High pathogenic avian influenza A(H5) viruses of clade 2.3.4.4b in Europe-Why trends of virus evolution are more difficult to predict.

Since 2016, A(H5Nx) high pathogenic avian influenza (HPAI) virus of clade 2.3.4.4b has become one of the most serious global threats not only to wild and domestic birds, but also to public health. In recent years, important changes in the ecology, epidemiology, and evolution of this virus have been reported, with an unprecedented global diffusion and variety of affected birds and mammalian species. After the two consecutive and devastating epidemic waves in Europe in 2020-2021 and 2021-2022, with the second one recognized as one of the largest epidemics recorded so far, this clade has begun to circulate endemically in European wild bird populations. This study used the complete genomes of 1,956 European HPAI A(H5Nx) viruses to investigate the virus evolution during this varying epidemiological outline. We investigated the spatiotemporal patterns of A(H5Nx) virus diffusion to/from and within Europe during the 2020-2021 and 2021-2022 epidemic waves, providing evidence of ongoing changes in transmission dynamics and disease epidemiology. We demonstrated the high genetic diversity of the circulating viruses, which have undergone frequent reassortment events, providing for the first time a complete overview and a proposed nomenclature of the multiple genotypes circulating in Europe in 2020-2022. We described the emergence of a new genotype with gull adapted genes, which offered the virus the opportunity to occupy new ecological niches, driving the disease endemicity in the European wild bird population. The high propensity of the virus for reassortment, its jumps to a progressively wider number of host species, including mammals, and the rapid acquisition of adaptive mutations make the trend of virus evolution and spread difficult to predict in this unfailing evolving scenario.

Susceptibility and Pathology in Juvenile Atlantic Cod Gadus morhua to a Marine Viral Haemorrhagic Septicaemia Virus Isolated from Diseased Rainbow Trout Oncorhynchus mykiss.

The first known outbreak caused by a viral haemorrhagic septicaemia virus (VHSV) strain of genotype III in rainbow trout occurred in 2007 at a marine farm in Storfjorden, Norway. The source of the virus is unknown, and cod and other marine fish around the farms are suspected as a possible reservoir. The main objective of this study was to test the susceptibility of juvenile Atlantic cod to the VHSV isolate from Storfjorden. As the pathology of VHS in cod is sparsely described, an additional aim of the study was to give a histopathological description of the disease. Two separate challenge experiments were carried out, using both intra peritoneal (ip) injection and cohabitation as challenge methods. Mortality in the ip injection experiment leveled at approximately 50% three weeks post challenge. Both immunohistochemical and rRT-PCR analysis of organs sampled from diseased and surviving fish confirmed VHSV infection. No VHSV was detected in the cohabitants. The results indicate that Atlantic cod has a low natural susceptibility to this VHSV genotype III strain. One of the most extensive pathological changes was degeneration of cardiac myocytes. Immunohistochemistry confirmed that the lesions were related to VHSV. In some fish, the hematopoietic tissue of spleen and kidney showed degeneration and immunostaining, classical signs of VHS, as described in rainbow trout. Positive immunostaining of the capillaries of the gills, suggests this organ as a useful alternative when screening for VHSV.

An Official Outbreak Investigation of Acute Haemorrhagic Diarrhoea in Dogs in Norway Points to Providencia alcalifaciens as a Likely Cause.

An outbreak investigation was initiated in September 2019, following a notification to the Norwegian Food Safety Authority (NFSA) of an unusually high number of dogs with acute haemorrhagic diarrhoea (AHD) in Oslo. Diagnostic testing by reporting veterinarians had not detected a cause. The official investigation sought to identify a possible common cause, the extent of the outbreak and prevent spread. Epidemiological data were collected through a survey to veterinarians and interviews with dog owners. Diagnostic investigations included necropsies and microbiological, parasitological and toxicological analysis of faecal samples and food. In total, 511 dogs with acute haemorrhagic diarrhoea were registered between 1 August and 1 October. Results indicated a common point source for affected dogs, but were inconclusive with regard to common exposures. A notable finding was that 134 of 325 faecal samples (41%) cultured positive for Providencia alcalifaciens. Whole genome sequencing (WGS) of 75 P. alcalifaciens isolates from 73 dogs revealed that strains from 51 dogs belonged to the same WGS clone. Findings point to P. alcalifaciens as implicated in the outbreak, but investigations are needed to reveal the pathogenic potential of P. alcalifaciens in dogs and its epidemiology.

First detection of highly pathogenic avian influenza virus in Norway.

BACKGROUND: Several outbreaks of highly pathogenic avian influenza (HPAI) caused by influenza A virus of subtype H5N8 have been reported in wild birds and poultry in Europe during autumn 2020. Norway is one of the few countries in Europe that had not previously detected HPAI virus, despite widespread active monitoring of both domestic and wild birds since 2005. RESULTS: We report detection of HPAI virus subtype H5N8 in a wild pink-footed goose (Anser brachyrhynchus), and several other geese, ducks and a gull, from south-western Norway in November and December 2020. Despite previous reports of low pathogenic avian influenza (LPAI), this constitutes the first detections of HPAI in Norway. CONCLUSIONS: The mode of introduction is unclear, but a northward migration of infected geese or gulls from Denmark or the Netherlands during the autumn of 2020 is currently our main hypothesis for the introduction of HPAI to Norway. The presence of HPAI in wild birds constitutes a new, and ongoing, threat to the Norwegian poultry industry, and compliance with the improved biosecurity measures on poultry farms should therefore be ensured. [MK1]Finally, although HPAI of subtype H5N8 has been reported to have very low zoonotic potential, this is a reminder that HPAI with greater zoonotic potential in wild birds may pose a threat in the future. [MK1]Updated with a sentence emphasizing the risk HPAI pose to poultry farms, both in the Abstract and in the Conclusion-section in main text, as suggested by Reviewer 1 (#7).

Bovine respiratory syncytial virus in experimentally exposed and rechallenged calves; viral shedding related to clinical signs and the potential for transmission.

BACKGROUND: Bovine respiratory syncytial virus (BRSV) is an important respiratory pathogen worldwide, detrimentally affecting the economy and animal welfare. To prevent and control BRSV infection, further knowledge on virus shedding and transmission potential in individual animals is required. This study aimed to detect viral RNA and infective virions during BRSV infection to evaluate duration of the transmission period and correlation with clinical signs of disease. The outcome of BRSV re-exposure on calves, their housing environment and effect of introduction of sentinel calves was also investigated. A live animal experiment including 10 calves was conducted over 61 days. Initially, two calves were inoculated with a non-passaged BRSV field isolate. Two days later, six naïve calves (EG: Exposed group) were introduced for commingling and four weeks later, another two naïve calves (SG: Sentinel group) were introduced. Seven weeks after commingling, EG animals were re-inoculated. Clinical examination was performed daily. Nasal swabs were collected regularly and analysed for viral RNA by RT-ddPCR, while virus isolation was performed in cell culture. BRSV serology was performed with ELISA. RESULTS: All the EG calves seroconverted and showed clinical signs of respiratory disease. Viral RNA was detected from days 1-27 after exposure, while the infective virus was isolated on day 6 and 13. On day 19, all animals were seropositive and virus could not be isolated. Total clinical score for respiratory signs corresponded well with the shedding of viral RNA. The SG animals, introduced 27 days after exposure, remained negative for BRSV RNA and stayed seronegative throughout the study. Inoculation of the EG calves seven weeks after primary infection did not lead to new shedding of viral RNA or clinical signs of disease. CONCLUSION: Viral RNA was detected in nasal swabs from the calves up to four weeks after exposure. The detection and amount of viral RNA corresponded well with the degree of respiratory signs. The calves were shedding infective virions for a considerable shorter period, and naïve calves introduced after four weeks were not infected. Infected calves were protected from reinfection for at least seven weeks. This knowledge is useful to prevent spread of BRSV.

Temporary carriage of bovine coronavirus and bovine respiratory syncytial virus by fomites and human nasal mucosa after exposure to infected calves.

BACKGROUND: In order to prevent spread of the endemic pathogens bovine coronavirus (BCoV) and bovine respiratory syncytial virus (BRSV) between herds, knowledge of indirect transmission by personnel and fomites is fundamental. The aims of the study were to determine the duration of viral RNA carriage and the infectivity of viral particles on fomites and human nasal mucosa after exposure to BCoV and BRSV. During two animal infection experiments, swabs were collected from personnel (nasal mucosa) and their clothes, boots and equipment after contact with calves shedding either virus. Viral RNA was quantified by RT-qPCR or droplet digital RT-PCR (RT-ddPCR), and selected samples with high levels of viral RNA were tested by cell culture for infectivity. RESULTS: For BCoV, 46% (n = 80) of the swabs from human nasal mucosa collected 30 min after exposure were positive by RT-qPCR. After two, four and six hours, 15%, 5% and 0% of the swabs were positive, respectively. Infective virions were not detected in mucosal swabs (n = 2). A high viral RNA load was detected on 97% (n = 44) of the fomites 24 h after exposure, and infective virions were detected in two of three swabs. For BRSV, 35% (n = 26) of the human nasal mucosa swabs collected 30 min after exposure, were positive by RT-ddPCR, but none were positive for infective virions. Of the fomites, 89% (n = 38) were positive for BRSV RNA 24 h after exposure, but all were negative for infective viruses. CONCLUSIONS: The results indicate that human nasal mucosa can carry both BCoV and BRSV RNA after exposure to virus shedding calves, but the carriage seems short-lived and the transmission potential is likely limited. High viral loads on contaminates fomites 24 h after exposure to infected animals, and detection of infective BCoV, indicate that contaminated fomites represent a significant risk for indirect transmission between herds.

Human to animal transmission of influenza A(H1N1)pdm09 in a turkey breeder flock in Norway.

Introduction: Routine surveillance samples disclosed seropositivity to influenza A virus (IAV) in a Norwegian turkey breeder flock. Simultaneous reports of influenza-like symptoms in farm workers and a laboratory confirmed influenza A(H1N1)pdm09 (H1N1pdm09) infection in one person led to the suspicion of a H1N1pdm09 infection in the turkeys. Animals and methods: H1N1pdm09 infection was confirmed by a positive haemaggutinin inhibition test using H1N1pdm09 antigens, and detection of H1N1pdm09 nucleic acid in reproductive organs of turkey hens. The flock showed no clinical signs except for a temporary drop in egg production. Previous reports of H1N1pdm09 infection in turkeys suggested human-to-turkey transmission (anthroponosis) during artificial insemination. Results and discussion: The flock remained seropositive to IAV and the homologous H1N1pdm09 antigen throughout the following 106 days, with decreasing seroprevalence over time. IAV was not detected in fertilised eggs or in turkey poults from the farm, however, maternally derived antibodies against H1N1pdm09 were found in egg yolks and in day-old poults. Genetic analyses of haemagglutinin gene sequences from one of the infected farm workers and turkeys revealed a close phylogenetic relationship, and confirmed human-to-turkey virus transmission.

Screening for viral hemorrhagic septicemia virus in marine fish along the Norwegian coastal line.

Viral hemorrhagic septicemia virus (VHSV) infects a wide range of marine fish species. To study the occurrence of VHSV in wild marine fish populations in Norwegian coastal waters and fjord systems a total of 1927 fish from 39 different species were sampled through 5 research cruises conducted in 2009 to 2011. In total, VHSV was detected by rRT-PCR in twelve samples originating from Atlantic herring (Clupea harengus), haddock (Melanogrammus aeglefinus), whiting (Merlangius merlangus) and silvery pout (Gadiculus argenteus). All fish tested positive in gills while four herring and one silvery pout also tested positive in internal organs. Successful virus isolation in cell culture was only obtained from one pooled Atlantic herring sample which shows that today's PCR methodology have a much higher sensitivity than cell culture for detection of VHSV. Sequencing revealed that the positive samples belonged to VHSV genotype Ib and phylogenetic analysis shows that the isolate from Atlantic herring and silvery pout are closely related. All positive fish were sampled in the same area in the northern county of Finnmark. This is the first detection of VHSV in Atlantic herring this far north, and to our knowledge the first detection of VHSV in silvery pout. However, low prevalence of VHSV genotype Ib in Atlantic herring and other wild marine fish are well known in other parts of Europe. Earlier there have been a few reports of disease outbreaks in farmed rainbow trout with VHSV of genotype Ib, and our results show that there is a possibility of transfer of VHSV from wild to farmed fish along the Norwegian coast line. The impact of VHSV on wild fish is not well documented.

Multiple introductions of salmonid alphavirus from a wild reservoir have caused independent and self-sustainable epizootics in aquaculture.

Salmonid alphavirus (SAV) causes infections in farmed Atlantic salmon and rainbow trout in Europe. Genetic diversity exists among SAV strains from farmed fish and six subtypes have been proposed based on genetic distance. Here, we used six full-genome sequences and 71 partial sequences of the structural ORF to estimate the evolutionary rate of SAV. The rate, 2.13×10(-4) nt substitutions per site per year, was further used to date evolutionary events in a Bayesian phylogenetic framework. The comparison of these dates with known historical events suggested that all six subtypes diverged prior to the twentieth century, earlier than the first attempts to introduce and farm rainbow trout in Europe. The subtypes must therefore have existed in a wild reservoir, as yet unidentified. The strains of each subtype, with the exception of subtype 2, have a common ancestor that existed after the 1970s - the start of modern farming of Atlantic salmon. These ancestors are likely to represent the independent introductions to farmed fish populations from the wild reservoir. The subtypes have developed subsequently into self-sustainable epizootics. The most parsimonious phylogeographic reconstruction suggested that the location of the wild reservoir is in or around the North Sea. After the initial introductions to aquaculture, further transmission of SAV was likely related to the industry infrastructure. This was exemplified by the finding of genetically identical subtype 2 and 3 strains separated by large geographical distances, as well as genetically distinct co-circulating lineages within the same geographical area.

Swine influenza in Norway: a distinct lineage of influenza A(H1N1)pdm09 virus.

BACKGROUND: Since the influenza A(H1N1)pdm09 virus was first introduced to the Norwegian pig population in September 2009, it has repeatedly been detected in pigs in Norway. No other subtypes of influenza virus are circulating in Norwegian pigs. OBJECTIVE: To follow the diversity of A(H1N1)pdm09 viruses circulating in pigs in Norway and to investigate the relationship between viruses circulating in Norwegian pigs and in humans. METHODS: Between January 2011 and January 2013, nasal swabs from 507 pigs were tested for A(H1N1)pdm09 virus by real-time RT-PCR. The hemagglutinin (HA) gene of virus-positive samples was sequenced and compared with publically available sequences from viruses circulating in humans at the time. RESULTS: Sequencing and phylogenetic analysis of the HA gene showed that the A(H1N1)pdm09 virus circulating in Norwegian pigs early in 2011 resembled the A(H1N1)pdm09 virus circulating in humans during this time. Viruses detected in pigs by the end of 2011 had acquired four characteristic amino acid substitutions (N31D, S84I S164F, and N473D) and formed a distinct phylogenetic group. CONCLUSIONS: A(H1N1)pdm09 virus detected in Norwegian pigs by the end of 2011 formed a distinct genetic lineage. Also, our findings indicate that reverse-zoonotic transmission from humans to pigs of the A(H1N1)pdm09 virus is still important.

Influenza A(H1N1)pdm09 virus infection in Norwegian swine herds 2009/10: the risk of human to swine transmission.

Influenza A viruses cause respiratory infection in humans and pigs, and some serotypes can be transmitted between these species. The emergence of influenza A(H1N1)pdm09 virus infections in the spring of 2009 quickly led to a worldwide pandemic in humans, with subsequent introduction of the virus to pig populations. Following a widespread infection in the human population in Norway, influenza A(H1N1)pdm09 virus was introduced to the influenza A naïve Norwegian pig population, and within a few months pigs in more than one third of Norwegian swine herds had antibodies against the virus. A cross-sectional study was performed on all swine nucleus and multiplier herds in Norway to analyze risk factors for introduction of infection, and the preventive effects of recommended biosecurity practices. A surveillance program provided information on infection status of the study herds, and a questionnaire was administered to all 118 nucleus and multiplier herds to collect information on herd variables. The surveillance program revealed that pigs in 42% of the herds had antibodies against influenza A(H1N1)pdm09 virus. The incidence of serologically positive pigs was similar in both multiplier herds (41%) and closed nucleus herds (43%). Multivariable logistic regression showed that presence of farm staff with influenza-like illness (ILI) (OR=4.15, CI 1.5-11.4, p=0.005) and herd size (OR=1.01, CI 1-1.02, p=0.009) were risk factors for infection. The rapid and widespread seroconversion for antibodies against influenza A(H1N1)pdm09 virus in the Norwegian pig population can be explained by the emergence of a novel virus that is readily transmitted between people and swine in a largely susceptible population of humans, and an entirely naïve population of pigs.

Clinical Impact of Infection with Pandemic Influenza (H1N1) 2009 Virus in Naïve Nucleus and Multiplier Pig Herds in Norway.

The Norwegian pig population has been free from influenza viruses until 2009. The pandemic influenza outbreak during the autumn 2009 provided an opportunity to study the clinical impact of this infection in an entirely naïve pig population. This paper describes the results of a case-control study on the clinical impact of pandemic influenza (H1N1) 2009 infection in the nucleus and multiplier herds in Norway. The infection spread readily and led to seroconversion of 42% of the Norwegian nucleus and multiplier herds within a year. Positive and negative herds were identified based on surveillance data from the Norwegian Veterinary Institute. Telephone interviews were conducted with pig herd owners or managers between November 2010 and January 2011. Pigs with clinical signs were reported from 40% of the case herds with varying morbidity and duration of respiratory disease and reduced reproductive performance. Clinical signs were reported in all age groups.

Pathological Findings and Distribution of Pandemic Influenza A (H1N1) 2009 Virus in Lungs from Naturally Infected Fattening Pigs in Norway.

The Norwegian pig population was considered free from influenza A virus infections until the first case of porcine pandemic influenza A (H1N1) 2009 virus infection in October 2009. Human to pig transmission of virus was suspected. Unusual lung lesions were observed in fattening pigs, with red, lobular, multifocal to coalescing consolidation, most frequently in the cranial, middle, and accessory lobes. The main histopathological findings were epithelial degeneration and necrosis, lymphocyte infiltration in the epithelial lining and lamina propria of small bronchi and bronchioles, and peribronchial and peribronchiolar lymphocyte infiltrations. Infection with pandemic influenza A (H1N1) 2009 virus was confirmed by real-time RT-PCR and immunohistochemical detection of influenza A virus nucleoprotein in the lesions. This investigation shows that natural infection with the pandemic influenza A (H1N1) 2009 virus induces lung lesions similar to lesions described in experimental studies and natural infections with other swine-adapted subtypes of influenza A viruses.

Molecular epidemiology of salmonid alphavirus (SAV) subtype 3 in Norway.

BACKGROUND: Pancreas disease (PD) is a viral fish disease which in recent years has significantly affected Norwegian salmonid aquaculture. In Norway, the aetiological agent salmonid alphavirus (SAV) has been found to be represented by the subtype 3 only. SAV subtype 3 has in previous analyses been found to show a lower genetic divergence than the subtypes found to cause PD in Ireland and Scotland. The aim of this study was to evaluate the nucleotide (nt) and amino acid divergence and the phylogenetic relationship of 33 recent SAV subtype 3 sequences. The samples from which the sequences were obtained originated from both PD endemic and non-endemic regions in an attempt to investigate agent origin/spread. Multiple samples throughout the seawater production phase from several salmonid populations were included to investigate genetic variation during an outbreak. The analyses were mainly based on partial sequences from the E2 gene. For some samples, additional partial 6 K and nsP3 gene sequences were available. RESULTS: The nucleotide divergence for all gene fragments ranged from total identity (0.0% divergence) to 0.45% (1103 nt fragment of E2), 1.11% (451 nt fragment of E2), 0.94% (6 K) and 0.28% (nsP3). This low nucleotide divergence corresponded well to previous reports on SAV 3 sequences; however the observed divergence for the short E2 fragment was higher than that previously reported. When compared to SAVH20/03 (AY604235), amino acid substitutions were detected in all assessed gene fragments however the in vivo significance of these on for example disease outbreak mortality could not be concluded on. The phylogenetic tree based on the 451 nt E2 fragment showed that the sequences divided into two clusters with low genetic divergence, representing only a single SAV subtype. CONCLUSIONS: The analysed sequences represented two clusters of a single SAV subtype; however some of the observed sequence divergence was higher than that previously reported by other researchers. Larger scale, full length sequence analyses should be instigated to allow further phylogenetic and molecular epidemiology investigations of SAV subtype 3.

Impact of natural sheep-goat transmission on detection and control of small ruminant lentivirus group C infections.

Dissemination of small ruminant lentivirus (SRLV) infections in Norway is affected by the different control strategies used for maedi-visna virus (MVV) infections in sheep and caprine arthritis-encephalitis virus (CAEV) infections in goats. Here we investigated SRLV phylogenetic group variants in sheep. CAEV-like isolates, belonging to phylogenetic group C, were found among both seropositive sheep and goats in mixed flocks, in which sheep and goats are kept together. Intra-herd clustering confirmed that mixed flock animals were infected by the same virus variant, suggesting ongoing interspecies transmission. Few sheep flocks were found to be infected with the MVV-like phylogenetic group A. The apparent absence of SRLV group A type in goats is probably due to the MVV control programme and animal management practices. SRLV group C targets lungs and mammary glands in sheep, and induces typical SRLV pathological lesions. SRLV group C isolated from the sheep mammary glands suggested a productive infection and potential for transmission to offspring. SRLV group C was most prevalent among goats. A lower PCR sensitivity in seropositive sheep suggested a lower load of SRLV group C provirus in sheep than in goats. Higher genetic divergence of group C than in other SRLV groups and extensive heterogeneity among group C isolates in the matrix C-terminal region demonstrate the need for identifying conserved target regions when developing PCR protocols for SRLV detection. As sheep and goats may serve as reservoirs for all SRLV genogroup types, successful control programmes require inclusion of both species.

Natural transmission and comparative analysis of small ruminant lentiviruses in the Norwegian sheep and goat populations.

Serological surveys for small ruminant lentivirus (SRLV) infections have revealed seropositive sheep in several mixed herds, where sheep are kept together with seropositive goats. Here we have examined the genetic relationships in LTR, pol and env surface unit (SU) and the growth patterns in goat (GSM) and sheep (FOS) synovial membrane cell cultures of SRLV isolates obtained from both mixed and single species herds. Phylogenetic analyses of pol and env SU revealed that Norwegian SRLVs derived from both goat and sheep in mixed herds are distributed into group C, while isolates obtained from unmixed sheep flocks cluster in group A, together with maedi-visna-like representatives of the A1 subtype. In this study, the direction of group C virus transmission is proposed to be from goat to sheep. The replication efficiency in GSM and FOS cultures and the cytopathic phenotype induced by the SRLV isolates gave no indication of any species-specific characteristics. No particular nucleotide sequences of the LTR-U3 region or env SU were identified that could be related to cytopathic phenotype. This study shows that sheep in Norway harbour SRLVs belonging to phylogenetic groups A and C, and this provides further evidence for cross-species infection being a regular characteristic of SRLVs, which may represent an important source for viral persistence.

Genetic diversity of small-ruminant lentiviruses: characterization of Norwegian isolates of Caprine arthritis encephalitis virus.

Small-ruminant lentiviruses (SRLVs), including Caprine arthritis encephalitis virus (CAEV) in goats and maedi-visna virus (MVV) in sheep, are lentiviruses that, despite overall similarities, show considerable genetic variation in regions of the SRLV genome. To gain further knowledge about the genetic diversity and phylogenetic relationships among field isolates of SRLVs occurring in geographically distinct areas, the full-length genomic sequence of a CAEV isolate (CAEV-1GA) and partial env sequences obtained from Norwegian CAEV-infected goats were determined. The genome of CAEV-1GA consisted of 8,919 bp. Alignment studies indicated significant diversity from published SRLV sequences. Deletions and hypervariability in the 5' part of the env gene have implications for the size of the proposed CAEV-1GA Rev protein and the encoded surface glycoprotein (SU). The variable regions in the C-terminal part of SU obtained from Norwegian CAEV isolates demonstrate higher sequence divergence than has been described previously for SRLVs. Phylogenetic analysis based on SU sequences gives further support for a unique group designation. The results described here reveal a distant genetic relationship between Norwegian CAEV and other SRLVs and demonstrate that there is more geographical heterogeneity among SRLVs than reported previously.