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In recent decades, waves of yellow fever virus (YFV) from the Amazon Rainforest have spread and caused outbreaks in other regions of Brazil, including the Cerrado, a savannah-like biome through which YFV usually moves before arriving at the Atlantic Forest. To identify the vectors involved in the maintenance of the virus in semiarid environments, an entomological survey was conducted after confirmation of yellow fever (YF) epizootics at the peak of the dry season in the Cerrado areas of the state of Minas Gerais. In total, 917 mosquitoes from 13 taxa were collected and tested for the presence of YFV. Interestingly, mosquitoes of the Sabethes genus represented 95% of the diurnal captured specimens, displaying a peak of biting activity never previously recorded, between 4:30 and 5:30 p.m. Molecular analysis identified three YFV-positive pools, two from Sabethes chloropterus—from which near-complete genomes were generated—and one from Sa. albiprivus, whose low viral load prevented sequencing. Sa. chloropterus was considered the primary vector due to the high number of copies of YFV RNA and the high relative abundance detected. Its bionomic characteristics allow its survival in dry places and dry time periods. For the first time in Brazil, Sa. albiprivus was found to be naturally infected with YFV and may have played a role as a secondary vector. Despite its high relative abundance, fewer copies of viral RNA were found, as well as a lower Minimum Infection Rate (MIR). Genomic and phylogeographic analysis showed that the virus clustered in the sub-lineage YFVPA-MG, which circulated in Pará in 2017 and then spread into other regions of the country. The results reported here contribute to the understanding of the epidemiology and mechanisms of YFV dispersion and maintenance, especially in adverse weather conditions. The intense viral circulation, even outside the seasonal period, increases the importance of surveillance and YFV vaccination to protect human populations in affected areas. [ABSTRACT FROM AUTHOR]

Emerging RNA virus infections are a growing concern among domestic poultry industries due to the severe impact they can have on flock health and economic livelihoods. Avian paramyxoviruses (APMV; avulaviruses, AaV) are pathogenic, negative-sense RNA viruses that cause serious infections in the respiratory and central nervous systems. APMV was detected in multiple avian species during the 2017 wild bird migration season in Ukraine and studied using PCR, virus isolation, and sequencing. Of 4090 wild bird samples collected, mostly from southern Ukraine, eleven isolates were grown in ovo and identified for APMV serotype by hemagglutinin inhibition test as: APMV-1, APMV-4, APMV-6, and APMV-7. To build One Health's capacity to characterize APMV virulence and analyze the potential risks of spillover to immunologically naïve populations, we sequenced virus genomes in veterinary research labs in Ukraine using a nanopore (MinION) platform. RNA was extracted and amplified using a multiplex tiling primer approach to specifically capture full-length APMV-1 (n = 5) and APMV-6 (n = 2) genomes at high read depth. All APMV-1 and APMV-6 fusion (F) proteins possessed a monobasic cleavage site, suggesting these APMVs were likely low virulence, annually circulating strains. Utilization of this low-cost method will identify gaps in viral evolution and circulation in this understudied but important critical region for Eurasia. [ABSTRACT FROM AUTHOR]

Bovine respiratory disease (BRD), which is the leading cause of morbidity and mortality in cattle, is caused by numerous known and unknown viruses and is responsible for the widespread use of broad-spectrum antibiotics despite the use of polymicrobial BRD vaccines. Viral metagenomics sequencing on the portable, inexpensive Oxford Nanopore Technologies MinION sequencer and sequence analysis with its associated user-friendly point-and-click Epi2ME cloud-based pathogen identification software has the potential for point-of-care/same-day/sample-to-result metagenomic sequence diagnostics of known and unknown BRD pathogens to inform a rapid response and vaccine design. We assessed this potential using in vitro viral cell cultures and nasal swabs taken from calves that were experimentally challenged with a single known BRD-associated DNA virus, namely, bovine herpes virus 1. Extensive optimisation of the standard Oxford Nanopore library preparation protocols, particularly a reduction in the PCR bias of library amplification, was required before BoHV-1 could be identified as the main virus in the in vitro cell cultures and nasal swab samples within approximately 7 h from sample to result. In addition, we observed incorrect assignment of the bovine sequence to bacterial and viral taxa due to the presence of poor-quality bacterial and viral genome assemblies in the RefSeq database used by the EpiME Fastq WIMP pathogen identification software. [ABSTRACT FROM AUTHOR]

During autumn/winter in 2016–2017 and 2020–2021, highly pathogenic avian influenza viruses (HPAIV) caused severe outbreaks in Germany and Europe. Multiple clade 2.3.4.4b H5 HPAI subtypes were responsible for increased mortality in wild birds and high mortality and massive losses in the poultry sector. To clarify putative entry sources and delineate interconnections between outbreaks in poultry holdings and wild birds, we applied whole-genome sequencing and phylodynamic analyses combined with the results of epidemiological outbreak investigations. Varying outbreak dynamics of the distinct reassortants allowed for the identification of individual, putatively wild bird-mediated entries into backyard holdings, several clusters comprising poultry holdings, local virus circulation for several weeks, direct farm-to-farm transmission and potential reassortment within a turkey holding with subsequent spill-over of the novel reassorted virus into the wild bird population. Whole-genome sequencing allowed for a unique high-resolution molecular epidemiology analysis of HPAIV H5Nx outbreaks and is recommended to be used as a standard tool. The presented detailed account of the genetic, temporal, and geographical characteristics of the recent German HPAI H5Nx situation emphasizes the role of poultry holdings as an important source of novel genetic variants and reassortants. [ABSTRACT FROM AUTHOR]

The Eurasian otter (Lutra lutra) is a piscivorous apex predator in aquatic habitats, and a flagship species of conservation biology throughout Europe. Despite the wide distribution and ecological relevance of the species, there is a considerable lack of knowledge regarding its virological and veterinary health context, especially in Central Europe. Canine morbillivirus (Canine distemper virus (CDV)) is a highly contagious viral agent of the family Paramyxoviridae with high epizootic potential and veterinary health impact. CDV is present worldwide among a wide range of animals; wild carnivores are at particular risk. As part of a retrospective study, lung-tissue samples (n = 339) from Eurasian otters were collected between 2000 and 2021 throughout Hungary. The samples were screened for CDV using a real-time RT-PCR method. Two specimens proved positive for CDV RNA. In one sample, the complete viral genome was sequenced using a novel, pan-genotype CDV-specific amplicon-based sequencing method with Oxford Nanopore sequencing technology. Both viral sequences were grouped to a European lineage based on the hemagglutinin-gene phylogenetic classification. In this article, we present the feasibility of road-killed animal samples for understanding the long-term dynamics of CDV among wildlife and provide novel virological sequence data to better understand CDV circulation and evolution. [ABSTRACT FROM AUTHOR]

Porcine circovirus type 2 (PCV2) is responsible for a number of porcine circovirus-associated diseases (PCVAD) that can severely impact domestic pig herds. For a non-enveloped virus with a small genome (1.7 kb ssDNA), PCV2 is remarkably diverse, with eight genotypes (a–h). New genotypes of PCV2 can spread through the migration of wild boar, which are thought to infect domestic pigs and spread further through the domestic pig trade. Despite a large swine population, the diversity of PCV2 genotypes in Ukraine has been under-sampled, with few PCV2 genome sequences reported in the past decade. To gain a deeper understanding of PCV2 genotype diversity in Ukraine, samples of blood serum were collected from wild boars (n = 107) that were hunted in Ukraine during the November–December 2012 hunting season. We found 34/107 (31.8%) prevalence of PCV2 by diagnostic PCR. For domestic pigs, liver samples (n = 16) were collected from a commercial market near Kharkiv in 2019, of which 6 out of 16 (37%) samples were positive for PCV2. We sequenced the genotyping locus ORF2, a gene encoding the PCV2 viral capsid (Cap), for 11 wild boar and six domestic pig samples in Ukraine using an Oxford Nanopore MinION device. Of 17 samples with resolved genotypes, the PCV2 genotype b was the most common in wild boar samples (10 out of 11, 91%), while the domestic pigs were infected with genotypes b and d. We also detected genotype b/d and b/a co-infections in wild boars and domestic pigs, respectively, and for the first time in Ukraine we detected genotype f in a wild boar from Poltava. Building a maximum-likelihood phylogeny, we identified a sublineage of PCV2 genotype b infections in both wild and domestic swine, suggesting a possible epizootic cluster and an ecological interaction between wild boar and domestic pig populations in northeastern Ukraine. [ABSTRACT FROM AUTHOR]

The adoption of Oxford Nanopore Technologies (ONT) sequencing as a tool in plant virology has been relatively slow despite its promise in more recent years to yield large quantities of long nucleotide sequences in real time without the need for prior amplification. The portability of the MinION and Flongle platforms combined with lowering costs and continued improvements in read accuracy make ONT an attractive method for both low- and high-scale virus diagnostics. Here, we provide a detailed step-by-step protocol using the ONT Flongle platform that we have developed for the routine application on a range of symptomatic post-entry quarantine and domestic surveillance plant samples. The aim of this methods paper is to highlight ONT's feasibility as a valuable component to the diagnostician's toolkit and to hopefully stimulate other laboratories towards the eventual goal of integrating high-throughput sequencing technologies as validated plant virus diagnostic methods in their own right. [ABSTRACT FROM AUTHOR]

As countries with endemic canine rabies progress towards elimination by 2030, it will become necessary to employ techniques to help plan, monitor, and confirm canine rabies elimination. Sequencing can provide critical information to inform control and vaccination strategies by identifying genetically distinct virus variants that may have different host reservoir species or geographic distributions. However, many rabies testing laboratories lack the resources or expertise for sequencing, especially in remote or rural areas where human rabies deaths are highest. We developed a low-cost, high throughput rabies virus sequencing method using the Oxford Nanopore MinION portable sequencer. A total of 259 sequences were generated from diverse rabies virus isolates in public health laboratories lacking rabies virus sequencing capacity in Guatemala, India, Kenya, and Vietnam. Phylogenetic analysis provided valuable insight into rabies virus diversity and distribution in these countries and identified a new rabies virus lineage in Kenya, the first published canine rabies virus sequence from Guatemala, evidence of rabies spread across an international border in Vietnam, and importation of a rabid dog into a state working to become rabies-free in India. Taken together, our evaluation highlights the MinION's potential for low-cost, high volume sequencing of pathogens in locations with limited resources. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Sequencing the viral genome as the outbreak progresses is important, particularly in the identification of emerging isolates with different pathogenic potential and to identify whether nucleotide changes in the genome will impair clinical diagnostic tools such as real-time PCR assays. Although single nucleotide polymorphisms and point mutations occur during the replication of coronaviruses, one of the biggest drivers in genetic change is recombination. This can manifest itself in insertions and/or deletions in the viral genome. Therefore, sequencing strategies that underpin molecular epidemiology and inform virus biology in patients should take these factors into account. A long amplicon/read length-based RT-PCR sequencing approach focused on the Oxford Nanopore MinION/GridION platforms was developed to identify and sequence the SARS-CoV-2 genome in samples from patients with or suspected of COVID-19. The protocol, termed Rapid Sequencing Long Amplicons (RSLAs) used random primers to generate cDNA from RNA purified from a sample from a patient, followed by single or multiplex PCRs to generate longer amplicons of the viral genome. The base protocol was used to identify SARS-CoV-2 in a variety of clinical samples and proved sensitive in identifying viral RNA in samples from patients that had been declared negative using other nucleic acid-based assays (false negative). Sequencing the amplicons revealed that a number of patients had a proportion of viral genomes with deletions. [ABSTRACT FROM AUTHOR]

Capripox viruses, with their members "lumpy skin disease virus (LSDV)", "goatpox virus (GTPV)" and "sheeppox virus (SPPV)", are described as the most serious pox diseases of production animals. A GTPV isolate and a SPPV isolate were sequenced in a combined approach using nanopore MinION sequencing to obtain long reads and Illumina high throughput sequencing for short precise reads to gain full-length high-quality genome sequences. Concomitantly, sheep and goats were inoculated with SPPV and GTPV strains, respectively. During the animal trial, varying infection routes were compared: a combined intravenous and subcutaneous infection, an only intranasal infection, and the contact infection between naïve and inoculated animals. Sheep inoculated with SPPV showed no clinical signs, only a very small number of genome-positive samples and a low-level antibody reaction. In contrast, all GTPV inoculated or in-contact goats developed severe clinical signs with high viral genome loads observed in all tested matrices. Furthermore, seroconversion was detected in nearly all goats and no differences concerning the severity of the disease depending on the inoculation route were observed. Conclusively, the employed SPPV strain has the properties of an attenuated vaccine strain, consistent with the genetic data, whereas the GTPV strain represents a highly virulent field strain. [ABSTRACT FROM AUTHOR]

Newcastle disease (ND) is one of the most challenging infectious diseases affecting poultry production in Africa, causing major economic losses. To date, Newcastle disease virus isolates from several African countries have been grouped into class II NDV genotypes I, IV, V, VI, VII, XI, XIII, XIV, XVII, XVIII and XXI. Although ND is endemic in many African countries, information on circulating genotypes is still scarce. In Tanzania, outbreaks with genotypes V and XIII have been reported. In West and Central Africa, genotypes XIV, XVII, and XVIII are the most predominant. To investigate other genotypes circulating in Tanzania and Ghana, we performed molecular genotyping on isolates from Tanzania and Ghana using the MinION, a third-generation portable sequencing device from Oxford Nanopore Technologies. Using the MinION, we successfully sequenced the NDV F gene hypervariable region of 24 isolates from Tanzania and four samples from Ghana. In Tanzania, genotypes V, VII and XIII were detected. All isolates from Ghana belonged to genotype XVIII. The data obtained in this study reflect the genetic diversity of NDV in Africa and highlight the importance of surveillance for monitoring the distribution of NDV genotypes and viral evolution. [ABSTRACT FROM AUTHOR]

The MinION sequencer is increasingly being used for the detection and outbreak surveillance of pathogens due to its rapid throughput. For RNA viruses, MinION's new direct RNA sequencing is the next significant development. Direct RNA sequencing studies are currently limited and comparisons of its diagnostic performance relative to different DNA sequencing approaches are lacking as a result. We sought to address this gap and sequenced six subtypes from the mycovirus CHV-1 using MinION's direct RNA sequencing and DNA sequencing based on a targeted viral amplicon. Reads from both techniques could correctly identify viral presence and species using BLAST, though direct RNA reads were more frequently misassigned to closely related CHV species. De novo consensus sequences were error prone but suitable for viral species identification. However, subtype identification was less accurate from both reads and consensus sequences. This is due to the high sequencing error rate and the limited sequence divergence between some CHV-1 subtypes. Importantly, neither RNA nor amplicon sequencing reads could be used to obtain reliable intra-host variants. Overall, both sequencing techniques were suitable for virus detection, though limitations are present due to the error rate of MinION reads. [ABSTRACT FROM AUTHOR]

A novel H5N8 highly pathogenic avian influenza virus (HPAIV) was detected in a greater white-fronted goose in January 2020 in Brandenburg, Germany, and, in February 2020, in domestic chickens belonging to a smallholding in Baden-Wuerttemberg, Germany. Full-genome sequencing was conducted on the MinION platform, enabling further phylogenetic analyses. The virus of clade 2.3.4.4b holds six segments from a Eurasian/Asian/African HPAIV H5N8 reassortant and two segments from low pathogenic avian influenza H3N8 subtype viruses recently detected in wild birds in Central Russia. These new entries continue to show the reassortment potential of the clade 2.3.4.4 H5Nx viruses, underlining the necessity for full-genome sequencing and continuous surveillance. [ABSTRACT FROM AUTHOR]

Prompt detection and effective control of porcine reproductive and respiratory syndrome virus (PRRSV) during outbreaks is important given its immense adverse impact on the swine industry. However, the diagnostic process can be challenging due to the high genetic diversity and high mutation rate of PRRSV. A diagnostic method that can provide more detailed genetic information about pathogens is urgently needed. In this study, we evaluated the ability of Oxford Nanopore MinION direct RNA sequencing to generate a PRRSV whole genome sequence and detect and discriminate virus at the strain-level. A nearly full length PRRSV genome was successfully generated from raw sequence reads, achieving an accuracy of 96% after consensus genome generation. Direct RNA sequencing reliably detected the PRRSV strain present with an accuracy of 99.9% using as few as 5 raw sequencing reads and successfully differentiated multiple co-infecting strains present in a sample. In addition, PRRSV strain information was obtained from clinical samples containing 104 to 106 viral copies or more within 6 hours of sequencing. Overall, direct viral RNA sequencing followed by bioinformatic analysis proves to be a promising approach for identification of the viral strain or strains involved in clinical infections, allowing for more precise prevention and control strategies during PRRSV outbreaks. [ABSTRACT FROM AUTHOR]

In the past ten years, several novel hantaviruses were discovered in shrews, moles, and bats, suggesting the dispersal of hantaviruses in many animal taxa other than rodents during their evolution. Interestingly, the coevolutionary analyses of most recent studies have raised the possibility that nonrodents may have served as the primordial mammalian host and harboured the ancestors of rodent-borne hantaviruses as well. The aim of our study was to investigate the presence of hantaviruses in bat lung tissue homogenates originally collected for taxonomic purposes in Malaysia in 2015. Hantavirus-specific nested RT-PCR screening of 116 samples targeting the L segment of the virus has revealed the positivity of two lung tissue homogenates originating from two individuals, a female and a male of the Murina aenea bat species collected at the same site and sampling occasion. Nanopore sequencing of hantavirus positive samples resulted in partial genomic data from S, M, and L genome segments. The obtained results indicate molecular evidence for hantaviruses in the M. aenea bat species. Sequence analysis of the PCR amplicon and partial genome segments suggests that the identified virus may represent a novel species in the Mobatvirus genus within the Hantaviridae family. Our results provide additional genomic data to help extend our knowledge about the evolution of these viruses. [ABSTRACT FROM AUTHOR]