Complete genome reconstruction of the global and European regional dispersal history of the lumpy skin disease virus.

IMPORTANCE: Lumpy skin disease virus (LSDV) has a complex epidemiology involving multiple strains, recombination, and vaccination. Its DNA genome provides limited genetic variation to trace outbreaks in space and time. Sequencing of LSDV whole genomes has also been patchy at global and regional scales. Here, we provide the first fine-grained whole genome sequence sampling of a constrained LSDV outbreak (southeastern Europe, 2015-2017), which we analyze along with global publicly available genomes. We formally evaluate the past occurrence of recombination events as well as the temporal signal that is required for calibrating molecular clock models and subsequently conduct a time-calibrated spatially explicit phylogeographic reconstruction. Our study further illustrates the importance of accounting for recombination events before reconstructing global and regional dynamics of DNA viruses. More LSDV whole genomes from endemic areas are needed to obtain a comprehensive understanding of global LSDV dispersal dynamics.

Combined Phylogeographic Analyses and Epidemiologic Contact Tracing to Characterize Atypically Pathogenic Avian Influenza (H3N1) Epidemic, Belgium, 2019.

The high economic impact and zoonotic potential of avian influenza call for detailed investigations of dispersal dynamics of epidemics. We integrated phylogeographic and epidemiologic analyses to investigate the dynamics of a low pathogenicity avian influenza (H3N1) epidemic that occurred in Belgium during 2019. Virus genomes from 104 clinical samples originating from 85% of affected farms were sequenced. A spatially explicit phylogeographic analysis confirmed a dominating northeast to southwest dispersal direction and a long-distance dispersal event linked to direct live animal transportation between farms. Spatiotemporal clustering, transport, and social contacts strongly correlated with the phylogeographic pattern of the epidemic. We detected only a limited association between wind direction and direction of viral lineage dispersal. Our results highlight the multifactorial nature of avian influenza epidemics and illustrate the use of genomic analyses of virus dispersal to complement epidemiologic and environmental data, improve knowledge of avian influenza epidemiologic dynamics, and enhance control strategies.

RNA sequencing of avian paramyxovirus (Paramyxoviridae, Avulavirinae) isolates from wild mallards in Belgium, 2021: complete genomes and coinfections.

We used untargeted RNA sequencing to characterize three Avulavirinae isolates from pooled samples obtained from wild mallards in Belgium in 2021. The complete genome sequences of two avian Orthoavulavirus-1 (AOAV-1) strains and one avian Paraavulavirus-4 (APMV-4) strain were determined confirming hemagglutination inhibition testing of the virus isolates. In addition, the applied sequencing strategy identified an avian influenza virus (AIV) coinfection in all three virus isolates, confirming weak-positive AIV realtime RT-PCR results from the original sample material. In one AOAV-1 isolate, partial sequences covering all genome segments of an AIV of subtype H11N9 could be de novo assembled from the sequencing data. Besides an AIV coinfection, RNA metagenomic data from the APMV-4 isolate also showed evidence of Alpharetrovirus and Megrivirus coinfection. In total, two AOAV-1 of Class II, genotype I.2 and one APMV-4 complete genome sequences were assembled and compared to publicly available sequences, highlighting the importance of surveillance for poultry pathogens in wild birds. Beyond the insights from full genome characterization of virus isolates, untargeted RNA sequencing strategies provide additional insights in the RNA virome of clinical samples as well as their derived virus isolates that are particularly useful when targeting wild avifauna reservoirs of poultry pathogens.

Metagenomic sequencing determines complete infectious bronchitis virus (avian Gammacoronavirus) vaccine strain genomes and associated viromes in chicken clinical samples.

Infectious bronchitis virus (IBV, genus Gammacoronavirus) causes an economically important and highly contagious disease in chicken. Random primed RNA sequencing was applied to two IBV positive clinical samples and one in ovo-passaged virus. The virome of a cloacal swab pool was dominated by IBV (82% of viral reads) allowing de novo assembly of a GI-13 lineage complete genome with 99.95% nucleotide identity to vaccine strain 793B. In addition, substantial read counts (16% of viral reads) allowed the assembly of a near-complete chicken astrovirus genome, while lower read counts identified the presence of chicken calicivirus and avian leucosis virus. Viral reads in a respiratory/intestinal tissue pool were distributed between IBV (22.53%), Sicinivirus (Picornaviridae, 24%), and avian leucosis virus (37.04%). A complete IBV genome with 99.95% nucleotide identity to vaccine strain H120 (lineage GI-1), as well as a near-complete avian leucosis virus genome and a partial Sicinivirus genome were assembled from the tissue sample data. Lower read counts identified chicken calicivirus, Avibirnavirus (infectious bursal disease virus, assembling to 98.85% of segment A and 69.66% of segment B closely related to D3976/1 from Germany, 2017) and avian orthoreovirus, while three avian orthoavulavirus 1 reads confirmed prior real-time RT-PCR result. IBV sequence variation analysis identified both fixed and minor frequency variations in the tissue sample compared to its in ovo-passaged virus. Metagenomic methods allow the determination of complete coronavirus genomes from clinical chicken samples while providing additional insights in RNA virus sequence diversity and coinfecting viruses potentially contributing to pathogenicity.

Atypical Pathogenicity of Avian Influenza (H3N1) Virus Involved in Outbreak, Belgium, 2019.

In 2019, an outbreak of avian influenza (H3N1) virus infection occurred among commercial poultry in Belgium. Full-genome phylogenetic analysis indicated a wild bird origin rather than recent circulation among poultry. Although classified as a nonnotifiable avian influenza virus, it was associated with reproductive tropism and substantial mortality in the field.

Increased viral read counts and metagenomic full genome characterization of porcine astrovirus 4 and Posavirus 1 in sows in a swine farm with unexplained neonatal piglet diarrhea.

Neonatal diarrhea in piglets may cause major losses in affected pig herds. The present study used random high-throughput RNA sequencing (metagenomic next generation sequencing, mNGS) to investigate the virome of sows from a farm with persistent neonatal piglet diarrhea in comparison to two control farms without diarrhea problems. A variety of known swine gastrointestinal viruses was detected in the control farms as well as in the problem farm (Mamastrovirus, Enterovirus, Picobirnavirus, Posavirus 1, Kobuvirus, Proprismacovirus). A substantial increase in normalized viral read counts was observed in the affected farm compared to the control farms. The increase was attributable to a single viral species in each of the sampled sows (porcine astrovirus 4 and Posavirus 1). The complete genomes of a porcine astrovirus 4 and two co-infecting Posavirus 1 were de novo assembled and characterized. The 6734 nt single-stranded RNA genome of porcine astrovirus 4 (PoAstV-4) strain Belgium/2019 contains three overlapping open reading frames (nonstructural protein 1ab, nonstructural protein 1a, capsid protein). Posavirus 1 strains Belgium/01/2019 and Belgium/02/2019 have a 9814 nt single-stranded positive-sense RNA genome encoding a single open reading frame (polyprotein precursor) containing the five expected Picornavirales-conserved protein domains. The study highlights the potential of mNGS workflows to study unexplained neonatal diarrhea in piglets and contributes to the scarce availability of both PoAstV-4 and Posavirus-1 whole genome sequences from Western Europe.

Complete coding sequence of a novel picorna-like virus in a blackbird infected with Usutu virus.

Using random high-throughput RNA sequencing, the complete coding sequence of a novel picorna-like virus (a 9,228-nt contig containing 212,202 reads) was determined from a blackbird (Turdus merula) infected with Usutu virus. This sequence shares only 36% amino acid sequence identity with its closest homolog, arivirus 1, (an unclassified member of the order Picornavirales), and shares its dicistronic genome arrangement. The new virus was therefore tentatively named "blackbird arilivirus" (ari-like virus). The nearly complete genome sequence consists of at least 9,228 nt and contains two open reading frames (ORFs) encoding the nonstructural polyprotein (2235 amino acids) and structural polyprotein (769 amino acids). Two TaqMan RT-qPCR assays specific for ORF1 confirmed the presence of high levels of this novel virus in the original sample. Nucleotide composition analysis suggests that blackbird arilivirus is of dietary (plant) origin.

Extensive genetic variability linked to IS26 insertions in the fljB promoter region of atypical monophasic variants of Salmonella enterica serovar Typhimurium.

Fifty-nine monophasic Salmonella enterica serovar Typhimurium isolates, collected in Belgium during the period from 2008 to 2011, have been serotyped as 4,[5]:i:- and shown to harbor an fljB coding sequence. The genetic differences between these strains and phenotypically biphasic Salmonella Typhimurium were analyzed through PCR and DNA sequencing. Genetic alterations in the fljB promoter region affecting expression of the phase 2 flagellin were observed in 53 isolates. Other genetic events in the invertible region carrying the fljB promoter were observed in 2 isolates. For the remaining 4 isolates, no molecular differences with a reference biphasic Salmonella Typhimurium strain could be observed. Next-generation sequencing of one representative isolate affected in the fljB promoter region revealed a 26-kb IS26 composite transposon insertion along with a local genomic rearrangement. Several other IS26 element-mediated alterations of this genomic region were observed. This group of monophasic Salmonella Typhimurium isolates was genetically heterogeneous, as revealed by multilocus variable-number tandem-repeat analysis (MLVA), PCR, and sequencing. Pigs and pork represented a major source of such monophasic isolates in Belgium, as reported in other countries. Three out of 5 isolates of human origin presented genetic profiles identical to those of food isolates, demonstrating the pathogenic potential of the newly characterized variants and potential dissemination along the food chain. This study highlighted the key role played by IS26 insertions in the loss of phase 2 flagellin expression and the subsequent generation of multiple monophasic variant lineages from biphasic Salmonella Typhimurium ancestors.

Differential Viral Fitness Between H1N1 and H3N8 Avian Influenza Viruses Isolated from Mallards (Anas platyrhynchos).

Homosubtypic and heterosubtypic immunity in mallards (Anas platyrhynchos) play an important role in the avian influenza virus (AIV) diversity. The mechanisms of AIV replication among wild birds and the role of immunity in AIV diversity have thus not been completely clarified. During the monitoring of AI circulation among wild waterfowl in 2007-2008, two viruses (H3N8 and H1N1) were isolated from ducks caught in a funnel trap located in La Hulpe wetland in Belgium. H3N8 viruses were revealed to be more prevalent in the mallard population than was H1N1, which might suggest a better adaptation to this species. In order to investigate this hypothesis, we characterized both isolated viruses biologically by experimental inoculation. Virus excretion and humoral response induced by both isolated viruses were evaluated in mallards after a first infection followed by a homo- or heterosubtypic reinfection under controlled experimental conditions. The H1N1 virus had a delayed peak of excretion of 4 days compared to the H3N8, but the virus shedding was more limited, earlier, and shorter after each reinfection. Moreover, the H3N8 virus could spread to all ducks after homo- or heterosubtypic reinfections and during a longer period. Although the humoral response induced by both viruses after infection and reinfection could be detected efficiently by competitive ELISA, only a minimal H1 antibody response and almost no H3-specific antibodies could be detected by the HI test. Our results suggest that the H3N8 isolate replicates better in mallards under experimental controlled conditions.

A thiazepino[4,5-a]benzimidazole derivative hampers the RNA replication of Eurasian serotypes of foot-and-mouth disease virus.

The stamping-out policy for the control of foot-and-mouth disease virus (FMDV) in countries that are free from FMD without vaccination has a dramatic socio-economic impact, huge animal welfare issues and may result in the loss of farm animal genetic resources. As an alternative to pre-emptive culling or emergency vaccination we further explore the possibility to use antiviral drugs in the event of an FMD outbreak. In the present study, we tested the in vitro cytotoxicity and anti-FMDV activity of 1,2,4,5-tetrahydro-[1,4]thiazepino[4,5-a]benzimidazole. The molecule was shown to inhibit the replication of reference strains of the Eurasian FMDV serotypes O, A, C and Asia but not the FMDV serotypes from the South African Territories (SAT) neither a related picornavirus, i.e. swine vesicular disease virus. The molecule can be added until 2h post inoculation in a 'single replication cycle experiment' without losing its antiviral activity. The genetic characterization of progressively selected resistant FMD viruses shows that the molecule presumably interacts with the non-structural 2C protein of FMDV. Further studies are required on the use of this molecule in vivo.

Phylogeographic analysis of avian influenza viruses isolated from Charadriiformes in Belgium confirms intercontinental reassortment in gulls.

Nine influenza viruses isolated from gulls and shorebirds in Belgium (2008-2010), including H3N8, H5N2, H6N1, H11N9, H13N6, H13N8, and H16N3 subtypes, were targeted using random amplification and next-generation sequencing. The gene segments of these viruses segregated into three phylogeographic lineage types: (1) segments circulating in waterfowl in Eurasia with sporadic introduction in other species and in the Americas ("Eurasian avian"), (2) segments circulating in American waterfowl with sporadic introduction to other species and regions ("American avian"), and (3) segments circulating exclusively in gulls and shorebirds and having increased connectivity between the two hemispheres ("Charadriiformes specific"). Notably, an H6N1 and an H5N2 isolated from L. argentatus had mainly Eurasian avian genes but shared a matrix segment of American avian origin (first documentation in European gulls of transhemispheric reassortment). These data support the growing evidence of an important role of Charadriiformes birds in the dynamic nature of avian influenza ecology.

Further evidence for the widespread co-circulation of lineages 4b and 7 velogenic Newcastle disease viruses in rural Nigeria.

Newcastle disease (ND) is an endemic disease in rural poultry of Western Africa. It may cause severe economic losses in the poultry sector and, as such, is listed as a notifiable disease by the World Organisation for Animal Health (OIE). Recently, a new genetic lineage of ND viruses was discovered in Western Africa. We determined the complete fusion (F) gene coding sequence of 12 ND viruses isolated from pigeons and rural chickens in six Nigerian states in 2007 and 2008. Phylogenetic analysis of the complete F coding sequence confirmed the circulation of genetically diverse ND isolates in a large geographic area in Nigeria. Next to isolates belonging to lineage 4b, viruses of the recently discovered lineage 7 (some of which were previously reported to escape routine real-time reverse transcriptase-polymerase chain reaction detection) were isolated in six states during the two-year period. The documented genetic variants occurred over a large geographic area, indicating an endemic circulation of these viruses. Three different velogenic fusion gene cleavage site motifs were observed. These findings confirm the endemic circulation and diversification of ND isolates in rural poultry and pigeons in Nigeria and highlight the importance of surveillance in developing countries to monitor the validity of rapid molecular diagnostic tools and of vaccination regimes.

Different replication profiles in specific-pathogen-free chickens of two H7 low pathogenic avian influenza viruses isolated from wild birds.

During an active wild bird survey conducted in Belgium from 2007 to 2011, two low pathogenic avian influenza (LPAI) H7 viruses were isolated from wild birds: an H7N1 virus from a common shelduck (Tadorna tadorna) and an H7N7 virus from a Canada goose (Branta canadensis). The H7 sequence analyses and intravenous pathogenicity indices indicated that they were both low pathogenic isolates and genetically related to other recent European H7 LPAIs isolated from wild birds. Interestingly, the two isolates showed different replication profiles in specific-pathogen-free (SPF) chickens, but poultry can be at risk from both. Indeed, the H7N1 isolated from the common shelduck had the ability to infect and to replicate efficiently in SPF chickens as indicated by high oropharyngeal and cloacal excretions compatible with efficient transmission as well as strong immune responses. On the other hand, the H7N7 isolated from the Canada goose presented a lower replication profile because the inoculated chickens excreted less virus, mostly via the oropharyngeal route, and only three chickens seroconverted. None of the chickens showed clinical signs during the entire infection. Our study using an SPF chicken model underlines that the mechanisms of adaptation of LPAIs in poultry remain unpredictable and are still poorly understood but it represents a powerful tool to gain a better evaluation of the risks of LPAI circulation in poultry.

A robust, cost-effective and widely applicable whole-genome sequencing protocol for capripoxviruses.

The diseases caused by capripoxviruses (CaPVs) are of major economic concern in sheep, goat and cattle as they are inexorably spreading into non-endemic regions. As CaPV strains are serologically indistinguishable and genetically highly homologous, typing closely related strains can only be achieved by whole genome sequencing. Unfortunately the number of publicly available genomes remains low as most sequencing methods rely on virus isolation. Therefore, we developed a robust, cost-effective and widely applicable method that allows to generate (nearly) complete CaPV genomes directly from clinical samples or commercial vaccine batches. A set of pan-CaPVs long-range PCRs spanning the entire genome was designed to generate PCR amplicons that can be sequenced on commonly used high-throughput sequencing platforms: MiSeq (Illumina), RSII (PacBio) and MinION (Oxford Nanopore Technologies). The robustness of the LR-PCR strategy was evaluated for all 3 members of CaPV directly from a variety of samples, including clinical samples (N = 7), vaccine batches (N = 6), and virus isolates (N = 2). The sequencing method described here allows to reconstruct (nearly) complete CaPV genomes in less than a week and will aid researchers studying closely-related CaPV strains worldwide.

Has Epizootic Become Enzootic? Evidence for a Fundamental Change in the Infection Dynamics of Highly Pathogenic Avian Influenza in Europe, 2021.

Phylogenetic evidence from the recent resurgence of high-pathogenicity avian influenza (HPAI) virus subtype H5N1, clade 2.3.4.4b, observed in European wild birds and poultry since October 2021, suggests at least two different and distinct reservoirs. We propose contrasting hypotheses for this emergence: (i) resident viruses have been maintained, presumably in wild birds, in northern Europe throughout the summer of 2021 to cause some of the outbreaks that are part of the most recent autumn/winter 2021 epizootic, or (ii) further virus variants were reintroduced by migratory birds, and these two sources of reintroduction have driven the HPAI resurgence. Viruses from these two principal sources can be distinguished by their hemagglutinin genes, which segregate into two distinct sublineages (termed B1 and B2) within clade 2.3.4.4b, as well as their different internal gene compositions. The evidence of enzootic HPAI virus circulation during the summer of 2021 indicates a possible paradigm shift in the epidemiology of HPAI in Europe.

Identification and complete genome sequencing of paramyxoviruses in mallard ducks (Anas platyrhynchos) using random access amplification and next generation sequencing technologies.

BACKGROUND: During a wildlife screening program for avian influenza A viruses (AIV) and avian paramyxoviruses (APMV) in Belgium, we isolated two hemagglutinating agents from pools of cloacal swabs of wild mallards (Anas platyrhynchos) caught in a single sampling site at two different times. AIV and APMV1 were excluded using hemagglutination inhibition (HI) testing and specific real-time RT-PCR tests. METHODS: To refine the virological identification of APMV2-10 realized by HI subtyping tests and in lack of validated molecular tests for APMV2-10, random access amplification was used in combination with next generation sequencing for the sequence independent identification of the viruses and the determination of their genomes. RESULTS: Three different APMVs were identified. From one pooled sample, the complete genome sequence (15054 nucleotides) of an APMV4 was assembled from the random sequences. From the second pooled sample, the nearly complete genome sequence of an APMV6 (genome size of 16236 nucleotides) was determined, as well as a partial sequence for an APMV4. This APMV4 was closely related but not identical to the APMV4 isolated from the first sample. Although a cross-reactivity with other APMV subtypes did not allow formal identification, the HI subtyping revealed APMV4 and APMV6 in the respective pooled samples but failed to identify the co-infecting APMV4 in the APMV6 infected pool. CONCLUSIONS: These data further contribute to the knowledge about the genetic diversity within the serotypes APMV4 and 6, and confirm the limited sensitivity of the HI subtyping test. Moreover, this study demonstrates the value of a random access nucleic acid amplification method in combination with massive parallel sequencing. Using only a moderate and economical sequencing effort, the characterization and full genome sequencing of APMVs can be obtained, including the identification of viruses in mixed infections.

Nearly Complete Genome Sequences of Two Bluetongue Viruses Isolated during the 2020 Outbreak in the Grand Duchy of Luxembourg.

Bluetongue is one of the major diseases of ruminants listed by the World Organisation for Animal Health. Bluetongue virus serotype 8 (BTV-8) has been considered enzootic in France since 2018. Here, we report the nearly complete genome sequences of two BTV-8 isolates from the 2020 outbreak in the Grand Duchy of Luxembourg.

Coding-Complete Sequences of Recombinant Lumpy Skin Disease Viruses Collected in 2020 from Four Outbreaks in Northern Vietnam.

Lumpy skin disease virus (LSDV) causes a severe, systemic, and economically important disease in cattle. Here, we report coding-complete sequences of recombinant LSDVs from four outbreaks in October and November 2020 in northeastern Vietnam.

Molecular and virological characterization of the first poultry outbreaks of Genotype VII.2 velogenic avian orthoavulavirus type 1 (NDV) in North-West Europe, BeNeLux, 2018.

After two decades free of Newcastle disease, Belgium encountered a velogenic avian orthoavulavirus type 1 epizootic in 2018. In Belgium, 20 cases were diagnosed, of which 15 occurred in hobby flocks, 2 in professional poultry flocks and 3 in poultry retailers. The disease also disseminated from Belgium towards the Grand Duchy of Luxembourg by trade. Independently, the virus was detected once in the Netherlands, almost simultaneously to the first Belgian detection. As such Newcastle disease emerged in the entire BeNeLux region. Both the polybasic sequence of the fusion gene cleavage site and the intracerebral pathotyping assay demonstrated the high pathogenicity of the strain. This paper represents the first notification of this specific VII.2 subgenotype in the North-West of Europe. Time-calibrated full genome phylogenetic analysis indicated the silent or unreported circulation of the virus prior to the emergence of three genetic clusters in the BeNeLux region without clear geographical or other epidemiological correlation. The Dutch strain appeared as an outgroup to the Belgian and Luxembourgian strains in the time-correlated genetic analysis and no epidemiological link could be identified between the Belgian and Dutch outbreaks. In contrast, both genetic and epidemiological outbreak investigation data linked the G.D. Luxembourg case to the Belgian outbreak. The genetic links between Belgian viruses from retailers and hobby flocks only partially correlated with epidemiological data. Two independent introductions into the professional poultry sector were identified, although their origin could not be determined. Animal experiments using 6-week- old specific pathogen-free chickens indicated a systemic infection and efficient transmission of the virus. The implementation of re-vaccination in the professional sector, affected hobby and retailers, as well as the restriction on assembly and increased biosecurity measures, possibly limited the epizootic and resulted in the disappearance of the virus. These findings emphasize the constant need for awareness and monitoring of notifiable viruses in the field.

Fine quantification of avian influenza H5N1 escape mutant quasispecies populations using mutation-specific real-time PCR.

Influenza viruses have a rapid replication cycle, using enzymes without proofreading capacity and generating multiple virus quasispecies during replication. The identification and quantification of these quasispecies populations require time-consuming and expensive cloning and sequencing approaches. In the present study, we developed mutation-specific real-time PCR (RT-PCR) tests for the fine quantification of mutations in a background of wild-type sequences. As a proof-of-concept model, we developed mutation-specific RT-PCR tests to quantify antibody escape mutations during passage under monoclonal antibody (mAb) selection pressure in quasispecies populations of HPAI A/crested eagle/Belgium/01/2004 (H5N1). Mutation-specific RT-PCRs were developed for two mutations (one in HA1 and one in HA2) and validated using plasmids representing either the wildtype sequence or the mutation. The approach achieves a precise and accurate estimation of mutation frequencies on mixed populations in the range of 1% to 99% and does not require standard curves or calibrators. For the HA1 mutations, a directional increase of % G over the passages towards fixation of the G mutation could be observed. On the contrary, as expected from the inaccessibility of the HA2 region to antibodies, the HA2 mutation increased in frequency by factors unrelated to mAb-driven selection. This approach allows in-depth analysis of quasispecies dynamics using large sample sizes. It may also be applied to the dynamics of hot spots of mutations in several genes, such as HA or PB2, and to the early detection of critical changes in the field situation.