Proteomic and Lipidomic Profiling of Calves Experimentally Co-Infected with Influenza D Virus and Mycoplasma bovis: Insights into the Host-Pathogen Interactions.

The role of Influenza D virus (IDV) in bovine respiratory disease remains unclear. An in vivo experiment resulted in increased clinical signs, lesions, and pathogen replication in calves co-infected with IDV and Mycoplasma bovis (M. bovis), compared to single-infected calves. The present study aimed to elucidate the host-pathogen interactions and profile the kinetics of lipid mediators in the airways of these calves. Bronchoalveolar lavage (BAL) samples collected at 2 days post-infection (dpi) were used for proteomic analyses by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Additionally, lipidomic analyses were performed by LC-MS/MS on BAL samples collected at 2, 7 and 14 dpi. Whereas M. bovis induced the expression of proteins involved in fibrin formation, IDV co-infection counteracted this coagulation mechanism and downregulated other acute-phase response proteins, such as complement component 4 (C4) and plasminogen (PLG). The reduced inflammatory response against M. bovis likely resulted in increased M. bovis replication and delayed M. bovis clearance, which led to a significantly increased abundance of oxylipids in co-infected calves. The identified induced oxylipids mainly derived from arachidonic acid; were likely oxidized by COX-1, COX-2, and LOX-5; and peaked at 7 dpi. This paper presents the first characterization of BAL proteome and lipid mediator kinetics in response to IDV and M. bovis infection in cattle and raises hypotheses regarding how IDV acts as a co-pathogen in bovine respiratory disease.

Identification of Astrovirus in the virome of the upper and lower respiratory tracts of calves with acute signs of bronchopneumonia.

IMPORTANCE: Astroviruses (AstV) are known suspects of enteric disease in humans and livestock. Recently, AstV have been linked to encephalitis in immunocompromised patients and other animals, such as cattle, minks, and swine. In our study, we also identified AstV in the respiratory samples of calves with signs of bronchopneumonia, suggesting that their tropism could be even broader. We obtained one bovine AstV (BAstV) complete genome sequence by next-generation sequencing and showed that respiratory and enteric AstV from different species formed a divergent genetic cluster with AstV isolated from encephalitis cases, indicating that tropism might be strain-specific. These data provide further insight into understanding the biology of these understudied pathogens and suggest BAstV as a potential new candidate for bovine respiratory disease.

Cross-Neutralization between Bovine Viral Diarrhea Virus (BVDV) Types 1 and 2 after Vaccination with a BVDV-1a Modified-Live-Vaccine.

Control of Bovine Viral Diarrhea Virus types 1 and 2 (BVDV-1 and BVDV-2) involves removing persistently infected animals from the herd, ensuring the biosecurity level of the farms and vaccination for the prevention of fetal infection. Given pestiviruses high genetic and antigenic diversities, one challenge for a BVDV vaccine is to provide the broadest possible heterologous protection against most genotypes and sub-genotypes. The Modified-Live Mucosiffa® vaccine, which contains the BVDV-1 sub-genotype 1a (BVDV-1a) cytopathic Oregon C24 strain, was shown to protect fetuses of pregnant heifers against a challenge with a BVDV-1f Han strain. In this study, we tested the cross-neutralizing antibody (NA) response of 9 heifers at 28, 203- and 363-days post-vaccination with Mucosiffa® against recent and circulating European strains of BVDV-1a, -1b, -1e, -1f and BVDV-2a. We showed that Mucosiffa® vaccination generates a stable over time NA response against all BVDV strains. NA response was greater against BVDV-1a and -1b, with no significant differences between these sub-genotypes. Interestingly the NA response against the two BVDV-2a strains was similar to that observed against the BVDV-1f Han strain, which was the challenge strain used in fetal protection studies to validate the Mucosiffa® vaccine. These results suggest that Mucosiffa® vaccination provides humoral cross-immunity, which may protect against BVDV-1 and BVDV-2a infection.

Molecular and genetic characterization of bovine parainfluenza type 3 European field and vaccine strains.

Bovine Parainfluenza Type 3 virus (BPIV-3) is an enveloped, non-segmented single-stranded, negative-sense RNA virus belonging to the Paramyxoviridae family (genus Respirovirus) with a well-known role in Bovine Respiratory Disease (BRD) onset. Being isolated for the first time in 1959, BPIV-3 currently circulates worldwide in cattle herds and is routinely tested in suspected BRD cases. Different commercial vaccines are available to prevent infection and/or to reduce the clinical signs associated with BPIV-3 infection, which are essential to prevent secondary infections. Despite years of molecular surveillance, a very limited number of complete genome sequences were made publicly available, preventing thus the understanding of the genetic diversity of the circulating strains in the field. In addition, no data about the genetic identity between field and vaccine strains is currently available. In this study, we sequenced the full-genome and genetically characterized BPIV-3 strains isolated from animals displaying respiratory illness in France and Sweden, as well as the vaccine strains contained in three different commercialized vaccines. Our results show that the sequences from France and Sweden belong to genotype C. However, a third sequence from Sweden from 2017 clustered within genotype A. The sequencing of vaccine strains revealed that two of the vaccine strains clustered within genotype C, whereas the third vaccine strain belonged to genotype A. Altogether, our findings suggest that both genotypes A and C circulate in Europe and that BPIV-3 field and vaccine strains are genetically divergent. Our sequencing results could be useful to better understand the genetic differences between the circulating field and vaccine BPIV-3 strains. This is crucial for a correct interpretation of diagnostic findings and for the assessment of BPIV-3 prevalence in cattle population.

Detection of Influenza D-Specific Antibodies in Bulk Tank Milk from Swedish Dairy Farms.

Influenza D virus (IDV) has been detected in bovine respiratory disease (BRD) outbreaks, and experimental studies demonstrated this virus's capacity to cause lesions in the respiratory tract. In addition, IDV-specific antibodies were detected in human sera, which indicated that this virus plays a potential zoonotic role. The present study aimed to extend our knowledge about the epidemiologic situation of IDV in Swedish dairy farms, using bulk tank milk (BTM) samples for the detection of IDV antibodies. A total of 461 and 338 BTM samples collected during 2019 and 2020, respectively, were analyzed with an in-house indirect ELISA. In total, 147 (32%) and 135 (40%) samples were IDV-antibody-positive in 2019 and 2020, respectively. Overall, 2/125 (2%), 11/157 (7%) and 269/517 (52%) of the samples were IDV-antibody-positive in the northern, middle and southern regions of Sweden. The highest proportion of positive samples was repeatedly detected in the south, in the county of Halland, which is one of the counties with the highest cattle density in the country. In order to understand the epidemiology of IDV, further research in different cattle populations and in humans is required.

Protection against Bovine Respiratory Syncytial Virus Afforded by Maternal Antibodies from Cows Immunized with an Inactivated Vaccine.

The passive protection afforded by the colostrum from cattle that were vaccinated prepartum with an inactivated combination vaccine against the bovine respiratory syncytial virus (BRSV) was evaluated after an experimental challenge of calves. Pregnant cows without or with a low ELISA and neutralizing BRSV antibody titers were twice vaccinated or not vaccinated, the last immunization being at one month prior to calving. Vaccination was followed by a rapid increase in BRSV antibody titers after the second immunization. Twenty-eightnewborn calves were fed during the 6 h following birth, with 4 L of colostrum sourced from vaccinated cows (14 vaccine calves) or non-vaccinated cows (14 control calves) and were challenged with BRSV at 21 days of age. We showed that maternal immunity to BRSV provides a significant reduction in the clinical signs of BRSV in calves, especially for severe clinical forms. This protection was correlated with reduced BRSV detection in the lower respiratory tract but not in nasal swabs, indicating an absence of protection against BRSV nasal excretion. Finally, transcriptomic assays in bronchoalveolar lavages showed no statistical differences between groups for chemokine and cytokine mRNA transcriptions, with the exception of the overexpression of IL-9 at days 6 and 10 post-challenge, and a severe downregulation of CXCL-1 at day 3 post-challenge, in the vaccine group.

The Activation of the RIG-I/MDA5 Signaling Pathway upon Influenza D Virus Infection Impairs the Pulmonary Proinflammatory Response Triggered by Mycoplasma bovis Superinfection.

Concurrent infections with multiple pathogens are often described in cattle with respiratory illness. However, how the host-pathogen interactions influence the clinical outcome has been only partially explored in this species. Influenza D virus (IDV) was discovered in 2011. Since then, IDV has been detected worldwide in different hosts. A significant association between IDV and bacterial pathogens in sick cattle was shown in epidemiological studies, especially with Mycoplasma bovis. In an experimental challenge, IDV aggravated M. bovis-induced pneumonia. However, the mechanisms through which IDV drives an increased susceptibility to bacterial superinfections remain unknown. Here, we used the organotypic lung model precision-cut lung slices to study the interplay between IDV and M. bovis coinfection. Our results show that a primary IDV infection promotes M. bovis superinfection by increasing the bacterial replication and the ultrastructural damages in lung pneumocytes. In our model, IDV impaired the innate immune response triggered by M. bovis by decreasing the expression of several proinflammatory cytokines and chemokines that are important for immune cell recruitment and the bacterial clearance. Stimulations with agonists of cytosolic helicases and Toll-like receptors (TLRs) revealed that a primary activation of RIG-I/MDA5 desensitizes the TLR2 activation, similar to what was observed with IDV infection. The cross talk between these two pattern recognition receptors leads to a nonadditive response, which alters the TLR2-mediated cascade that controls the bacterial infection. These results highlight innate immune mechanisms that were not described for cattle so far and improve our understanding of the bovine host-microbe interactions and IDV pathogenesis. IMPORTANCE Since the spread of the respiratory influenza D virus (IDV) infection to the cattle population, the question about the impact of this virus on bovine respiratory disease (BRD) remains still unanswered. Animals affected by BRD are often coinfected with multiple pathogens, especially viruses and bacteria. In particular, viruses are suspected to enhance secondary bacterial superinfections. Here, we use an ex vivo model of lung tissue to study the effects of IDV infection on bacterial superinfections. Our results show that IDV increases the susceptibility to the respiratory pathogen Mycoplasma bovis. In particular, IDV seems to activate immune pathways that inhibit the innate immune response against the bacteria. This may allow M. bovis to increase its proliferation and to delay its clearance from lung tissue. These results suggest that IDV could have a negative impact on the respiratory pathology of cattle.

Evaluating the potential of whole-genome sequencing for tracing transmission routes in experimental infections and natural outbreaks of bovine respiratory syncytial virus.

Bovine respiratory syncytial virus (BRSV) is a major cause of respiratory disease in cattle. Genomic sequencing can resolve phylogenetic relationships between virus populations, which can be used to infer transmission routes and potentially inform the design of biosecurity measures. Sequencing of short (<2000 nt) segments of the 15 000-nt BRSV genome has revealed geographic and temporal clustering of BRSV populations, but insufficient variation to distinguish viruses collected from herds infected close together in space and time. This study investigated the potential for whole-genome sequencing to reveal sufficient genomic variation for inferring transmission routes between herds. Next-generation sequencing (NGS) data were generated from experimental infections and from natural outbreaks in Jämtland and Uppsala counties in Sweden. Sufficient depth of coverage for analysis of consensus and sub-consensus sequence diversity was obtained from 47 to 20 samples respectively. Few (range: 0-6 polymorphisms across the six experiments) consensus-level polymorphisms were observed along experimental transmissions. A much higher level of diversity (146 polymorphic sites) was found among the consensus sequences from the outbreak samples. The majority (144/146) of polymorphisms were between rather than within counties, suggesting that consensus whole-genome sequences show insufficient spatial resolution for inferring direct transmission routes, but might allow identification of outbreak sources at the regional scale. By contrast, within-sample diversity was generally higher in the experimental than the outbreak samples. Analyses to infer known (experimental) and suspected (outbreak) transmission links from within-sample diversity data were uninformative. In conclusion, analysis of the whole-genome sequence of BRSV from experimental samples discriminated between circulating isolates from distant areas, but insufficient diversity was observed between closely related isolates to aid local transmission route inference.

Evolutionary and temporal dynamics of emerging influenza D virus in Europe (2009-22).

Influenza D virus (IDV) is an emerging influenza virus that was isolated for the first time in 2011 in the USA from swine with respiratory illness. Since then, IDV has been detected worldwide in different animal species, and it was also reported in humans. Molecular epidemiological studies revealed the circulation of two major clades, named D/OK and D/660. Additional divergent clades have been described but have been limited to specific geographic areas (i.e. Japan and California). In Europe, IDV was detected for the first time in France in 2012 and subsequently also in Italy, Luxembourg, Ireland, the UK, Switzerland, and Denmark. To understand the time of introduction and the evolutionary dynamics of IDV on the continent, molecular screening of bovine and swine clinical samples was carried out in different European countries, and phylogenetic analyses were performed on all available and newly generated sequences. Until recently, D/OK was the only clade detected in this area. Starting from 2019, an increase in D/660 clade detections was observed, accompanied by an increase in the overall viral genetic diversity and genetic reassortments. The time to the most recent common ancestor (tMRCA) of all existing IDV sequences was estimated as 1995-16 years before its discovery, indicating that the virus could have started its global spread in this time frame. Despite the D/OK and D/660 clades having a similar mean tMRCA (2007), the mean tMRCA for European D/OK sequences was estimated as January 2013 compared to July 2014 for European D/660 sequences. This indicated that the two clades were likely introduced on the European continent at different time points, as confirmed by virological screening findings. The mean nucleotide substitution rate of the hemagglutinin-esterase-fusion (HEF) glycoprotein segment was estimated as 1.403 × 10-3 substitutions/site/year, which is significantly higher than the one of the HEF of human influenza C virus (P < 0.0001). IDV genetic drift, the introduction of new clades on the continent, and multiple reassortment patterns shape the increasing viral diversity observed in the last years. Its elevated substitution rate, diffusion in various animal species, and the growing evidence pointing towards zoonotic potential justify continuous surveillance of this emerging influenza virus.

Understanding the mechanisms of viral and bacterial coinfections in bovine respiratory disease: a comprehensive literature review of experimental evidence.

Bovine respiratory disease (BRD) is one of the most important diseases impacting the global cattle industry, resulting in significant economic loss. Commonly referred to as shipping fever, BRD is especially concerning for young calves during transport when they are most susceptible to developing disease. Despite years of extensive study, managing BRD remains challenging as its aetiology involves complex interactions between pathogens, environmental and host factors. While at the beginning of the twentieth century, scientists believed that BRD was only caused by bacterial infections ("bovine pasteurellosis"), we now know that viruses play a key role in BRD induction. Mixtures of pathogenic bacteria and viruses are frequently isolated from respiratory secretions of animals with respiratory illness. The increased diagnostic screening data has changed our understanding of pathogens contributing to BRD development. In this review, we aim to comprehensively examine experimental evidence from all existing studies performed to understand coinfections between respiratory pathogens in cattle. Despite the fact that pneumonia has not always been successfully reproduced by in vivo calf modelling, several studies attempted to investigate the clinical significance of interactions between different pathogens. The most studied model of pneumonia induction has been reproduced by a primary viral infection followed by a secondary bacterial superinfection, with strong evidence suggesting this could potentially be one of the most common scenarios during BRD onset. Different in vitro studies indicated that viral priming may increase bacterial adherence and colonization of the respiratory tract, suggesting a possible mechanism underpinning bronchopneumonia onset in cattle. In addition, a few in vivo studies on viral coinfections and bacterial coinfections demonstrated that a primary viral infection could also increase the pathogenicity of a secondary viral infection and, similarly, dual infections with two bacterial pathogens could increase the severity of BRD lesions. Therefore, different scenarios of pathogen dynamics could be hypothesized for BRD onset which are not limited to a primary viral infection followed by a secondary bacterial superinfection.

Development and assessment of a new bioassay for accurate quantification of Type I interferons induced by bovine respiratory viruses.

Type I interferon (IFN-I) plays a major role in antiviral and inflammatory processes of the infected host. In the bovine industry, the bovine respiratory disease complex is a major cause of economic and health problems. This disease is caused by interactions of pathogens, together with environmental and host factors. Several pathogens have been identified as causal agents of respiratory diseases in cattle. To better understand how primary infections by viruses predispose animals to further infections by pathogenic bacteria, tools to accurately detect antiviral and immunoregulatory cytokines are needed. To facilitate the detection and quantification of bovine IFN-I, we have established a new specific and sensitive bioassay studies in the bovine host. This assay is based on a Madin-Darby Bovine Kidney (MDBK) cell line that carries a luciferase gene under the control of the IFN-I inducible bovine Mx1 promoter. Specific luciferase activity was measured after stimulation with serial dilutions of recombinant bovine alpha and beta IFNs and human IFN-α. With this novel bioassay we have successfully measured IFN-I production in supernatant from MDBK cells after stimulation of Toll-like receptors (TLR3, TLR7 and TLR8) and RIG-I-like receptors (RIG-I and MDA5), after viral infection with bovine respiratory pathogens, but also in samples from infected calves. Finally, this new bioassay is an easy-to-use and low cost tool to measure the production of bovine Type-I Interferon.

Influenza D virus in respiratory disease in Canadian, province of Québec, cattle: Relative importance and evidence of new reassortment between different clades.

BACKGROUND: Influenza D virus (IDV), a segmented single-stranded negative-sense ribonucleic acid (RNA) virus, belongs to the new Delta influenza virus genus of the Orthomyxoviridae family. Cattle were proposed as the natural reservoir of IDV in which infection was associated with mild-to-moderate respiratory clinical signs (i.e. cough, nasal discharge and dyspnoea). METHODS AND PRINCIPAL FINDINGS: In order to investigate the role of IDV in bovine respiratory disease, during the period 2017-2020, 883 nasal or naso-pharyngeal swabs from Canadian cattle with respiratory signs (cough and/or dyspnoea) were tested by (RT-)qPCR for IDV and other major bovine viral (bovine herpesvirus 1, bovine viral diarrhoea virus, bovine respiratory syncytial virus, bovine parainfluenza virus 3 and bovine coronavirus) and bacterial (Mannheimia haemolytica, Pasteurella multocida, Histophilus somni and Mycoplasma bovis) respiratory pathogens. In addition, whole-genome sequencing and phylogenetic analyses were carried out on five IDV-positive samples. The prevalence of IDV RT-qPCR (with cut-off: Cq < 38) at animal level was estimated at 5.32% (95% confidence interval: 3.94-7.02). Positive result of IDV was significantly associated with (RT-)qPCR-positive results for bovine respiratory syncytial virus and Mycoplasma bovis. While phylogenetic analyses indicate that most segments belonged to clade D/660, reassortment between clades D/660 and D/OK were evidenced in four samples collected in 2018-2020. CONCLUSIONS AND SIGNIFICANCE: Relative importance of influenza D virus and associated pathogens in bovine respiratory disease of Canadian dairy cattle was established. Whole-genome sequencing demonstrated evidence of reassortment between clades D/660 and D/OK. Both these new pieces of information claim for more surveillance of IDV in cattle production worldwide.

Enhanced Pathogenesis Caused by Influenza D Virus and Mycoplasma bovis Coinfection in Calves: a Disease Severity Linked with Overexpression of IFN-γ as a Key Player of the Enhanced Innate Immune Response in Lungs.

Bovine respiratory disease (BRD) is a major disease of young cattle whose etiology lies in complex interactions between pathogens and environmental and host factors. Despite a high frequency of codetection of respiratory pathogens in BRD, data on the molecular mechanisms and pathogenesis associated with viral and bacterial interactions are still limited. In this study, we investigated the effects of a coinfection with influenza D virus (IDV) and Mycoplasma bovis in cattle. Naive calves were infected by aerosol with a French IDV strain and an M. bovis strain. The combined infection shortened the incubation period, worsened the disease, and led to more severe macroscopic and microscopic lesions compared to these parameters in calves infected with only one pathogen. In addition, IDV promoted colonization of the lower respiratory tract (LRT) by M. bovis and increased white cell recruitment to the airway lumen. The transcriptomic analysis highlighted an upregulation of immune genes in the lungs of coinfected calves. The gamma interferon (IFN-γ) gene was shown to be the gene most statistically overexpressed after coinfection at 2 days postinfection (dpi) and at least until 7 dpi, which correlated with the high level of lymphocytes in the LRT. Downregulation of the PACE4 and TMPRSS2 endoprotease genes was also highlighted, being a possible reason for the faster clearance of IDV in the lungs of coinfected animals. Taken together, our coinfection model with two respiratory pathogens that when present alone induce moderate clinical signs of disease was shown to increase the severity of the disease in young cattle and a strong transcriptomic innate immune response in the LRT, especially for IFN-γ. IMPORTANCE Bovine respiratory disease (BRD) is among the most prevalent diseases in young cattle. BRD is due to complex interactions between viruses and/or bacteria, most of which have a moderate individual pathogenicity. In this study, we showed that coinfection with influenza D virus (IDV) and Mycoplasma bovis increased the severity of the respiratory disease in calves in comparison with IDV or M. bovis infection. IDV promoted M. bovis colonization of the lower respiratory tract and increased white cell recruitment to the airway lumen. The transcriptomic analysis highlighted an upregulation of immune genes in the lungs of coinfected calves. The IFN-γ gene in particular was highly overexpressed after coinfection, correlated with the disease severity, immune response, and white cell recruitment in the lungs. In conclusion, we showed that IDV facilitates coinfections within the BRD complex by modulating the local innate immune response, providing new insights into the mechanisms involved in severe respiratory diseases.

Vaccination of Sheep with Bovine Viral Diarrhea Vaccines Does Not Protect against Fetal Infection after Challenge of Pregnant Ewes with Border Disease Virus.

Border Disease (BD) is a major sheep disease characterized by immunosuppression, congenital disorders, abortion, and birth of lambs persistently infected (PI) by Border Disease Virus (BDV). Control measures are based on the elimination of PI lambs, biosecurity, and frequent vaccination which aims to prevent fetal infection and birth of PI. As there are no vaccines against BDV, farmers use vaccines directed against the related Bovine Viral Diarrhea Virus (BVDV). To date, there is no published evidence of cross-effectiveness of BVDV vaccination against BDV infection in sheep. We tested three commonly used BVDV vaccines, at half the dose used in cattle, for their efficacy of protection against a BDV challenge of ewes at 52 days of gestation. Vaccination limits the duration of virus-induced leukopenia after challenge, suggesting partial protection in transient infection. Despite the presence of BDV neutralizing antibodies in vaccinated ewes on the day of the challenge, fetuses of vaccinated and unvaccinated sheep were, two months after, highly positive for BDV RNA loads and seronegative for antibodies. Therefore, BVDV vaccination at half dose was not sufficient to prevent ovine fetal infection by BDV in a severe challenge model and can only be reconsidered as a complementary mean in BD control.

Emerging Influenza D virus infection in European livestock as determined in serology studies: Are we underestimating its spread over the continent?

Influenza D virus (IDV) is a novel orthomyxovirus that was first isolated in 2011 in the United States from a swine exhibiting influenza-like disease. To date, its detection is extended to all continents and in a broad host range: IDV is circulating in cattle, swine, feral swine, camelids, small ruminants and horses. Evidence also suggests a possible species jump to humans, underlining the issue of zoonotic potential. In Europe, serological investigations in cattle have partially allowed the understanding of the virus diffusion in different countries such as Italy, France, Luxembourg and Ireland. The infection is widespread in cattle but limited in other investigated species, consolidating the assumption of cattle as IDV primary host. We hypothesize that commercial livestock trade could play a role in the observed differences in IDV seroprevalence among these areas. Indeed, the overall level of exposure in cattle and swine in destination countries (e.g. Italy) is higher than in origin countries (e.g. France), leading to the hypothesis of a viral shedding following the transportation of young cattle abroad and thus contributing to larger diffusion at countries of destination. IDV large geographic circulation in cattle from Northern to more Southern European countries also supports the hypothesis of a viral spread through livestock trade. This review summarizes available data on IDV seroprevalence in Europe collected so far and integrates unpublished data from IDV European surveillance framework of the last decade. In addition, the possible role of livestock trade and biosecurity measures in this pathogen's spread is discussed.

Formal estimation of the seropositivity cut-off of the hemagglutination inhibition assay in field diagnosis of influenza D virus in cattle and estimation of the associated true prevalence in Morocco.

The influenza D virus (IDV) was discovered less than ten years ago. Increased interest in this virus is due to its nature (RNA virus with high mutation rate), its worldwide circulation in livestock species, its probable role in bovine respiratory disease and its zoonotic potential. Until currently, the establishment of positivity cut-off of the hemagglutination inhibition (HI) assay was not formalized in field conditions for the detection of antibodies directed against IDV in cattle (i.e. the proposed reservoir). In this study, the positivity cut-off of the HI assays was formally established (titre = 10) using a receiver operating characteristic (ROC) curve. This information was used to estimate the sensitivity (68.04 to 73.20%) and the specificity (94.17 to 96.12%) of two different HI assays (HI1 and HI2 , with two different IDV antigens) relatively to virus micro-neutralization test (VNT) as reference test. Based on the above characteristics, the true prevalence of IDV was then estimated in Morocco using a stochastic approach. Irrespective of the HI assays used, the estimation of the true prevalence was statistically equivalent (between 48.44% and 48.73%). In addition, the Spearman rank correlation between HI titres and VNT titres was statistically good (0.76 and 0.81 for HA1 and HA2 , respectively). The positive (0.82 and 0.79 for HA1 and HA2 , respectively) and the negative (0.86 and 0.85 for HA1 and HA2 , respectively) agreement indices between results of HI assays and VNT were good and similar. This study allowed for a formal establishment of a positivity cut-off in HI assays for the detection of antibodies directed against IDV. This information is of prime importance to estimate the diagnostic sensitivity and specificity of the test relatively to the VNT (i.e. the reference test). Using these characteristics, the true prevalence of IDV should be determined in a country.

Limited Cross-Protection Provided by Prior Infection Contributes to High Prevalence of Influenza D Viruses in Cattle.

Since its detection in swine, influenza D virus (IDV) has been shown to be present in multiple animal hosts, and bovines have been identified as its natural reservoir. However, it remains unclear how IDVs emerge, evolve, spread, and maintain in bovine populations. Through multiple years of virological and serological surveillance in a single order-buyer cattle facility in Mississippi, we showed consistently high seroprevalence of IDVs in cattle and recovered a total of 32 IDV isolates from both healthy and sick animals, including those with antibodies against IDV. Genomic analyses of these isolates along with those isolated from other areas showed that active genetic reassortment occurred in IDV and that five reassortants were identified in the Mississippian facility. Two antigenic groups were identified through antigenic cartography analyses for these 32 isolates and representative IDVs from other areas. Remarkably, existing antibodies could not protect cattle from experimental reinfection with IDV. Additional phenotypic analyses demonstrated variations in growth dynamics and pathogenesis in mice between viruses independent of genomic constellation. In summary, this study suggests that, in addition to epidemiological factors, the ineffectiveness of preexisting immunity and cocirculation of a diverse viral genetic pool could facilitate its high prevalence in animal populations.IMPORTANCE Influenza D viruses (IDVs) are panzootic in multiple animal hosts, but the underlying mechanism is unclear. Through multiple years of surveillance in the same order-buyer cattle facility, 32 IDV isolates were recovered from both healthy and sick animals, including those with evident antibodies against IDV. Active reassortment occurred in the cattle within this facility and in those across other areas, and multiple reassortants cocirculated in animals. These isolates are shown with a large extent of phenotypic diversity in replication efficiency and pathogenesis but little in antigenic properties. Animal experiments demonstrated that existing antibodies could not protect cattle from experimental reinfection with IDV. This study suggests that, in addition to epidemiological factors, limited protection from preexisting immunity against IDVs in cattle herds and cocirculation of a diverse viral genetic pool likely facilitate the high prevalence of IDVs in animal populations.

Global Transmission, Spatial Segregation, and Recombination Determine the Long-Term Evolution and Epidemiology of Bovine Coronaviruses.

Bovine coronavirus (BCoV) is widespread in cattle and wild ruminant populations throughout the world. The virus causes neonatal calf diarrhea and winter dysentery in adult cattle, as well as upper and lower respiratory tract infection in young cattle. We isolated and deep sequenced whole genomes of BCoV from calves with respiratory distress in the south-west of France and conducted a comparative genome analysis using globally collected BCoV sequences to provide insights into the genomic characteristics, evolutionary origins, and global diversity of BCoV. Molecular clock analyses allowed us to estimate that the BCoV ancestor emerged in the 1940s, and that two geographically distinct lineages diverged from the 1960s-1970s. A recombination event in the spike gene (breakpoint at nt 1100) may be at the origin of the genetic divergence sixty years ago. Little evidence of genetic mixing between the spatially segregated lineages was found, suggesting that BCoV genetic diversity is a result of a global transmission pathway that occurred during the last century. However, we found variation in evolution rates between the European and non-European lineages indicating differences in virus ecology.

Seroprevalence of influenza D virus in selected sample groups of Irish cattle, sheep and pigs.

Influenza D virus (IDV) is a new member of the Orthomyxoviridae family. It was first reported in swine in 2011 and isolated from bovine samples received for routine respiratory disease diagnosis in Ireland during 2014-2016. The goal of this study was to determine the seroprevalence in selected populations of IDV in cattle, pigs and sheep. Results showed a high prevalence of IDV in cattle sampled at slaughter (94.6%) or for diagnostic reasons (64.9%), whereas prevelance in samples taken for diagnostic reasons from sheep (4.5%) and pigs (5.8%) was much lower. This study suggests that IDV is widespread in Irish cattle.

Serological Evidence of Influenza D Virus Circulation Among Cattle and Small Ruminants in France.

Influenza D virus (IDV) has first been identified in 2011 in the USA and was shown to mainly circulate in cattle. While IDV is associated with mild respiratory signs, its prevalence is still unknown. In the present study we show that IDV has been circulating throughout France in cattle and small ruminants, with 47.2% and 1.5% seropositivity, respectively. The high prevalence and moderate pathogenicity of IDV in cattle suggest that it may play an initiating role in the bovine respiratory disease complex.