Influenza D virus infection in China, 2022-2023.

Influenza D virus (IDV) plays an important role in the bovine respiratory disease (BRD) complex. Its potential for the zoonotic transmission is of particular concern. In China, IDV has previously been identified in agricultural animals by molecular surveys with no live virus isolates reported. In this study, live IDVs were successfully isolated from cattle in China, which prompted us to further investigate the national prevalence, antigenic property, and infection biology of the virus. IDV RNA was detected in 11.1% (51/460) of cattle throughout the country in 2022-2023. Moreover, we conducted the first IDV serosurveillance in China, revealing a high seroprevalence (91.4%, 393/430) of IDV in cattle during the 2022-2023 winter season. Notably, all the 16 provinces from which cattle originated possessed seropositive animals, and 3 of them displayed the 100% IDV-seropositivity rate. In contrast, a very low seroprevalence of IDV was observed in pigs (3%, 3/100) and goats (1%, 1/100) during the same period of investigation. Furthermore, besides D/Yama2019 lineage-like IDVs, we discovered the D/660 lineage-like IDV in Chinese cattle, which has not been detected to date in Asia. Finally, the Chinese IDVs replicated robustly in diverse cell lines but less efficiently in the swine cell line. Considering the nationwide distribution, high seroprevalence, and appreciably genetic diversity, further studies are required to fully evaluate the risk of Chinese IDVs for both animal and human health in China, which can be evidently facilitated by IDV isolates reported in this study.

Detection of Influenza D Antibodies in Dogs, Apulia Region, Italy, 2016 and 2023.

Dogs are known to be susceptible to influenza A viruses, although information on influenza D virus (IDV) is limited. We investigated the seroprevalence of IDV in 426 dogs in the Apulia region of Italy during 2016 and 2023. A total of 14 samples were positive for IDV antibodies, suggesting exposure to IDV in dogs.

Influenza A and D Viruses in Non-Human Mammalian Hosts in Africa: A Systematic Review and Meta-Analysis.

We conducted a systematic review and meta-analysis to investigate the prevalence and current knowledge of influenza A virus (IAV) and influenza D virus (IDV) in non-human mammalian hosts in Africa. PubMed, Google Scholar, Wiley Online Library and World Organisation for Animal Health (OIE-WAHIS) were searched for studies on IAV and IDV from 2000 to 2020. Pooled prevalence and seroprevalences were estimated using the quality effects meta-analysis model. The estimated pooled prevalence and seroprevalence of IAV in pigs in Africa was 1.6% (95% CI: 0-5%) and 14.9% (95% CI: 5-28%), respectively. The seroprevalence of IDV was 87.2% (95% CI: 24-100%) in camels, 9.3% (95% CI: 0-24%) in cattle, 2.2% (95% CI: 0-4%) in small ruminants and 0.0% (95% CI: 0-2%) in pigs. In pigs, H1N1 and H1N1pdm09 IAVs were commonly detected. Notably, the highly pathogenic H5N1 virus was also detected in pigs. Other subtypes detected serologically and/or virologically included H3N8 and H7N7 in equids, H1N1, and H3N8 and H5N1 in dogs and cats. Furthermore, various wildlife animals were exposed to different IAV subtypes. For prudent mitigation of influenza epizootics and possible human infections, influenza surveillance efforts in Africa should not neglect non-human mammalian hosts. The impact of IAV and IDV in non-human mammalian hosts in Africa deserves further investigation.

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.

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.

Emergence of new phylogenetic lineage of Influenza D virus with broad antigenicity in California, United States.

Influenza D virus (IDV), with bovines as a primary host, circulates widely in cattle populations across North America and Eurasia. Here we report the identification of a novel IDV group with broad antigenicity in U.S. bovine herds, which is genetically different from previously known lineages of IDV.

Influenza D virus.

Influenza D is the only type of influenza virus that mainly affects cattle with frequent spillover to other species. Since the initial description of influenza D virus (IDV) in 2011, the virus has been found to circulate among cattle and swine populations worldwide. Research conducted during the past several years has led to an increased understanding of this novel influenza virus with bovines as a reservoir. In this review, we describe the current knowledge of epidemiology and host range of IDV followed by discussion of infection biology and animal model development for IDV. Finally, we review progress towards understanding of the pathogenesis and host response of IDV as well as developing preventive vaccines for IDV.

Genetic and Antigenic Characterization and Retrospective Surveillance of Bovine Influenza D Viruses Identified in Hokkaido, Japan from 2018 to 2020.

Influenza D virus (IDV), which is a new member of the Orthomyxoviridae family, is potentially involved in bovine respiratory diseases (BRDs). Bovine IDVs (BIDVs) from Japan have been distributed nationwide since 2010 and are genetically distinct from foreign IDVs. We isolated BIDVs from three BRD outbreaks, in Hokkaido during 2018-2020, to understand their genetic and antigenic characteristics. Retrospective surveillance was performed using sera collected throughout the last decade in Hokkaido to investigate BIDV existence. Three BIDVs were isolated using cell culture. Comparative and phylogenetic analyses using sequence data of the three BIDVs and IDVs from Japan and other countries available in GenBank demonstrated that Japanese BIDVs, including the three BIDV isolates, were genetically distinct from other IDVs. Genotype classifications based on the rotavirus genotype classification revealed multiple genotypes of RNA segments 1-7. Two BIDVs were of a new genotype, different from those of other Japanese BIDVs. Neutralization assays against two BIDVs with different genotypes using sera collected in acute and recovery phases of BRD revealed differences in cross-reactivity to heterogenous BIDVs. Retrospective surveillance suggested that BIDV existed in Hokkaido, in 2009. Our findings suggest that BIDVs of different genotypes and antigenicity are distributed and maintained in Hokkaido and provide new insights into molecular characteristics and the evolution of IDVs.

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.

Assessment of Influenza D Virus in Domestic Pigs and Wild Boars in France: Apparent Limited Spread within Swine Populations Despite Serological Evidence of Breeding Sow Exposure.

In order to assess influenza D virus (IDV) infections in swine in France, reference reagents were produced in specific pathogen free pigs to ensure serological and virological analyses. Hemagglutination inhibition (HI) assays were carried out on 2090 domestic pig sera collected in 2012-2018 in 102 farms. Only 31 sera from breeding sows sampled in 2014-2015 in six farrow-to-finish herds with respiratory disorders contained IDV-specific antibodies. In two of them, within-herd percentage of positive samples (73.3% and 13.3%, respectively) and HI titers (20-160) suggested IDV infections, but virus persistence was not confirmed following new sampling in 2017. All growing pigs tested seronegative, whatever their age and the sampling year. Moreover, PB1-gene RT-qPCR performed on 452 nasal swabs taken in 2015-2018 on pigs with acute respiratory syndrome (137 farms) gave negative results. In Corse, a Mediterranean island where pigs are mainly bred free-range, 2.3% of sera (n = 177) sampled on adult pigs in 2013-2014 obtained low HI titers. Finally, 0.5% of sera from wild boars hunted in 2009-2016 (n = 644) tested positive with low HI titers. These results provide the first serological evidence that sows were exposed to IDV in France but with a limited spread within the swine population.

Contribution of Host Immune Responses Against Influenza D Virus Infection Toward Secondary Bacterial Infection in a Mouse Model.

Influenza D viruses (IDV) are known to co-circulate with viral and bacterial pathogens in cattle and other ruminants. Currently, there is limited knowledge regarding host responses to IDV infection and whether IDV infection affects host susceptibility to secondary bacterial infections. To begin to address this gap in knowledge, the current study utilized a combination of in vivo and in vitro approaches to evaluate host cellular responses against primary IDV infection and secondary bacterial infection with Staphylococcus aureus (S. aureus). Primary IDV infection in mice did not result in clinical signs of disease and it did not enhance the susceptibility to secondary S. aureus infection. Rather, IDV infection appeared to protect mice from the usual clinical features of secondary bacterial infection, as demonstrated by improved weight loss, survival, and recovery when compared to S. aureus infection alone. We found a notable increase in IFN-ß expression following IDV infection while utilizing human alveolar epithelial A549 cells to analyze early anti-viral responses to IDV infection. These results demonstrate for the first time that IDV infection does not increase the susceptibility to secondary bacterial infection with S. aureus, with evidence that anti-viral immune responses during IDV infection might protect the host against these potentially deadly outcomes.

Serosurvey for Influenza D Virus Exposure in Cattle, United States, 2014-2015.

Influenza D virus has been detected predominantly in cattle from several countries. In the United States, regional and state seropositive rates for influenza D have previously been reported, but little information exists to evaluate national seroprevalence. We performed a serosurveillance study with 1,992 bovine serum samples collected across the country in 2014 and 2015. We found a high overall seropositive rate of 77.5% nationally; regional rates varied from 47.7% to 84.6%. Samples from the Upper Midwest and Mountain West regions showed the highest seropositive rates. In addition, seropositive samples were found in 41 of the 42 states from which cattle originated, demonstrating that influenza D virus circulated widely in cattle during this period. The distribution of influenza D virus in cattle from the United States highlights the need for greater understanding about pathogenesis, epidemiology, and the implications for animal health.

Therapeutic efficacy of favipiravir against Bourbon virus in mice.

Bourbon virus (BRBV) is an emerging tick-borne RNA virus in the orthomyxoviridae family that was discovered in 2014. Although fatal human cases of BRBV have been described, little is known about its pathogenesis, and no antiviral therapies or vaccines exist. We obtained serum from a fatal case in 2017 and successfully recovered the second human infectious isolate of BRBV. Next-generation sequencing of the St. Louis isolate of BRBV (BRBV-STL) showed >99% nucleotide identity to the original reference isolate. Using BRBV-STL, we developed a small animal model to study BRBV-STL tropism in vivo and evaluated the prophylactic and therapeutic efficacy of the experimental antiviral drug favipiravir against BRBV-induced disease. Infection of Ifnar1-/- mice lacking the type I interferon receptor, but not congenic wild-type animals, resulted in uniformly fatal disease 6 to 10 days after infection. RNA in situ hybridization and viral yield assays demonstrated a broad tropism of BRBV-STL with highest levels detected in liver and spleen. In vitro replication and polymerase activity of BRBV-STL were inhibited by favipiravir. Moreover, administration of favipiravir as a prophylaxis or as post-exposure therapy three days after infection prevented BRBV-STL-induced mortality in immunocompromised Ifnar1-/- mice. These results suggest that favipiravir may be a candidate treatment for humans who become infected with BRBV.

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.

Mx1 in Hematopoietic Cells Protects against Thogoto Virus Infection.

Myxovirus resistance 1 (Mx1) is an interferon-induced gene that encodes a GTPase that plays an important role in the defense of mammalian cells against influenza A and other viruses. The Mx1 protein can restrict a number of viruses independently of the expression of other interferon-induced genes. Mx genes are therefore considered to be an important part of the innate antiviral immune response. However, the possible impact of Mx expression in the hematopoietic cellular compartment has not been investigated in detail in the course of a viral infection. To address this, we performed bone marrow chimera experiments using congenic B6.A2G Mx1+/+ and B6.A2G Mx1-/- mice to study the effect of Mx1 expression in cells of hematopoietic versus nonhematopoietic origin. Mx1+/+ mice were protected and Mx1-/- mice were susceptible to influenza A virus challenge infection, regardless of the type of bone marrow cells (Mx1+/+ or Mx1-/- ) the animals had received. Infection with Thogoto virus, however, revealed that Mx1-/- mice with a functional Mx1 gene in the bone marrow compartment showed reduced liver pathology compared with Mx1-/- mice that had been grafted with Mx1-/- bone marrow. The reduced pathology in these mice was associated with a reduction in Thogoto virus titers in the spleen, lung, and serum. Moreover, Mx1+/+ mice with Mx1-/- bone marrow failed to control Thogoto virus replication in the spleen. Mx1 in the hematopoietic cellular compartment thus contributes to protection against Thogoto virus infection.IMPORTANCE Mx proteins are evolutionarily conserved in vertebrates and can restrict a wide range of viruses in a cell-autonomous way. The contribution to antiviral defense of Mx1 expression in hematopoietic cells remains largely unknown. We show that protection against influenza virus infection requires Mx1 expression in the nonhematopoietic cellular compartment. In contrast, Mx1 in bone marrow-derived cells is sufficient to control disease and virus replication following infection with a Thogoto virus. This indicates that, in addition to its well-established antiviral activity in nonhematopoietic cells, Mx1 in hematopoietic cells can also play an important antiviral function. In addition, cells of hematopoietic origin that lack a functional Mx1 gene contribute to Thogoto virus dissemination and associated disease.

Pathogenesis, Host Innate Immune Response, and Aerosol Transmission of Influenza D Virus in Cattle.

The recently discovered influenza D virus (IDV) of the Orthomyxoviridae family has been detected in swine and ruminants with a worldwide distribution. Cattle are considered to be the primary host and reservoir, and previous studies suggested a tropism of IDV for the upper respiratory tract and a putative role in the bovine respiratory disease complex. This study aimed to characterize the pathogenicity of IDV in naive calves as well as the ability of this virus to transmit by air. Eight naive calves were infected by aerosol with a recent French isolate, D/bovine/France/5920/2014. Results show that IDV replicates not only in the upper respiratory tract but also in the lower respiratory tract (LRT), inducing moderate bronchopneumonia with restricted lesions of interstitial pneumonia. Inoculation was followed by IDV-specific IgG1 production as early as 10 days postchallenge and likely both Th1 and Th2 responses. Study of the innate immune response in the LRT of IDV-infected calves indicated the overexpression of pathogen recognition receptors and of chemokines CCL2, CCL3, and CCL4, but without overexpression of genes involved in the type I interferon pathway. Finally, virological examination of three aerosol-sentinel animals, housed 3 m apart from inoculated calves (and thus subject to infection by aerosol transmission), and IDV detection in air samples collected in different areas showed that IDV can be airborne transmitted and infect naive contact calves on short distances. This study suggests that IDV is a respiratory virus with moderate pathogenicity and probably a high level of transmission. It consequently can be considered predisposing to or a cofactor of respiratory disease.IMPORTANCE Influenza D virus (IDV), a new genus of the Orthomyxoviridae family, has a broad geographical distribution and can infect several animal species. Cattle are so far considered the primary host for IDV, but the pathogenicity and the prevalence of this virus are still unclear. We demonstrated that under experimental conditions (in a controlled environment and in the absence of coinfecting pathogens), IDV is able to cause mild to moderate disease and targets both the upper and lower respiratory tracts. The virus can transmit by direct as well as aerosol contacts. While this study evidenced overexpression of pathogen recognition receptors and chemokines in the lower respiratory tract, IDV-specific IgG1 production as early as 10 days postchallenge, and likely both Th1 and Th2 responses, further studies are warranted to better understand the immune responses triggered by IDV and its role as part of the bovine respiratory disease complex.

Influenza D Virus: Serological Evidence in the Italian Population from 2005 to 2017.

Influenza D virus is a novel influenza virus, which was first isolated from an ailing swine in 2011 and later detected in cattle, suggesting that these animals may be a primary natural reservoir. To date, few studies have been performed on human samples and there is no conclusive evidence on the ability of the virus to infect humans. The aim of this serological study was to assess the prevalence of antibodies against influenza D virus in human serum samples collected in Italy from 2005 to 2017. Serum samples were analysed by haemagglutination inhibition and virus neutralization assays. The results showed that the prevalence of antibodies against the virus increased in the human population in Italy from 2005 to 2017, with a trend characterized by a sharp increase in some years, followed by a decline in subsequent years. The virus showed the ability to infect and elicit an immune response in humans. However, prevalence peaks in humans appear to follow epidemics in animals and not to persist in the human population.

Viral targeting of TFIIB impairs de novo polymerase II recruitment and affects antiviral immunity.

Viruses have evolved a plethora of mechanisms to target host antiviral responses. Here, we propose a yet uncharacterized mechanism of immune regulation by the orthomyxovirus Thogoto virus (THOV) ML protein through engaging general transcription factor TFIIB. ML generates a TFIIB depleted nuclear environment by re-localizing it into the cytoplasm. Although a broad effect on gene expression would be anticipated, ML expression, delivery of an ML-derived functional domain or experimental depletion of TFIIB only leads to altered expression of a limited number of genes. Our data indicate that TFIIB is critically important for the de novo recruitment of Pol II to promoter start sites and that TFIIB may not be required for regulated gene expression from paused promoters. Since many immune genes require de novo recruitment of Pol II, targeting of TFIIB by THOV represents a neat mechanism to affect immune responses while keeping other cellular transcriptional activities intact. Thus, interference with TFIIB activity may be a favourable site for therapeutic intervention to control undesirable inflammation.

Influenza D Virus Infection in Feral Swine Populations, United States.

Influenza D virus (IDV) has been identified in domestic cattle, swine, camelid, and small ruminant populations across North America, Europe, Asia, South America, and Africa. Our study investigated seroprevalence and transmissibility of IDV in feral swine. During 2012-2013, we evaluated feral swine populations in 4 US states; of 256 swine tested, 57 (19.1%) were IDV seropositive. Among 96 archived influenza A virus-seropositive feral swine samples collected from 16 US states during 2010-2013, 41 (42.7%) were IDV seropositive. Infection studies demonstrated that IDV-inoculated feral swine shed virus 3-5 days postinoculation and seroconverted at 21 days postinoculation; 50% of in-contact naive feral swine shed virus, seroconverted, or both. Immunohistochemical staining showed viral antigen within epithelial cells of the respiratory tract, including trachea, soft palate, and lungs. Our findings suggest that feral swine might serve an important role in the ecology of IDV.

A DNA Vaccine Expressing Consensus Hemagglutinin-Esterase Fusion Protein Protected Guinea Pigs from Infection by Two Lineages of Influenza D Virus.

Two lineages of influenza D virus (IDV) have been found to infect cattle and promote bovine respiratory disease complex, one of the most commonly diagnosed causes of morbidity and mortality within the cattle industry. Furthermore, IDV can infect other economically important domestic livestock, including pigs, and has the potential to infect humans, which necessitates the need for an efficacious vaccine. In this study, we designed a DNA vaccine expressing consensus hemagglutinin-esterase fusion (HEF) protein (FluD-Vax) and tested its protective efficacy against two lineages of IDV (D/OK and D/660) in guinea pigs. Animals that received FluD-Vax (n = 12) developed appreciable titers of neutralizing antibodies against IDV lineage representatives, D/OK and D/660. Importantly, vaccinated animals were protected against intranasal challenge with IDV [3 × 105 50% tissue culture infective dose(s) (TCID50)] D/OK (n = 6) or D/600 (n = 6), based on the absence of viral RNA in necropsied tissues (5 and 7 days postchallenge) using quantitative reverse transcription-PCR and in situ hybridization. In contrast, animals that received a sham DNA vaccine (n = 12) had no detectable neutralizing antibodies against IDV, and viral RNA was readily detectable in respiratory tract tissues after intranasal challenge (3 × 105 TCID50) with IDV D/OK (n = 6) or D/660 (n = 6). Using a TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay, we found that IDV D/OK and D/600 infections induced apoptosis in epithelial cells lining alveoli and bronchioles, as well as nonepithelial cells in lung tissues. Our results demonstrate for the first time that the consensus IDV HEF DNA vaccine can elicit complete protection against infection from two lineages of IDV in the guinea pig model.IMPORTANCE Influenza D virus (IDV) infection has been associated with bovine respiratory disease complex, one of the most devastating diseases of the cattle population. Moreover, with broad host range and high environmental stability, IDV has the potential to further gain virulence or even infect humans. An efficacious vaccine is needed to prevent infection and stop potential cross-species transmission. In this study, we designed a DNA vaccine encoding the consensus hemagglutinin-esterase fusion (HEF) protein of two lineages of IDV (D/OK and D/660) and tested its efficacy in a guinea pig model. Our results showed that the consensus DNA vaccine elicited high-titer neutralizing antibodies and achieved sterilizing protection against two lineage-representative IDV intranasal infections. To our knowledge, this is the first study showing that a DNA vaccine expressing consensus HEF is efficacious in preventing different lineages of IDV infections.