Low Pathogenic Avian Influenza H9N2 Viruses in Morocco: Antigenic and Molecular Evolution from 2021 to 2023.

Avian influenza viruses pose significant threats to both the poultry industry and public health worldwide. Among them, the H9N2 subtype has gained substantial attention due to its high prevalence, especially in Asia, the Middle East, and Africa; its ability to reassort with other influenza viruses; and its potential to infect humans. This study presents a comprehensive phylogenetic and molecular analysis of H9N2 avian influenza viruses circulating in Morocco from 2021 to 2023. Through an active epidemiological survey, a total of 1140 samples (trachea and lungs) and oropharyngeal swabs pooled into 283 pools, collected from 205 farms located in 7 regions of Morocco known for having a high density of poultry farms, were analyzed. Various poultry farms were investigated (159 broiler farms, 24 layer farms, 10 breeder farms, and 12 turkey breeder farms). A total of 21 AI H9N2 strains were isolated, and in order to understand the molecular evolution of the H9N2 avian influenza virus, their genetic sequences were determined using the Sanger sequencing technique. Phylogenetic analysis was performed using a dataset comprising global H9N2 sequences to determine the genetic relatedness and evolutionary dynamics of the Moroccan strains. The results revealed the continued circulation and diversification of H9N2 avian influenza viruses in Morocco during the study period. Real-time RT-PCR showed a positivity rate of 35.6% (73/205), with cycle threshold values ranging from 19.2 to 34.9. The phylogenetic analysis indicated that all Moroccan strains belonged to a G1-like lineage and regrouped into two distinct clusters. Our newly detected isolates aggregated distinctly from the genotypes previously isolated in Morocco, North and West Africa, and the Middle East. This indicats the potential of virus evolution resulting from both national circulation and cross-border transmission. A high genetic diversity at both nucleotide and amino-acid levels was observed among all the strains isolated in this study, as compared to H9N2 strains isolated in Morocco since 2016, which suggests the co-circulation of genetically diverse H9N2 variants. Newly discovered mutations were detected in hemagglutinin positions 226, 227, and 193 (H3 numbering), which highlights the genetic evolution of the H9N2 AIVs. These findings contribute to our understanding of the evolution and epidemiology of H9N2 in the region and provide valuable insights for the development of effective prevention and control strategies against this emerging avian influenza subtype.

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.

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.

Molecular characterisation of human adenoviruses associated with respiratory infections in Uganda.

Human adenoviruses (HAdV) are a diverse group of viruses causing a broad range of infections of the respiratory, urogenital and gastrointestinal tracts and keratoconjunctivitis. There are seven species of human adenoviruses with 113 genotypes which may contain multiple genetic variants. This study characterised respiratory human adenoviruses and associated factors in samples collected from selected hospitals in Uganda. A total of 2,298 nasopharyngeal samples were collected between the period of 2008 to 2016 from patients seeking health care at tertiary hospitals for influenza-like illness. They were screened by polymerase chain reaction (PCR) to determine the prevalence of HAdV. HAdV was cultured in A549 cell lines and the hexon gene was sequenced for genotyping. Of the 2,298 samples tested, 225 (9.8%) were adenovirus-positive by PCR. Age was found to be significantly associated with HAdV infections (p = 0.028) with 98% (220/225) of the positives in children aged 5 years and below and none in adults above 25 years of age. The sequenced isolates belonged to species HAdV-B and HAdV-C with most isolates identified as genotype B3. The results showed a high prevalence and genetic diversity in respiratory HAdV circulating in Ugandan population. Deeper genomic characterization based on whole genome sequencing may be necessary to further elucidate possible transmission and impact of current adenovirus-vectored vaccines in Africa.

Epidemiology and molecular characterization of avian influenza A viruses H5N1 and H3N8 subtypes in poultry farms and live bird markets in Bangladesh.

Avian influenza virus (AIV) remains a global threat, with waterfowl serving as the primary reservoir from which viruses spread to other hosts. Highly pathogenic avian influenza (HPAI) H5 viruses continue to be a devastating threat to the poultry industry and an incipient threat to humans. A cross-sectional study was conducted in seven districts of Bangladesh to estimate the prevalence and subtypes (H3, H5, and H9) of AIV in poultry and identify underlying risk factors and phylogenetic analysis of AIVs subtypes H5N1 and H3N8. Cloacal and oropharyngeal swab samples were collected from 500 birds in live bird markets (LBMs) and poultry farms. Each bird was sampled by cloacal and oropharyngeal swabbing, and swabs were pooled for further analysis. Pooled samples were analyzed for the influenza A virus (IAV) matrix (M) gene, followed by H5 and H9 molecular subtyping using real-time reverse transcription-polymerase chain reaction (rRT-PCR). Non-H5 and Non-H9 influenza A virus positive samples were sequenced to identify possible subtypes. Selected H5 positive samples were subjected to hemagglutinin (HA) and neuraminidase (NA) gene sequencing. Multivariable logistic regression was used for risk factor analysis. We found that IAV M gene prevalence was 40.20% (95% CI 35.98-44.57), with 52.38%, 46.96%, and 31.11% detected in chicken, waterfowl, and turkey, respectively. Prevalence of H5, H3, and H9 reached 22%, 3.4%, and 6.9%, respectively. Waterfowl had a higher risk of having AIV (AOR: 4.75), and H5 (AOR: 5.71) compared to chicken; more virus was detected in the winter season than in the summer season (AOR: 4.93); dead birds had a higher risk of AIVs and H5 detection than healthy birds, and the odds of H5 detection increased in LBM. All six H5N1 viruses sequenced were clade 2.3.2.1a-R1 viruses circulating since 2015 in poultry and wild birds in Bangladesh. The 12 H3N8 viruses in our study formed two genetic groups that had more similarity to influenza viruses from wild birds in Mongolia and China than to previous H3N8 viruses from Bangladesh. The findings of this study may be used to modify guidelines on AIV control and prevention to account for the identified risk factors that impact their spread.

Bayesian phylodynamics reveals the transmission dynamics of avian influenza A(H7N9) virus at the human-live bird market interface in China.

In 2013 to 2017, avian influenza A(H7N9) virus has caused five severe epidemic waves of human infections in China. The role of live bird markets (LBMs) in the transmission dynamics of H7N9 remains unclear. Using a Bayesian phylodynamic approach, we shed light on past H7N9 transmission events at the human-LBM interface that were not directly observed using case surveillance data-based approaches. Our results reveal concurrent circulation of H7N9 lineages in Yangtze and Pearl River Delta regions, with evidence of local transmission during each wave. Our results indicate that H7N9 circulated in humans and LBMs for weeks to months before being first detected. Our findings support the seasonality of H7N9 transmission and suggest a high number of underreported infections, particularly in LBMs. We provide evidence for differences in virus transmissibility between low and highly pathogenic H7N9. We demonstrate a regional spatial structure for the spread of H7N9 among LBMs, highlighting the importance of further investigating the role of local live poultry trade in virus transmission. Our results provide estimates of avian influenza virus (AIV) transmission at the LBM level, providing a unique opportunity to better prepare surveillance plans at LBMs for response to future AIV epidemics.

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.

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.

Disentangling the role of poultry farms and wild birds in the spread of highly pathogenic avian influenza virus in Europe.

In winter 2016-7, Europe was severely hit by an unprecedented epidemic of highly pathogenic avian influenza viruses (HPAIVs), causing a significant impact on animal health, wildlife conservation, and livestock economic sustainability. By applying phylodynamic tools to virus sequences collected during the epidemic, we investigated when the first infections occurred, how many infections were unreported, which factors influenced virus spread, and how many spillover events occurred. HPAIV was likely introduced into poultry farms during the autumn, in line with the timing of wild birds' migration. In Germany, Hungary, and Poland, the epidemic was dominated by farm-to-farm transmission, showing that understanding of how farms are connected would greatly help control efforts. In the Czech Republic, the epidemic was dominated by wild bird-to-farm transmission, implying that more sustainable prevention strategies should be developed to reduce HPAIV exposure from wild birds. Inferred transmission parameters will be useful to parameterize predictive models of HPAIV spread. None of the predictors related to live poultry trade, poultry census, and geographic proximity were identified as supportive predictors of HPAIV spread between farms across borders. These results are crucial to better understand HPAIV transmission dynamics at the domestic-wildlife interface with the view to reduce the impact of future epidemics.

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.

Highly Pathogenic Avian Influenza A(H5N1) Clade 2.3.4.4b Virus in Poultry, Benin, 2021.

In August 2021, we detected highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b viruses in poultry in southern Benin. The isolates were genetically similar to H5N1 viruses of clade 2.3.4.4b isolated during the same period in Africa and Europe. We also found evidence for 2 separate introductions of these viruses into Benin.

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.

Genetic Evolution of Avian Influenza A (H9N2) Viruses Isolated from Domestic Poultry in Uganda Reveals Evidence of Mammalian Host Adaptation, Increased Virulence and Reduced Sensitivity to Baloxavir.

A (H9N2) avian influenza A viruses were first detected in Uganda in 2017 and have since established themselves in live bird markets. The aim of this study was to establish the subsequent genetic evolution of H9N2 viruses in Uganda. Cloacal samples collected from live bird market stalls in Kampala from 2017 to 2019 were screened by RT-PCR for influenza A virus and H9N2 viruses were isolated in embryonated eggs. One hundred and fifty H9N2 isolates were subjected to whole genome sequencing on the Illumina MiSeq platform. The sequence data analysis and comparison with contemporary isolates revealed that the virus was first introduced into Uganda in 2014 from ancestors in the Middle East. There has since been an increase in nucleotide substitutions and reassortments among the viruses within and between live bird markets, leading to variations in phylogeny of the different segments, although overall diversity remained low. The isolates had several mutations such as HA-Q226L and NS-I106M that enable mammalian host adaptation, NP-M105V, PB1-D3V, and M1-T215A known for increased virulence/pathogenicity and replication, and PA-E199D, NS-P42S, and M2-S31N that promote drug resistance. The PA-E199D substitution in particular confers resistance to the endonuclease inhibitor Baloxavir acid, which is one of the new anti-influenza drugs. Higher EC50 was observed in isolates with a double F105L+E199D substitution that may suggest a possible synergistic effect. These H9N2 viruses have established an endemic situation in live bird markets in Uganda because of poor biosecurity practices and therefore pose a zoonotic threat. Regular surveillance is necessary to further generate the needed evidence for effective control strategies and to minimize the threats.

Hydroxychloroquine inhibits proteolytic processing of endogenous TLR7 protein in human primary plasmacytoid dendritic cells.

Toll-like receptor 7 (TLR7) triggers antiviral immune responses through its capacity to recognize ssRNA. Proteolytic cleavage of TLR7 protein is required for its functional maturation in the endosomal compartment. Structural studies demonstrated that the N- and C-terminal domains of TLR7 are connected and involved in ligand binding after cleavage. Hydroxychloroquine (HCQ), an antimalarial drug, has been studied for its antiviral effects. HCQ increases pH in acidic organelles and has been reported to potently inhibit endosomal TLR activation. Whether HCQ can prevent endogenous TLR7 cleavage in primary immune cells, such as plasmacytoid DCs (pDCs), had never been examined. Here, using a validated anti-TLR7 antibody suitable for biochemical detection of native TLR7 protein, we show that HCQ treatment of fresh PBMCs, CAL-1 leukemic, and primary human pDCs inhibits TLR7 cleavage and results in accumulation of full-length protein. As a consequence, we observe an inhibition of pDC activation in response to TLR7 stimulation with synthetic ligands and viruses including inactivated SARS-CoV2, which we show herein activates pDCs through TLR7-signaling. Together, our finding suggests that the major pathway by which HCQ inhibits ssRNA sensing by pDCs may rely on its capacity to inhibit endosomal acidification and the functional maturation of TLR7 protein.

Influenza A viruses are likely highly prevalent in South African swine farms.

Growth in pork production during the last decade in South Africa has escalated the risk of zoonotic pathogen emergence. This cross-sectional study was conducted to evaluate evidence for transmission of influenza A virus between pigs and swine workers. Between February and October 2018, samples from swine workers and pigs were collected from three farms in KwaZulu-Natal Province, South Africa. Workers nasal washes and serum samples, and swine oral secretion samples (rope sampling method) were studied for evidence of swine influenza A virus infection using molecular and serological methods. Among 84 human nasal washes and 51 swine oral secretion specimens, 44 (52.4%) and 6 (11.8%) had molecular evidence of influenza A virus. Microneutralization assays with enrolled workers' sera against swine H1N1 and H3N2 viruses revealed a high prevalence of elevated antibodies. Multivariate risk factor analysis showed that male workers from the age-group quartile 23-32 years, who self-reported a recent history of exposure to someone with influenza disease and seldom use of personal protective equipment were at highest risk of molecular detection of influenza A virus. These pilot study data suggest that influenza A viruses are likely highly prevalent in South African swine farms. South Africa would benefit from periodic surveillance for novel influenza viruses in swine farms as well as education and seasonal influenza vaccine programmes for swine workers.

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.

Immunogenicity and Protective Potential of Mucosal Vaccine Formulations Based on Conserved Epitopes of Influenza A Viruses Fused to an Innovative Ring Nanoplatform in Mice and Chickens.

Current inactivated vaccines against influenza A viruses (IAV) mainly induce immune responses against highly variable epitopes across strains and are mostly delivered parenterally, limiting the development of an effective mucosal immunity. In this study, we evaluated the potential of intranasal formulations incorporating conserved IAV epitopes, namely the long alpha helix (LAH) of the stalk domain of hemagglutinin and three tandem repeats of the ectodomain of the matrix protein 2 (3M2e), as universal mucosal anti-IAV vaccines in mice and chickens. The IAV epitopes were grafted to nanorings, a novel platform technology for mucosal vaccination formed by the nucleoprotein (N) of the respiratory syncytial virus, in fusion or not with the C-terminal end of the P97 protein (P97c), a recently identified Toll-like receptor 5 agonist. Fusion of LAH to nanorings boosted the generation of LAH-specific systemic and local antibody responses as well as cellular immunity in mice, whereas the carrier effect of nanorings was less pronounced towards 3M2e. Mice vaccinated with chimeric nanorings bearing IAV epitopes in fusion with P97c presented modest LAH- or M2e-specific IgG titers in serum and were unable to generate a mucosal humoral response. In contrast, N-3M2e or N-LAH nanorings admixed with Montanide™ gel (MG) triggered strong specific humoral responses, composed of serum type 1/type 2 IgG and mucosal IgG and IgA, as well as cellular responses dominated by type 1/type 17 cytokine profiles. All mice vaccinated with the [N-3M2e + N-LAH + MG] formulation survived an H1N1 challenge and the combination of both N-3M2e and N-LAH nanorings with MG enhanced the clinical and/or virological protective potential of the preparation in comparison to individual nanorings. Chickens vaccinated parenterally or mucosally with N-LAH and N-3M2e nanorings admixed with Montanide™ adjuvants developed a specific systemic humoral response, which nonetheless failed to confer protection against heterosubtypic challenge with a highly pathogenic H5N8 strain. Thus, while the combination of N-LAH and N-3M2e nanorings with Montanide™ adjuvants shows promise as a universal mucosal anti-IAV vaccine in the mouse model, further experiments have to be conducted to extend its efficacy to poultry.

Serological Surveillance of Influenza D Virus in Ruminants and Swine in West and East Africa, 2017-2020.

Influenza D virus (IDV) was first isolated in 2011 in Oklahoma, USA from pigs presenting with influenza-like symptoms. IDV is known to mainly circulate in ruminants, especially cattle. In Africa, there is limited information on the epidemiology of IDV, although the virus has likely circulated in the region since 2012. In the present study, we investigated the seropositivity of IDV among domestic ruminants and swine in West and East Africa from 2017 to 2020. Serum samples were analyzed using the hemagglutination inhibition (HI) assay. Our study demonstrated that IDV is still circulating in Africa, with variations in seropositivity among countries and species. The highest seropositivity was detected in cattle (3.9 to 20.9%). Our data highlights a need for extensive surveillance of IDV in Africa in order to better understand the epidemiology of the virus in the region.

Protective Efficacy Evaluation of Four Inactivated Commercial Vaccines Against Low Pathogenic Avian Influenza H9N2 Virus Under Experimental Conditions in Broiler Chickens.

Avian influenza vaccines are commonly used in the poultry industry. The objective of this study was to compare, under experimental conditions, the protective efficacy of four imported commercial inactivated H9N2 vaccines (A, B, C, and D) in broiler chickens. A total of 150 one-day-old chicks were divided into six groups: four experimental groups, each containing 30 chicks, received one of the vaccines (A, B, C, or D) delivered in a 0.3-ml dose subcutaneously at 1 day of age, whereas the control, Group T, was not vaccinated but challenged and Group E was kept unvaccinated and unchallenged. At 21 days postvaccination, Groups A, B, C, D, and T were challenged with 107 embryo infective dose 50% of A/Chicken/Morocco/01/2016 (H9N2). All chicks were observed daily for clinical signs during the 12 days postchallenge (dpc). At 5 and 12 dpc, chicks were euthanatized for necropsy examination. Blood samples were collected weekly for serologic analysis and oropharyngeal swabs were collected for virus detection by real-time RT-PCR. Respiratory signs started at 48 hr pc and maximum severity was observed on 9 dpc. Chiefly, the birds vaccinated with vaccine B showed significantly more respiratory signs than did their counterparts. Serologic analysis revealed that the sera of Groups A and D birds showed a decrease in antibody (Ab) levels up to day 26; then a slight increase of Ab level was observed until day 31, while Group B and C birds showed a stabilization of the titers from day 21 until the end of the experiment. The viral shedding rate was significantly lower in Groups C and A (40%-50% of the birds shed virus for <7 days) compared with other challenged groups (60%-75% of the birds shed virus for ≥9 days). This experiment illustrated that vaccination applied on the first day in the hatchery with the four vaccines tested did not provide an acceptable protection against H9N2 in comparison with the controls that did not receive any vaccine. However, at first glance, we might favor vaccines A and C for their ability to reduce and shorten viral shedding as compared with vaccines B and D.

Evaluación de la eficacia protectora de cuatro vacunas comerciales inactivadas contra el virus de la influenza aviar H9N2 de baja patogenicidad bajo condiciones experimentales en pollos de engorde. Las vacunas contra la influenza aviar se utilizan comúnmente en la industria avícola. El objetivo de este estudio fue comparar, en condiciones experimentales, la eficacia protectora de cuatro vacunas H9N2 inactivadas comerciales importadas (A, B, C y D) en pollos de engorde. Un total de 150 pollitos de un día se dividieron en seis grupos: cuatro grupos experimentales, cada uno con 30 pollitos, recibieron una de las vacunas (A, B, C o D) administradas en una dosis de 0.3 ml por vía subcutánea al día. de edad, mientras que el control, Grupo T, que no fue vacunado y desafiado y el Grupo E que se mantuvo sin vacunar y sin desafiar. A los 21 días después de la vacunación, los Grupos A, B, C, D y T fueron desafiados con 107 dosis infecciosas de embriones al 50% del virus A/Chicken/Marruecos/01/2016 (H9N2). Todos los pollos fueron observados diariamente para detectar signos clínicos durante los 12 días posteriores al desafío (dpc). A los cinco y 12 días después del desafío, los polluelos fueron sacrificados humanitariamente para un examen de necropsia. Se recolectaron muestras de sangre semanalmente para análisis serológicos y se recolectaron hisopos orofaríngeos para la detección de virus mediante RT-PCR en tiempo real. Los signos respiratorios comenzaron a las 48 horas después del desafío y la severidad máxima se observó a los nueve días después del desafío. Principalmente, las aves vacunadas con la vacuna B mostraron significativamente más signos respiratorios que sus contrapartes. El análisis serológico reveló que los sueros de las aves de los Grupos A y D mostraron una disminución en los niveles de anticuerpos (Ab) hasta el día 26; luego se observó un ligero aumento del nivel de anticuerpos hasta el día 31, mientras que las aves de los Grupos B y C mostraron una estabilización de los títulos desde el día 21 hasta el final del experimento. La tasa de excreción viral fue significativamente menor en los Grupos C y A (40% -50% de las aves excretaron el virus durante <7 días) en comparación con otros grupos desafiados (60% -75% de las aves excretaron el virus durante ≥9 días). Este experimento ilustró que la vacunación aplicada el primer día en la incubadora con las cuatro vacunas probadas no proporcionó una protección aceptable contra el virus H9N2 en comparación con los controles que no recibieron ninguna vacuna. Sin embargo, a primera vista, podríamos favorecer las vacunas A y C por su capacidad para reducir y acortar la diseminación viral en comparación con las vacunas B y D.