The effect of enzymatic and viability dye treatment in combination with long-range PCR on assessing Tulane virus infectivity.

Human norovirus (HuNoV) is regularly involved in food-borne infections. To detect infectious HuNoV in food, RT-qPCR remains state of the art but also amplifies non-infectious virus. The present study combines pre-treatments, RNase and propidium monoazide, with three molecular analyses, including long-range PCR, to predominantly detect infectious Tulane virus (TuV), a culturable HuNoV surrogate. TuV was exposed to inactivating conditions to assess which molecular method most closely approximates the reduction in infectious virus determined by cell culture (TCID50). After thermal treatments (56 °C/5 min, 70 °C/5 min, 72 °C/20 min), TCID50 reductions of 0.3, 4.4 and 5.9 log10 were observed. UV exposure (40/100/1000 mJ/cm2) resulted in 1.1, 2.5 and 5.9 log10 reductions. Chlorine (45/100 mg/L for 1 h) reduced infectious TuV by 2.0 and 3.0 log10. After thermal inactivation standard RT-qPCR, especially with pre-treatments, showed the smallest deviation from TCID50. On average, RT-qPCR with pre-treatments deviated by 1.1-1.3 log10 from TCID50. For UV light, long-range PCR was closest to TCID50 results. Long-range reductions deviated from TCID50 by ≤0.1 log10 for mild and medium UV-conditions. However, long-range analyses often resulted in qPCR non-detects. At higher UV doses, RT-qPCR with pre-treatments differed by ≤1.0 log10 from TCID50. After chlorination the molecular methods repeatedly deviated from TCID50 by >1.0 log10, Overall, each method needs to be further optimized for the individual types of inactivation treatment.

An international inter-laboratory study to compare digital PCR with ISO standardized qPCR assays for the detection of norovirus GI and GII in oyster tissue.

An optimized digital RT-PCR (RT-dPCR) assay for the detection of human norovirus GI and GII RNA was compared with ISO 15216-conform quantitative real-time RT-PCR (RT-qPCR) assays in an interlaboratory study (ILS) among eight laboratories. A duplex GI/GII RT-dPCR assay, based on the ISO 15216-oligonucleotides, was used on a Bio-Rad QX200 platform by six laboratories. Adapted assays for Qiagen Qiacuity or ThermoFisher QuantStudio 3D were used by one laboratory each. The ILS comprised quantification of norovirus RNA in the absence of matrix and in oyster tissue samples. On average, results of the RT-dPCR assays were very similar to those obtained by RT-qPCR assays. The coefficient of variation (CV%) of norovirus GI results was, however, much lower for RT-dPCR than for RT-qPCR in intra-laboratory replicates (eight runs) and between the eight laboratories. The CV% of norovirus GII results was in the same range for both detection formats. Had in-house prepared dsDNA standards been used, the CV% of norovirus GII could have been in favor of the RT-dPCR assay. The ratio between RT-dPCR and RT-qPCR results varied per laboratory, despite using the distributed RT-qPCR dsDNA standards. The study indicates that the RT-dPCR assay is likely to increase uniformity of quantitative results between laboratories.

Effectiveness of environmental surveillance of SARS-CoV-2 as an early-warning system: Update of a systematic review during the second year of the pandemic.

The aim of this updated systematic review was to offer an overview of the effectiveness of environmental surveillance (ES) of SARS-CoV-2 as a potential early-warning system (EWS) for COVID-19 and new variants of concerns (VOCs) during the second year of the pandemic. An updated literature search was conducted to evaluate the added value of ES of SARS-CoV-2 for public health decisions. The search for studies published between June 2021 and July 2022 resulted in 1,588 publications, identifying 331 articles for full-text screening. A total of 151 publications met our inclusion criteria for the assessment of the effectiveness of ES as an EWS and early detection of SARS-CoV-2 variants. We identified a further 30 publications among the grey literature. ES confirms its usefulness as an EWS for detecting new waves of SARS-CoV-2 infection with an average lead time of 1-2 weeks for most of the publication. ES could function as an EWS for new VOCs in areas with no registered cases or limited clinical capacity. Challenges in data harmonization and variant detection require standardized approaches and innovations for improved public health decision-making. ES confirms its potential to support public health decision-making and resource allocation in future outbreaks.

Gene expression profile of HCT-8 cells following single or co-infections with Cryptosporidium parvum and bovine coronavirus.

Among the causative agents of neonatal diarrhoea in calves, two of the most prevalent are bovine coronavirus (BCoV) and the intracellular parasite Cryptosporidium parvum. Although several studies indicate that co-infections are associated with greater symptom severity, the host-pathogen interplay remains unresolved. Here, our main objective was to investigate the modulation of the transcriptome of HCT-8 cells during single and co-infections with BCoV and C. parvum. For this, HCT-8 cells were inoculated with (1) BCoV alone, (2) C. parvum alone, (3) BCoV and C. parvum simultaneously. After 24 and 72 h, cells were harvested and analyzed using high-throughput RNA sequencing. Following differential expression analysis, over 6000 differentially expressed genes (DEGs) were identified in virus-infected and co-exposed cells at 72 hpi, whereas only 52 DEGs were found in C. parvum-infected cells at the same time point. Pathway (KEGG) and gene ontology (GO) analysis showed that DEGs in the virus-infected and co-exposed cells were mostly associated with immune pathways (such as NF-κB, TNF-α or, IL-17), apoptosis and regulation of transcription, with a more limited effect exerted by C. parvum. Although the modulation observed in the co-infection was apparently dominated by the virus, over 800 DEGs were uniquely expressed in co-exposed cells at 72 hpi. Our findings provide insights on possible biomarkers associated with co-infection, which could be further explored using in vivo models.

Implementing risk-based approaches to improve drinking water quality in small water supplies in the Nordic region - barriers and solutions.

Small water supplies face similar problems worldwide, regardless of ownership or management type. Non-compliance with water quality regulations is more frequent in small supplies than in large ones, as are waterborne disease outbreaks. The new European Union Drinking Water Directive requires risk-based approach (RBA) to secure water safety as is recommended in the World Health Organization's Guidelines for drinking water quality through 'water safety plans'. This is already in regulation in the Nordic countries, although less used in small supplies. In this research, we explore the challenges, barriers and possible solutions to implementing RBA and improving compliance in small supplies. This was achieved by conducting and analysing interviews with 53 stakeholders from all eight Nordic countries to produce recommendations for action by the different implicated actors. Our findings suggest the centrality of governmental policy, including support for continuous training, provision of simple RBA guidelines and increasing cooperation in the water sector. The Nordic experience reflects global challenges with small water supplies and the trend towards systematic preventive management epitomized in the framework for drinking water safety advocated by the World Health Organization since 2004.

Airborne SARS-CoV-2 is more frequently detected in environments related to children and elderly but likely non-infectious, Norway, 2022.

This study investigates the presence of SARS-CoV-2 in indoor and outdoor environments in two cities in Norway between April and May 2022. With the lifting of COVID-19 restrictions in the country and a focus on vaccination, this research aims to shed light on the potential for virus transmission in various settings. Air sampling was conducted in healthcare and non-healthcare facilities, covering locations frequented by individuals across different age groups. The study found that out of 31 air samples, only four showed the presence of SARS-CoV-2 RNA by RT-qPCR, with no viable virus detected after RNAse pre-treatment. These positive samples were primarily associated with environments involving children and the elderly. Notably, sequencing revealed mutations associated with increased infectivity in one of the samples. The results highlight the importance of considering children as potential sources of virus transmission, especially in settings with prolonged indoor exposure. As vaccination coverage increases globally, and with children still representing a substantial unvaccinated population, the study emphasizes the need to re-implement mask-wearing mandates indoors and in public transport to reduce virus transmission. The findings have implications for public health strategies to control COVID-19, particularly in the face of new variants and the potential for increased transmission during the autumn and winter seasons.

Freshwater plastispheres as a vector for foodborne bacteria and viruses.

There is growing evidence that plastic particles can accumulate microorganisms that are pathogenic to humans or animals. In the current study, the composition of the plastispheres that accumulated on polypropylene (PP), polyvinyl chloride (PVC), and high-density polyethylene (HDPE) pieces submerged in a river in the southeast Norway was characterized by 16S rRNA amplicon sequencing. Seasonal and geographical effects on the bacterial composition of the plastisphere were identified, in addition to the detection of potential foodborne pathogenic bacteria and viruses as part of the plastisphere. The diversity and taxonomic composition of the plastispheres were influenced by the number of weeks in the river, the season, and the location. The bacterial diversity differed significantly in the plastisphere from June and September, with a generally higher diversity in June. Also, the community composition of the plastisphere was significantly influenced by the geographical location, while the type of plastic had less impact. Plastics submerged in river water assembled a variety of microorganisms including potentially pathogenic bacteria and viruses (noro- and adenovirus) detected by qPCR. Cultivation methods detected viable bacteria such as Escherichia coli and Listeria monocytogenes. The results highlight the need for additional research on the risk of contaminating food with plastic particles colonized with human pathogens through irrigation water.

Bovine Enteroids as an In Vitro Model for Infection with Bovine Coronavirus.

Bovine coronavirus (BCoV) is one of the major viral pathogens of cattle, responsible for economic losses and causing a substantial impact on animal welfare. Several in vitro 2D models have been used to investigate BCoV infection and its pathogenesis. However, 3D enteroids are likely to be a better model with which to investigate host-pathogen interactions. This study established bovine enteroids as an in vitro replication system for BCoV, and we compared the expression of selected genes during the BCoV infection of the enteroids with the expression previously described in HCT-8 cells. The enteroids were successfully established from bovine ileum and permissive to BCoV, as shown by a seven-fold increase in viral RNA after 72 h. Immunostaining of differentiation markers showed a mixed population of differentiated cells. Gene expression ratios at 72 h showed that pro-inflammatory responses such as IL-8 and IL-1A remained unchanged in response to BCoV infection. Expression of other immune genes, including CXCL-3, MMP13, and TNF-α, was significantly downregulated. This study shows that the bovine enteroids had a differentiated cell population and were permissive to BCoV. Further studies are necessary for a comparative analysis to determine whether enteroids are suitable in vitro models to study host responses during BCoV infection.

Temperature Dependent Depuration of Norovirus GII and Tulane Virus from Oysters (Crassostrea gigas).

Raw oysters are considered a culinary delicacy but are frequently the culprit in food-borne norovirus (NoV) infections. As commercial depuration procedures are currently unable to efficiently eliminate NoV from oysters, an optimisation of the process should be considered. This study addresses the ability of elevated water temperatures to enhance the elimination of NoV and Tulane virus (TuV) from Pacific oysters (Crassostrea gigas). Both viruses were experimentally bioaccumulated in oysters, which were thereafter depurated at 12 °C and 17 °C for 4 weeks. Infectious TuV and viral RNA were monitored weekly for 28 days by TCID50 and (PMAxx-) RT-qPCR, respectively. TuV RNA was more persistent than NoV and decreased by < 0.5 log10 after 14 days, while NoV reductions were already > 1.0 log10 at this time. For RT-qPCR there was no detectable benefit of elevated water temperatures or PMAxx for either virus (p > 0.05). TuV TCID50 decreased steadily, and reductions were significantly different between the two temperatures (p < 0.001). This was most evident on days 14 and 21 when reductions at 17 °C were 1.3-1.7 log10 higher than at 12 °C. After 3 weeks, reductions > 3.0 log10 were observed at 17 °C, while at 12 °C reductions did not exceed 1.9 log10. The length of depuration also had an influence on virus numbers. TuV reductions increased from < 1.0 log10 after seven days to > 4.0 log10 after 4 weeks. This implies that an extension of the depuration period to more than seven days, possibly in combination with elevated water temperatures, may be beneficial for the inactivation and removal of viral pathogens.

Effectiveness of environmental surveillance of SARS-CoV-2 as an early warning system during the first year of the COVID-19 pandemic: a systematic review.

Since infected persons shed SARS-CoV-2 in faeces before symptoms appear, environmental surveillance (ES) may serve as an early warning system (EWS) for COVID-19 and new variants of concern. The ES of SARS-CoV-2 has been widely reviewed; however, its effectiveness as an EWS for SARS-CoV-2 in terms of timeliness, sensitivity and specificity has not been systematically assessed. We conducted a systematic review to identify and synthesise evidence on the ES of SARS-CoV-2 as an EWS to evaluate the added value for public health. Of 1,014 studies identified, we considered 29 for a qualitative synthesis of the timeliness of ES as an EWS for COVID-19, while six studies were assessed for the ability to detect new variants and two for both aims. The synthesis indicates ES may serve as an EWS of 1-2 weeks. ES could complement clinical surveillance for SARS-CoV-2; however, its cost-benefit value for public health decisions needs to be assessed based on the stage of the pandemic and resources available. Studies focusing methodological knowledge gaps as well as how to use and interpret ES signals for public health actions are needed, as is the sharing of knowledge within countries/areas with long experience of such surveillance.

Interactions between Cryptosporidium parvum and bovine corona virus during sequential and simultaneous infection of HCT-8 cells.

Neonatal diarrhoea in calves is one of the major health problems in the cattle industry. Although co-infections are often associated with greater severity of disease, there is limited information on any impact on the pathogens themselves. Herein, we studied Cryptosporidium parvum and bovine coronavirus (BCoV) in human HCT-8 cells, inoculated either sequentially or simultaneously, to investigate any influence from the co-infections. Quantitative results from (RT)-qPCR showed that prior inoculation with either of the two pathogens had no influence on the other. However, the results from simultaneous co-inoculation showed that entry of viral particles was higher when C. parvum sporozoites were present, although elevated virus copy numbers were no longer evident after 24 h. The attachment of BCoV to the sporozoites was probably due to specific binding, as investigations with bovine norovirus or equine herpes virus-1 showed no attachment between sporozoites and these viruses. Flow cytometry results at 72 h post inoculation revealed that C. parvum and BCoV infected 1-11% and 10-20% of the HCT-8 cells, respectively, with only 0.04% of individual cells showing double infections. The results from confocal microscopy corroborated those results, showing an increase in foci of infection from 24 to 72 h post inoculation for both pathogens, but with few double infected cells.

Microbiological Food Safety of Seaweeds.

The use of seaweeds in the human diet has a long history in Asia and has now been increasing also in the western world. Concurrent with this trend, there is a corresponding increase in cultivation and harvesting for commercial production. Edible seaweed is a heterogenous product category including species within the green, red, and brown macroalgae. Moreover, the species are utilized on their own or in combinatorial food products, eaten fresh or processed by a variety of technologies. The present review summarizes available literature with respect to microbiological food safety and quality of seaweed food products, including processing and other factors controlling these parameters, and emerging trends to improve on the safety, utilization, quality, and storability of seaweeds. The over- or misuse of antimicrobials and the concurrent development of antimicrobial resistance (AMR) in bacteria is a current worldwide health concern. The role of seaweeds in the development of AMR and the spread of antimicrobial resistance genes is an underexplored field of research and is discussed in that context. Legislation and guidelines relevant to edible seaweed are also discussed.

Early detection of salmonid alphavirus in seawater from marine farm sites of Atlantic salmon Salmo salar.

The traditional strategy for national surveillance of salmonid alphavirus (SAV) infection in Norwegian fish farms relies on a costly, time-consuming, and resource-demanding approach based on the monthly sampling of fish from all marine farms with salmonids. In order to develop an alternative surveillance method, a water filtration method was tested in parallel with the ongoing surveillance program at 7 Norwegian marine farm sites of Atlantic salmon Salmo salar L. with no current suspicion of SAV infection. During the period from May 2019 to January 2020, seawater samples were collected from the top layer water inside all net-pens at these 7 sites. The samples were concentrated for SAV by filtration through an MF-Millipore™ electronegative membrane filter, followed by rinsing with NucliSENS® Lysis Buffer, before RNA extraction and analysis by RT-qPCR. SAV was detected from seawater at an earlier stage compared to traditional sampling methods, at all sites where the fish tested positive for SAV. A significant negative relationship was observed at all sites between the SAV concentration found in seawater samples and the number of days until SAV was detected in the fish. This means that the fewer the SAV particles in the seawater, the more days it took until SAV was detected in the fish samples. Based on this, sampling of seawater every month for the surveillance of SAV has a great potential as an alternative method for early detection of SAV in Atlantic salmon farms.

Making Waves: Collaboration in the time of SARS-CoV-2 - rapid development of an international co-operation and wastewater surveillance database to support public health decision-making.

The presence of SARS-CoV-2 RNA in wastewater was first reported in March 2020. Over the subsequent months, the potential for wastewater surveillance to contribute to COVID-19 mitigation programmes has been the focus of intense national and international research activities, gaining the attention of policy makers and the public. As a new application of an established methodology, focused collaboration between public health practitioners and wastewater researchers is essential to developing a common understanding on how, when and where the outputs of this non-invasive community-level approach can deliver actionable outcomes for public health authorities. Within this context, the NORMAN SCORE "SARS-CoV-2 in sewage" database provides a platform for rapid, open access data sharing, validated by the uploading of 276 data sets from nine countries to-date. Through offering direct access to underpinning meta-data sets (and describing its use in data interpretation), the NORMAN SCORE database is a resource for the development of recommendations on minimum data requirements for wastewater pathogen surveillance. It is also a tool to engage public health practitioners in discussions on use of the approach, providing an opportunity to build mutual understanding of the demand and supply for data and facilitate the translation of this promising research application into public health practice.

Filtration, concentration and detection of salmonid alphavirus in seawater during a post-smolt salmon (Salmo salar) cohabitant challenge.

Currently, the prevalence of salmonid alphavirus (SAV) in Norwegian Atlantic salmon farms is largely surveyed via sacrificing fish and sampling of organ tissue on a monthly basis. However, a more cost-efficient, straightforward, rapid, reliable, reproducible and animal welfare friendly method based on the detection of SAV in water could be considered as an alternative method. In the present study, such a method was developed and optimized through a 6 wk cohabitant challenge trial, using post-smolt Atlantic salmon Salmo salar L challenged with high or low doses of SAV subtype 3 (SAV3). Tank water and tissue samples from cohabitant fish were collected at 16 time points. SAV3 was concentrated from the water by filtration, using either electronegative or electropositive membrane filters, which were subsequently rinsed with one of 4 different buffer solutions. SAV3 was detected first in tank water (7 d post-challenge, DPC), and later in cohabitant fish organ tissue samples (12 DPC). The electronegative filter (MF-Millipore™) and rinsing with NucliSENS® easyMAG® Lysis Buffer presented the best SAV3 recovery. A significant positive correlation was found between SAV3 in the tank water concentrates and the mid-kidney samples. Based on these results, detection of SAV3 in filtrated seawater is believed to have the potential to serve as an alternative method for surveillance of SAV in Atlantic salmon farms.

Development and evaluation of a method for concentration and detection of salmonid alphavirus from seawater.

Waterborne viral infections represent a major threat to fish health. For many viruses, understanding the interplay between pathogens, host and environment presents a major hurdle for transmission. Salmonid alphavirus (SAV) can infect and cause pancreas disease (PD) in farmed salmonids in seawater. During infection, SAV is excreted from infected fish to the seawater. We evaluated two types of filters and four different eluents, for concentration of SAV3. One L of seawater was spiked with SAV3, followed by filtration and virus elution from membrane filters. For the negatively charged MF hydrophilic membrane filter (MF-) combined with NucliSENS® lysis buffer the SAV3 recovery was 39.5 ±â€¯1.8 % by RT-ddPCR and 25.9 ±â€¯5.7 % by RT-qPCR. The recovery using the positively charged 1 MDS Zeta Plus® Virosorb® membrane filter (MD+), combined with NucliSENS® lysis buffer was 19.0 ±â€¯0.1 % by RT-ddPCR and 13.3 ±â€¯3.8 % by RT-qPCR. The limits of quantification (LOQ) and detection (LOD) were estimated to be 5.18 × 103 and 2.0 × 102 SAV3 copies/L of natural seawater, by RT-ddPCR. SAV3 recovery from small volumes of seawater, and the requirement for standard laboratory equipment, suggest the MF-filter combined with NucliSENS® lysis buffer would be a candidate for further validation in experimental trials.

Refined experimental design may increase the value of murine models for estimation of bluetongue virus virulence.

Bluetongue is a serious non-contagious vector-borne viral disease in ruminants, causing poor animal welfare and economic consequences globally. Concern has been raised about the development of novel bluetongue virus (BTV) strains and their possibly altered virulence through the process of viral reassortment. Virulence is traditionally estimated in lethal dose 50 (LD50) studies in murine models, but agreement with both in vitro and virulence in ruminants is questionable, and a refined experimental design is needed. Specific reassortants between wild-type and vaccine strains of BTV-1, -6 and -8 have previously been developed by reverse genetics. The aim of the present study was to rank the in vivo virulence of these parental and reassortant BTV strains by calculating LD50 in a murine model by using an experimental design that is new to virology: a between-patient optimised three-level response surface pathway design. The inoculation procedure was intracranial. Fifteen suckling mice were used to establish LD50 for each strain. Three parental and five reassortant virus strains were included. The LD50s varied from of 0.1 (95% confidence interval (CI) 0-0.20) to 3.3 (95% CI 2.96-3.72) tissue culture infectious dose 50/ml. The results support the hypothesis that reassortment in BTV may lead to increased virulence in mice with potential negative consequences for the natural ruminant host. The ranking showed low agreement with in vitro properties and virulence in ruminants according to existing literature. Refined design such as response surface pathway design was found suitable for use in virology, and it introduces significant ethical and scientific improvements.

Status of risk-based approach and national framework for safe drinking water in small water supplies of the Nordic water sector.

Reliable safe water supply is a pillar of society and a key to public health. The Nordic countries have an abundance of clean fresh water as a source for drinking water supplies. They have followed developments in safeguarding water, both the recommendations of the World Health Organization framework for safe drinking water and European legislation. Worldwide, including the Nordic countries, small water supplies are less compliant with water safety regulation. The forthcoming EU directive on drinking water require risk-based approaches and improved transparency on water quality. This research looks at the Nordic frameworks for safe water supply, with emphasis on risk-based approaches and smaller systems. We analyzed the legal frameworks for safe water, the structure of the water sector across the Nordic countries and explored how prepared these countries are to meet these requirements. Our findings show that, while legal requirements are mostly in place, delivery of information to the public needs to be improved. Most Nordic countries are in the process of implementing risk-based management in large and medium size water supplies, whereas small supplies are lagging. We conclude that a key to success is increased training and support for small supplies. We suggest wider adoption of the Nordic model of cooperation with benchmarking of safe water for all to transfer knowledge between the countries. This work provides insights into challenges and opportunities for the Nordic countries and provides insights relevant to countries worldwide in their effort towards realization of SDG Target 6.1.

Using Biosecurity Measures to Combat Respiratory Disease in Cattle: The Norwegian Control Program for Bovine Respiratory Syncytial Virus and Bovine Coronavirus.

Bovine respiratory disease (BRD) cause important health problems in all cattle husbandry systems. It contributes substantially to the use of antimicrobial substances and compromises animal welfare and the sustainability of the cattle industry. The existing preventive measures of BRD focus at the individual animal or herd level and include vaccination, mass treatment with antimicrobials and improvement of the animal's environment and general health status. Despite progress in our understanding of disease mechanism and technological development, the current preventive measures are not sufficiently effective. Thus, there is a need for alternative, sustainable strategies to combat the disease. Some of the primary infectious agents in the BRD complex are viruses that are easily transmitted between herds such as bovine respiratory syncytial virus (BRSV) and bovine coronavirus (BCoV). This conceptual analysis presents arguments for combatting BRD through improved external biosecurity in the cattle herds. As an example of a population-based approach to the control of BRD, the Norwegian BRSV/BCoV control-program is presented. The program is voluntary and launched by the national cattle industry. The core principle is classification of herds based on antibody testing and subsequent prevention of virus-introduction through improved biosecurity measures. Measures include external herd biosecurity barriers and regulations in the organization of animal trade to reduce direct and indirect transmission of virus. Improved biosecurity in a large proportion of herds will lead to a considerable effect at the population level. Positive herds are believed to gain freedom by time if new introduction is avoided. Vaccination is not used as part of the program. Dissemination of information to producers and veterinarians is essential. We believe that reducing the incidence of BRD in cattle is essential and will lead to reduced antimicrobial usage while at the same time improving animal health, welfare and production. Alternative approaches to the traditional control measures are needed.

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.