呼吸道在抵御病原感染方面发挥着关键作用。作为上呼吸道的重要组成部分，鼻腔是许多病原首次接触和入侵的主要靶位。已有研究表明，鼻腔菌群的组成与宿主对病原的易感性密切相关，但其具体机制尚不清楚。犬作为与人类密切接触的伴侣动物，其家庭地位越来越重要，研究犬鼻腔菌群不仅对犬类健康管理至关重要，还可为理解微生物与宿主的复杂关系、防控人兽共患病提供新视角。现阶段关于犬的鼻腔菌群组成特征及特定菌群介导的机体免疫调节机制研究尚未见报道。

近年来，流感病毒已成为影响犬呼吸道健康的重要病原。目前关于流感病毒与呼吸道菌群之间的关系，是医学微生物学关注的重点研究领域之一，但是犬流感病毒（Canine influenza virus，CIV）感染与呼吸道菌群组成及二者之间的关系尚属研究空白。有鉴于此，本研究利用16S rRNA测序表征了健康比格犬呼吸道菌群的组成，在此基础上，结合多组学分析以及细胞水平和机体水平的试验验证，系统地解析了CIV感染后犬鼻腔及肺脏的病毒滴度、宿主基因表达谱与菌群动态变化的潜在联系，并筛选出一株犬源乳酸菌，证实其可通过激活CIV感染细胞的I型干扰素信号通路并调控自噬流进而抑制CIV复制。该研究结果揭示了犬鼻腔菌群在 CIV 感染中的关键作用及其免疫调控机制。

1  犬鼻腔菌群失调对流感病毒感染致病特性的影响及鼻腔菌群组成和功能分析

利用两种广谱抗生素软膏（莫匹罗星和新霉素）处理，构建比格犬鼻腔菌群失调模型，结合16S rRNA测序和功能预测分析了犬鼻腔菌群组成以及菌群动态。同时，探讨了菌群失调对犬流感病毒感染的影响。结果显示，相比正常对照组（Nor），抗生素处理显著改变了鼻腔菌群结构（p < 0.05），并降低了菌群 α 多样性（Chao1 指数和Richness 指数，p < 0.01）。菌群 β 多样性分析表明，Bray-Curtis 相异度指数显著不同（p < 0.01）。鼻腔菌群组成分析显示，抗生素处理后，高峰度菌群（相对丰度 > 0.5%）的数量由原来的 37 个减少至 28 个。进一步分析表明，鼻腔菌群失调伴随变形杆菌门相对丰度的显著增加（p < 0.05）。此外，乳杆菌属（Lactobacillus）、普雷沃菌属（Prevotella）、巨单胞菌属（Prevotella）和 Lachnoclostridium 的相对丰度则显著下降。而在病毒感染后，抗生素处理鼻腔 CIV 感染比格鼻腔菌群中莫拉菌属（Moraxella）相对丰度显著上升（p < 0.05）。

基于菌群功能预测的 KEGG 通路分析结果显示，抗生素处理后共有 161 条菌群功能通路发生显著变化（p < 0.05），其中 8 条与病毒感染相关通路差异极显著（p < 0.0001）。此外，鼻腔菌群失调的犬在感染高剂量（10⁷ PFU/mL）CIV后，表现出更为严重的临床症状，包括体重显著下降和持续性的高烧（最高温度达39.6℃）。采集 CIV 感染后第 1、3、6和 8 天的鼻腔样本及第8天的肺脏样本，利用空斑测定 CIV 滴度，结果表明，抗生素处理后感染 CIV 组（Abx）鼻腔病毒滴度除第1 天外始终高于未经抗生素处理的 CIV 感染组（WT）（p < 0.05）。间接免疫荧光试验结果表明，Abx组第 8 天肺脏组织中 CIV 的 NP 蛋白阳性荧光值也显著高于 WT 组。

鼻腔组织差异转录组学分析结果表明，WT组与 Abx 共有 1031 个差异表达基因（DEGs）。相比 WT 组，Abx 组鼻腔组织中炎症相关基因（Nlrp3、Il1b）显著上调，而先天免疫相关基因（Mx1、Oasl、Oas1、Oas2、Oas3、Isg15、Isg20、Ifih1）则显著下调（p < 0.05）。对鼻腔以及肺脏的病理观察发现，高剂量的 CIV 感染可以导致犬鼻腔以及肺脏出现严重损伤，然而Abx组比格犬在感染 CIV 后其鼻腔及气管黏膜损伤更加严重，并且肺脏出现更加严重的间质性肺炎，包括肺泡壁增生显著，并伴随大量炎性细胞浸润。然而，维持屏障相关基因在转录水平上并没有表现出与之相一致的的表达趋势。间接免疫荧光试验表明，相比于 Abx 组，WT 组鼻腔组织的 ZO-1 在感染 CIV 后出现了代偿性表达。研究表明，由抗生素介导鼻腔菌群异常失调通过影响一系列宿主机制来加重流感病毒感染。这些潜在机制包括调节宿主炎症阈值，影响屏障维持和修复以及 IFN 通路激活。

2  犬鼻腔菌群失调对流感病毒感染犬肺脏菌群及免疫应答的影响

采集 Nor、WT 和 Abx 组肺脏样本，通过 16S rRNA 测序和功能预测分析犬肺脏菌群组成以及菌群动态。结果显示，Nor 组犬肺脏菌群主要由双歧杆菌属（Bifidobacterium）、乳杆菌属、芽孢杆菌属（Bacillus）、莫拉菌属、链球菌属（Streptococcus）、拟杆菌属（Bacteroides）以及硝酸盐还原菌属（Nitratireductor）构成(相对丰度> 0.5% )。WT 组与 Nor 组肺脏菌群结构类似，而 Abx 组在感染 CIV 后肺脏菌群的多样性显著低于 Nor 和 WT 组。3 组肺脏菌群组成差异分析结果表明，相比于 WT 组，Abx 组中莫拉菌属、硝酸盐还原菌属（Nitratireductor）、中根瘤菌属（Mesorhizobium）、马文菌属（Marvinbryantia）和分枝杆菌属（Mycobacterium）的相对丰度显著增加（p < 0.01），而乳杆菌属和臭味杆菌属（Odoribacter）的相对丰度显著减少（p < 0.01）。Abx 组与 Nor 组肺脏菌群中的莫拉菌属和乳杆菌属的相对丰度也存在较大的差异（p < 0.01）。WT 组与 Nor 组在属水平上近乎没有差异菌群。三组之间菌群功能变化趋势与鼻腔菌群变化功能趋势一致，均在感染相关通路出现显著富集。

相比于鼻腔转录组结果，肺脏转录组结果表明，抗生素处理后的 Abx 组在感染流感病毒后，其肺脏组织也表现出比 WT 组更为严重的高水平炎症转录特征，同时伴随较低水平的干扰素刺激基因（ISGs）表达。Abx 组肺脏组织中 Toll样受体（TLRs）基因（Tlr1、Tlr2、Tlr3、Tlr6、Tlr7 和 Tlr8） 的转录水平显著高于 WT 组。此外，Abx 组在炎症因子（Il1b、Il6、Il17b、Il18、Nlrp3、Ccl2、Cxcl8 和 Cxcl14）相比于 WT 组均出现显著上调。此外，荧光定量PCR结果显示，Abx 组血液中 Ifnb1 及抗病毒蛋白基因 Isg15、Mx1及 Oas1 的转录水平显著低于 WT 组，而促炎因子 Il1b、Il6、Tnfa 以及 Casp3 的转录水平则显著高于 WT 组。这些结果表明，鼻腔菌群失调在一定程度上影响了流感病毒感染过程中机体的先天免疫抗病毒反应及炎症反应。

3  犬源乳酸菌介导宿主抗犬流感病毒感染的作用机制

将鼻腔和肺脏差异菌群及其功能通路与病毒滴度、宿主差异表达基因进行关联分析发现，某些菌群相对丰度变化与流感易感性密切相关，其中，乳杆菌属相对丰度与鼻腔及肺脏中的流感病毒滴度呈显著的负相关（p < 0.01）。鼻腔及肺脏菌群中，莫拉菌属和乳杆菌属的相对丰度变化与菌群功能通路的变化存在一定的关联性。具体表现为病毒感染相关通路与乳杆菌属的相对丰度呈显著负相关（p < 0.01），而与莫拉菌属的相对丰度呈显著正相关（p < 0.01）。此外，在肺脏菌群中，乳杆菌属的相对丰度与肺组织中Ifnb1 和Myd88 的转录水平呈显著正相关（p < 0.01），而与 Tnfa 和 Casp3 的转录水平呈显著负相关（p < 0.01）。而莫拉菌属的变化趋势与乳杆菌属则完全相反。这些数据表明，鼻腔菌群中乳杆菌属和莫拉菌属的动态变化与宿主对流感的易感性密切相关。

从比格犬体内分离获得 15 株犬源乳酸菌。其中，有 10 株乳酸菌与细胞共孵育不会影响细胞正常生长的活性。药敏试验表明，复合抗生素（莫匹罗星和新霉素）可以完全抑制 15 株犬源乳酸菌的生长。荧光素酶报告试验评估CIV感染细胞后的 IFNβ 启动子活性及 NF-κB 通路活性结果表明，植物乳杆菌（L.plantarum）C123 可以显著激活 CIV 感染细胞后的 IFNβ 启动子活性但不影响 NF-κB 通路活性。体外抗病毒试验结果表明，植物乳杆菌 C123 具有显著的抗病毒活性。在小鼠模型中，鼻腔接种植物乳杆菌 C123 可在一定程度上抑制流感病毒在鼻腔和肺脏的复制，并减轻 CIV 感染引起的病理损伤。此外，植物乳杆菌 C123 与 CIV 共感染细胞试验结果显示，该菌可通过 IFN-TBK1 信号通路激活 IFN 并影响病毒感染引起的自噬流抑制作用来发挥抗病毒作用。

综上，本研究探究了犬鼻腔菌群组成及其动态变化在流感病毒感染中的潜在作用，研究结果为开发基于益生菌干预的呼吸道病毒感染防控策略提供了新的思路，并为揭示鼻腔菌群调控宿主免疫介导的抗病毒机制奠定了基础。

The respiratory microbiota plays a crucial role in defending against pathogenic infections. As a key component of the upper respiratory tract, the nasal cavity serves as a primary site for initial contact and invasion by many pathogens. The composition of the nasal microbiota is closely linked to host susceptibility to infection, yet the underlying mechanisms remain unclear. Despite the close contact between dogs and humans, systematic investigations into the composition of the canine nasal microbiota and its potential immunomodulatory roles are still lacking. In recent years, canine influenza virus (CIV) has emerged as a major pathogen affecting canine respiratory health. However, the interaction between CIV infection and the composition and temporal dynamics of the canine respiratory microbiota has not been reported. The role of the nasal microbiota in mediating resistance to CIV and its associated immune mechanisms remains to be elucidated.Here, we characterized the respiratory microbiota of healthy beagle dogs using 16S rRNA gene sequencing. We then integrated multi-omics analyses with experimental validation to investigate the relationships among viral loads in the nasal cavity and lungs, host gene expression profiles, and microbial community dynamics following CIV infection. Notably, we identified a canine-derived Lactobacillus strain that suppresses CIV replication by activating the host type I interferon signaling pathway and modulating autophagic flux. These findings provide initial evidence for the key role of specific nasal microbiota in regulating host immune responses during CIV infection.

1  Impact of nasal microbiota dysbiosis on the pathogenicity of influenza virus infection and functional profiling of the canine nasal microbiome

A nasal microbiota dysbiosis model was established in Beagle dogs using two broad-spectrum antibiotic ointments (mupirocin and neomycin). The microbiota composition and dynamics were analyzed through 16S rRNA sequencing and functional prediction analysis. The effect of microbiota dysbiosis on CIV infection was explored. The results showed that compared to the normal control group (Nor), antibiotic treatment significantly altered the nasal microbiota structure (p < 0.05) and reduced microbiota α - diversity (Chao1 index and Richness index, p < 0.01). β - diversity analysis revealed a significant difference in the Bray-Curtis dissimilarity index (p < 0.01). Analysis of nasal microbiota composition showed that the number of highly abundant microbiota (relative abundance > 0.5%) decreased from 37 to 28 following antibiotic treatment. Further analysis indicated a significant increase in the relative abundance of Proteobacteria (p < 0.05). Moreover, the relative abundance of Lactobacillus, Prevotella, Moraxella, and Lachnoclostridium significantly decreased. After viral infection, the relative abundance of Moraxella in the antibiotic-treated CIV-infected Beagle nasal microbiota significantly increased (p < 0.05). KEGG pathway analysis based on functional predictions showed that 161 microbial functional pathways were significantly altered (p < 0.05) following antibiotic treatment, with eight pathways strongly associated with viral infection showing highly significant differences (p< 0.0001). Furthermore, dogs with dysbiotic nasal microbiota infected with a high dose (10⁷ PFU/mL) of CIV exhibited more severe clinical symptoms, including significant weight loss and persistent high fever (up to 39.6°C). Nasal samples collected on days 1, 3, 6, and 8 post-infection and lung samples on day 8 were used to determine CIV titers by plaque assay. The results indicated that CIV titers in the nasal cavities of antibiotic-treated dogs (Abx) were consistently higher than in the untreated CIV-infected group (WT) after day 1 (p < 0.05). Indirect immunofluorescence assays showed that CIV NP protein positivity in lung tissues on day 8 was significantly higher in the Abx group compared to the WT group.

Differential transcriptomic analysis of nasal tissue revealed 1031 differentially expressed genes (DEGs) between the WT and Abx groups. Compared to the WT group, the Abx group exhibited a significant upregulation of inflammation-related genes (Nlrp3, Il1b), and a significant downregulation of innate immunity-related genes (Mx1, Oasl, Oas1, Oas2, Oas3, Isg15, Isg20, Ifih1) (p < 0.05). Pathological observations of the nasal and lung tissues indicated that high-dose CIV infection caused severe damage to both the nasal and lung tissues. However, the Abx group exhibited even more severe nasal and tracheal mucosal damage and more pronounced interstitial pneumonia in the lungs, including significant alveolar wall hyperplasia and extensive inflammatory cell infiltration. Interestingly, barrier-related genes did not show expression patterns consistent with these tissue damages at the transcriptional level. Indirect immunofluorescence assays revealed compensatory expression of ZO-1 in the nasal tissue of the WT group after CIV infection, in contrast to the Abx group. The study suggests that antibiotic-induced nasal microbiota dysbiosis exacerbates CIV infection through the modulation of host mechanisms, including altering the inflammation threshold, affecting barrier maintenance and repair, and modulating IFN pathway activation.

2  Impact of nasal microbiota dysbiosis on lung microbiota composition and immune response during influenza virus infection in dogs

Nasal and lung samples from the Nor, WT, and Abx groups were collected and analyzed for microbiota composition and dynamics using 16S rRNA sequencing and functional prediction analysis. The results showed that the lung microbiota of the Nor group was primarily composed of Bifidobacterium, Lactobacillus, Bacillus, Moraxella, Streptococcus, Bacteroides, and Nitratireductor (relative abundance > 0.5%). The lung microbiota structure of the WT group was similar to that of the Nor group, while the diversity of the lung microbiota in the Abx group was significantly lower than that in the Nor and WT groups after CIV infection. Microbiota composition analysis revealed that, compared to the WT group, the Abx group exhibited a significant increase in the relative abundance of Moraxella, Nitratireductor, Mesorhizobium, Marvinbryantia, and Mycobacterium (p < 0.01), whereas the relative abundance of Lactobacillus and Odoribacter significantly decreased (p < 0.01). Significant differences in the relative abundance of Moraxella and Lactobacillus were also observed between the Abx and Nor groups (p < 0.01). No significant differences in microbiota composition were observed at the genus level between the WT and Nor groups. The functional changes in the microbiota of the three groups followed a similar trend to that observed in the nasal microbiota, with significant enrichment in infection-related pathways.

Compared to the nasal transcriptome results, the lung transcriptome analysis revealed that, after antibiotic treatment, the Abx group exhibited more severe inflammatory transcriptional features in lung tissues following CIV infection compared to the WT group, along with lower expression levels of interferon-stimulated genes (ISGs). The transcription levels of Toll-like receptor (TLR) genes (Tlr1, Tlr2, Tlr3, Tlr6, Tlr7, and Tlr8) were significantly higher in the Abx group compared to the WT group. Additionally, the expression of inflammatory factors (Il1b, Il6, Il17b, Il18, Nlrp3, Ccl2, Cxcl8, and Cxcl14) was significantly upregulated in the Abx group compared to the WT group. Moreover, quantitative PCR results showed that the transcription levels of Ifnb1 and antiviral protein genes Isg15, Mx1, and Oas1 were significantly lower in the Abx group compared to the WT group, whereas pro-inflammatory factors Il1b, Il6, Tnfa, and Casp3 were significantly higher in the Abx group. These results suggest that nasal microbiota dysbiosis may, to some extent, influence the innate immune antiviral response and inflammatory response during CIV infection.

3  Mechanism of host antiviral activity mediated by canine-derived lactic acid bacteria against canine influenza virus infection

Correlation analysis of the differential microbiota and functional pathways in the nasal cavity and lungs with viral titers and host differentially expressed genes revealed that certain microbiota changes were closely associated with influenza susceptibility. Specifically, the relative abundance of Lactobacillus was significantly negatively correlated with influenza virus titers in the nasal cavity and lungs (p < 0.01). In the nasal and lung microbiota, the relative abundance changes of Moraxella and Lactobacillus were associated with changes in microbiota functional pathways, with virus infection-related pathways showing a significant negative correlation with Lactobacillus (p < 0.01) and a significant positive correlation with Moraxella (p < 0.01). Additionally, in the lung microbiota, the relative abundance of Lactobacillus was significantly positively correlated with the transcription levels of Ifnb1 and Myd88 in lung tissues (p < 0.01), while it was significantly negatively correlated with the transcription levels of Tnfa and Casp3 (p < 0.01). In contrast, the trends for Moraxella were completely opposite to those of Lactobacillus. These data suggest that the dynamic changes in Lactobacillus and Moraxella in the nasal microbiota are closely related to the host's susceptibility to influenza.

Fifteen strains of canine-derived lactic acid bacteria (LAB) were isolated from Beagle dogs. Of these, 10 strains did not affect cell viability when co-incubated with cells. Antibiotic susceptibility testing showed that a combination of antibiotics (mupirocin and neomycin) completely inhibited the growth of all 15 strains of canine-derived lactic acid bacteria. Luciferase reporter assays for the IFNβ promoter and NF-κB activity indicated that L.plantarum C123 significantly activated IFNβ activity but had no effect on NF-κB pathway activity. In vitro antiviral tests showed that L.plantarum C123 exhibited significant antiviral activity. In the mouse model, intranasal inoculation of L.plantarum C123 inhibited influenza virus replication in the nasal cavity and lungs to some extent and alleviated the pathological damage caused by CIV infection. Moreover, co-infection experiments with L.plantarum C123 and CIV in cells showed that this bacterium exerted its antiviral effect by activating IFN through the IFN-TBK1 signaling pathway and influencing autophagic flux inhibition caused by viral infection.

In conclusion, this study provides a preliminary investigation into the composition and dynamic changes of the nasal microbiota in dogs during influenza virus infection. Our findings offer new insights for developing probiotic-based strategies to prevent and control respiratory viral infections and lay the groundwork for elucidating the nasal microbiota-host immune interactions that mediate antiviral defense.

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