine makes it imperative to detect emerging novel strains and track their circulation. We analyzed in our review the sequencing technologies used for subtyping and characterizing swine IAV genomes. Google Scholar, PubMed, and International Nucleotide Sequence Database Collaboration (INSDC) database searches identified 216 studies that have utilized Sanger, second-, and third-generation sequencing techniques to subtype and characterize swine IAV genomes up to 31 March 2021. Sanger dideoxy sequencing was by far the most widely used sequencing technique for generating either full-length (43.0%) or partial (31.0%) IAV genomes in swine globally; however, in the last decade, other sequencing platforms such as Illumina have emerged as serious competitors for the generation of whole-genome sequences of swine IAVs. Although partial HA and NA gene sequences were sufficient to determine swine IAV subtypes, whole-genome sequences were critical for determining reassortments and identifying unusual or less frequently occurring IAV subtypes. The combination of Sanger and second-generation sequencing technologies also greatly improved swine IAV characterization. In addition, the rapidly evolving third-generation sequencing platform, MinION, appears promising for on-site, real-time sequencing of complete swine IAV genomes. With a higher raw read accuracy, the use of the MinION could enhance the scalability of swine IAV testing in the field and strengthen the swine IAV disease outbreak response.

Deciphering transmission dynamics and spillover of avian influenza viruses from avian species to swine populations globally.

Genome sequences of eleven avian influenza virus (AIV) subtypes have been reported in swine populations from seven countries until August 2020. To unravel the transmission dynamics and spillover events of AIVs from avian reservoirs to swine, full-length hemagglutinin (HA) sequences of AIV subtypes (n = 11) reported from various avian species and swine were retrieved from the 'Influenza Research Database'. Phylogenetic analysis identified closely related avian and swine AIV sequences suggesting potential spillover events from multiple domestic and wild avian species, including chicken, duck, pigeon, goose, quail, and aquatic birds to swine. Furthermore, N-linked glycosylation analysis of these closely related AIV sequences supported the possibility of multiple spillover events of highly pathogenic H5N1 and low pathogenic H9N2 viruses from various avian species to swine. The principal coordinate analysis further validated these findings for H5N1 and H9N2 viruses; however, spillover events of the other nine AIV subtypes were limited. Interestingly, the presence of potential mammalian adaptation markers, particularly in some of the swine H5N1, H7N9, and H9N2 viruses, suggested that these viruses may have already adapted in swine. The occurrence and circulation of these AIVs in swine, especially the H5N1 and H9N2 viruses with numerous spillover events from the avian reservoirs to swine, pose a significant threat in terms of their reassortment with endemic swine viruses or circulating human influenza viruses within the swine which may facilitate the emergence of a novel influenza virus strain with pandemic potential.

A Systematic Review Analyzing the Prevalence and Circulation of Influenza Viruses in Swine Population Worldwide.

The global anxiety and a significant threat to public health due to the current COVID-19 pandemic reiterate the need for active surveillance for the zoonotic virus diseases of pandemic potential. Influenza virus due to its wide host range and zoonotic potential poses such a significant threat to public health. Swine serve as a "mixing vessel" for influenza virus reassortment and evolution which as a result may facilitate the emergence of new strains or subtypes of zoonotic potential. In this context, the currently available scientific data hold a high significance to unravel influenza virus epidemiology and evolution. With this objective, the current systematic review summarizes the original research articles and case reports of all the four types of influenza viruses reported in swine populations worldwide. A total of 281 articles were found eligible through screening of PubMed and Google Scholar databases and hence were included in this systematic review. The highest number of research articles (n = 107) were reported from Asia, followed by Americas (n = 97), Europe (n = 55), Africa (n = 18), and Australia (n = 4). The H1N1, H1N2, H3N2, and A(H1N1)pdm09 viruses were the most common influenza A virus subtypes reported in swine in most countries across the globe, however, few strains of influenza B, C, and D viruses were also reported in certain countries. Multiple reports of the avian influenza virus strains documented in the last two decades in swine in China, the United States, Canada, South Korea, Nigeria, and Egypt provided the evidence of interspecies transmission of influenza viruses from birds to swine. Inter-species transmission of equine influenza virus H3N8 from horse to swine in China expanded the genetic diversity of swine influenza viruses. Additionally, numerous reports of the double and triple-reassortant strains which emerged due to reassortments among avian, human, and swine strains within swine further increased the genetic diversity of swine influenza viruses. These findings are alarming hence active surveillance should be in place to prevent future influenza pandemics.

Systematic Review of Important Viral Diseases in Africa in Light of the 'One Health' Concept.

Emerging and re-emerging viral diseases are of great public health concern. The recent emergence of Severe Acute Respiratory Syndrome (SARS) related coronavirus (SARS-CoV-2) in December 2019 in China, which causes COVID-19 disease in humans, and its current spread to several countries, leading to the first pandemic in history to be caused by a coronavirus, highlights the significance of zoonotic viral diseases. Rift Valley fever, rabies, West Nile, chikungunya, dengue, yellow fever, Crimean-Congo hemorrhagic fever, Ebola, and influenza viruses among many other viruses have been reported from different African countries. The paucity of information, lack of knowledge, limited resources, and climate change, coupled with cultural traditions make the African continent a hotspot for vector-borne and zoonotic viral diseases, which may spread globally. Currently, there is no information available on the status of virus diseases in Africa. This systematic review highlights the available information about viral diseases, including zoonotic and vector-borne diseases, reported in Africa. The findings will help us understand the trend of emerging and re-emerging virus diseases within the African continent. The findings recommend active surveillance of viral diseases and strict implementation of One Health measures in Africa to improve human public health and reduce the possibility of potential pandemics due to zoonotic viruses.

Molecular modeling, docking and protein-protein interaction analysis of MAPK signalling cascade involved in Camalexin biosynthesis in Brassica rapa.

Phytoalexins are small antimicrobial molecules synthesized and accumulated by plants upon exposure to pathogens. Camalexin is an indole-derived phytoalexin, which is accumulated in plants including Arabidopsis thaliana, and other Brassicaceae, which plays a major role in disease resistance against fungal pathogens. The productivity of Brassica crops is adversely affected by Alternaria blight disease, which is caused by Alternaria brassicae. In Arabidopsis thaliana, MAP kinase signalling cascade is known to be involved in synthesis of camalexin, which contributes to disease resistance against a necrtrophic fungal pathogen, Botrytis cinerea. In the present study, MAPK signalling cascade leading to biosynthesis of camalexin that triggers defense responses in B. rapa upon exposure to the most devastating nectrophic fungus, Alternaria brassicae has been elucidated with the help of previously reported MAPK cascade in Arabidopsis thaliana, Molecular modelling, docking, and protein-protein interaction analysis of MAP kinases retrieved from Brassica rapa genome have been carried out to reveal the above cascade. The tertiary structure prediction of MAPKs obtained through molecular modelling revealed that all the protein models fulfil the criteria of being the stable structures. The molecular docking of predicted models for elucidating potential partners of MAPKs revealed strong interactions between MKK1, MKK4, MKK5, MAPK3 and MAPK6 with MKK9. The MAPK signalling cascade also shows different genes that express and play major role in camalexin biosynthesis in B. rapa during defense response to A. brassicae. The understanding of MAPK defense signaling pathway in B. rapa against devastating fungal pathogen Alternaria brassicae would help in devising strategies to develop disease resistance in Brassica crops.

Complete genome sequence of nine isolates of canna yellow streak virus reveals its relationship to the sugarcane mosaic virus (SCMV) subgroup of potyviruses.

Complete genome sequences were obtained from nine isolates of canna yellow streak virus (CaYSV). CaYSV belongs to the sugarcane mosaic virus (SCMV) subgroup of potyviruses with johnsongrass mosaic virus (JGMV) as its closest relative. Multiple sequence alignments showed a pattern of amino acid substitutions in the CP sequences, which enabled us to relate these isolates to South East Asian or European isolates. Biological characterization of CaYSV identified Nicotiana benthamiana, Chenopodium quinoa and Phaseolus vulgaris as experimental hosts. Given the popularity and global trade of cannas, a clear picture of the genetic diversity of CaYSV is critical to disease management.

A Reliable and Rapid Multiplex RT-PCR Assay for Detection of Two Potyviruses and a Pararetrovirus Infecting Canna Plants.

Canna plants are subject to serious virus diseases. The three most common viruses identified in canna plants are Bean yellow mosaic virus, Canna yellow mottle virus, and Canna yellow streak virus. Recent studies indicate that canna plants are commonly infected with more than one virus. Thus, the efficient control of these viruses in canna plants requires the availability of a reliable method for detecting mixed virus infection. This report presents a two-step multiplex reverse-transcription polymerase chain reaction (RT-PCR) that was developed to simultaneously detect two potyviruses and one pararetrovirus genome. We optimized the method for nucleic acid isolation for managing a large population of samples, and the primer concentrations to ensure sensitivity and reliability of the assay, and determined the detection limit in simplex and multiplex RT-PCR assays using plasmid controls and nucleic acids isolated from virus-infected plants. Combined with an automated method for total nucleic acid isolation, this multiplex RT-PCR procedure could be routinely used for virus detection in research and diagnostic laboratories.

Reliable Detection for Bean yellow mosaic virus, Canna yellow streak virus, and Canna yellow mottle virus in Canna Varieties with Red Foliage.

Cannas grow from rhizomes to produce colorful foliage that ranges from deep burgundy, bronze, green, purple veined, and variegated. Bean yellow mosaic virus (BYMV), Canna yellow streak virus (CaYSV), and Canna yellow mottle virus (CaYMV) are problematic viruses infecting cannas. Their disease characteristics have been reported in green-leaved varieties. This study investigated if rhizome planting stocks can be a source of virus infection. PCR and RT-PCR tests identified BYMV, CaYSV, and CaYMV sequences in 20 canna rhizomes and newly emerging leaves. Immunosorbent electron microscopy tests identified filamentous potyvirus particles in rhizome and leaf tissue. In addition, disease characteristics were examined in a subset of red-leaved varieties 'Australia', 'Burning Ember', and 'Red Futurity' planted in pots in the greenhouse. Plants were assigned identifying codes, visual disease ratings, and samples were taken for RT-PCR and PCR virus detection assays. Statistical analysis was carried out to compare disease ratings with RT-PCR and PCR test results. Visual assessment was found to be not a reliable indicator of virus infection in 'Australia' and 'Burning Ember' plants. 'Red Futurity' produced the most obvious pattern of mosaic disease and virus symptoms were easier to identify in this variety. This study demonstrated that visual assessment was an ineffective method for disease identification for the red-leaved varieties. Growers would be well advised to utilize molecular testing to identify infected plants to aid in the clean-up of the crop.