Influenza D virus infection in China, 2022–2023

ABSTRACT

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.

KEYWORDS:

• Influenza D virus
• phylogenetic lineage
• seroprevalence
• antigenic characterization

Introduction

Influenza D virus (IDV) is the fourth influenza virus species (Deltainfluenzavirus genus, Orthomyxoviridae family) that was discovered initially in swine in Oklahoma, USA in 2011 [1]. Since then, it has been frequently detected in cattle with respiratory symptoms, and hence cattle are proposed as the natural reservoir for IDV [2,3]. Among the four types of influenza viruses, IDV is genetically close to the influenza C virus (ICV) [1], while it behaves like the influenza A virus (IAV) that has a broad host range and worldwide distribution. Now, besides swine and cattle, IDV infections have been reported in goats, sheep, camels, horses, buffalo, and deer [4–6]. Moreover, IDV has spread to more than 20 countries in North and South America (United States, Canada, Mexico, and Brazil), Europe (France, Italy, Luxembourg, Ireland, United Kingdom, Sweden, Denmark, and Turkey), Asia (China, Japan, and Korea) and Africa (Ethiopia, Togo, Morocco, Benin, Kenya, Uganda, Côte d'Ivoire, and Namibia) [4,7–13]. Influenza viruses from the animal reservoir carry genetic traits which may increase their host range and virulence, and genetic changes are often introduced when they cross the species barrier and infect humans [14]. It has been reported that IDV may pose a zoonotic risk due to the detection of IDV-specific antibodies in sera from occupational populations (swine veterinarians and cattle-exposed workers) as well as general populations [15–17]. Efficient replication and transmission of IDV without adaptative mutations in ferrets, mice, and guinea pigs, surrogate models for human influenza viruses [18,19], and the presence of IDV genome in bioaerosol samples in indoor public places (an international airport setting and a hospital emergency room setting) [20,21] also further strengthen this notion.

IDV infections cause mild to moderate respiratory disease in animals from both laboratory and field experiments [22–26]. Several metagenomic and molecular surveillance studies on cattle with bovine respiratory disease (BRD) show that IDV is implicated in BRD complex [8,27–30]. Despite that clinical signs and susceptibility to secondary bacterial infection with Mannheimia haemolytica in calves are not increased by a primary infection with IDV [26], calves co-infected with IDV and Mycoplasma bovis suffer from a more severe respiratory disease than those infected with either at the same condition [31], implying that IDV may facilitate certain coinfections within the BRD complex. Further research found that IDV was likely to trigger immune pathways that inhibited the innate immune response against the bacteria, which resulted in an increased susceptibility to the secondary bacterial infection in cattle [32].

To date, IDV strains, based on the hemagglutinin-esterase fusion (HEF) gene sequences, can be divided into at least five phylogenetic lineages: D/OK, D/660, D/Yama2016, D/Yama2019, and D/CA2019 [33,34]. The D/OK and D/660 are two dominant IDV lineages mainly circulating in Europe and North America [4]. IDV strains from the D/CA2019 lineage have been found only in the United States [35], while the D/Yama2016 and D/Yama2019 lineages are confined largely to the Asian countries such as China, Japan, and Korea [9,36,37]. When two different influenza viruses infect the same cell simultaneously, their negative-sense RNA genome segments can be swapped during the virion assembly, a process known as reassortment. Indeed, reassortment events occur frequently between different IDV lineages [8,38–40], leading to a possible impact on genetic diversity, pathogenicity, and host range of IDV reassortants. Currently, there is an upward trend in the genetic and antigenic landscape of IDV in global bovine populations. As a result, more studies are required to comprehensively understand the importance, prevalence, and even zoonotic threat of the IDV.

Several studies have demonstrated the presence of IDV in China primarily through the detection of IDV RNA genome in clinical samples from diseased bovines and pigs [37,41–43]. A limited genetic analysis showed that most of IDV strains from China exhibit a distinct phylogenetic pattern, forming a subclade (D/China sub-lineage) within the D/OK lineage [37,41]. Recently, D/Yama2019 lineage-like IDV strains have also been detected in China, highlighting the genetic diversity of IDV in this country [37]. In this study, we describe the first successful isolation of live IDVs in China, examine the replication fitness and antigenicity of Chinese IDVs. This work also involves the nationwide virologic and serologic surveys in farm animals towards better understanding of the national distribution and genetic diversity of IDV in China.

Materials and methods

Cells and viruses

Human embryonic kidney 293 T (HEK-293 T) cells (ATCC), Madin-Darby Canine Kidney (MDCK) cells (ATCC), Swine Testis (ST) cells (ATCC), Madin-Darby Bovine Kidney (MDBK) cells (ATCC), and Human Rectal Tumour (HRT-18G) cells (ATCC) were cultured in high glucose Dulbecco’s modified Eagle medium (DMEM) (Cytiva HyClone, USA) supplemented with 10% (v/v) fetal bovine serum (FBS, Procell) and 100 U/mL penicillin–streptomycin (Life Technologies, USA). All the cells grew at 37°C with 5% CO₂. The recombinant D/OK lineage-representative virus D/swine/Oklahoma/1314/2011 (rD/OK/11), chimeric virus rD/OK^660-HEF with the HEF from the D/660 lineage-representative virus D/bovine/Oklahoma/660/2013 (D/660/13) and the other 6 segments from the D/OK/11 virus, and recombinant human influenza C/Victoria/2/2012 (rC/Vic/12) virus were rescued by the reverse genetics system. The protocol for the rescue of these viruses was similar to that described in our previous publications [44,45]. These recombinant viruses were propagated in MDCK cells in virus growth medium (VGM) at 37°C. The VGM is serum-free DMEM supplemented with 0.2∼1 µg/mL TPCK-trypsin (Sigma-Aldrich, St Louis) and 100 U/mL penicillin–streptomycin.

Sampling

During the winter of 2022 and the spring of 2023, a total of 460 nasal swab samples from apparently healthy cattle were obtained, among which 360 samples were collected at the slaughterhouse in the city of Zhongshan, China, and 100 samples were collected at cattle farms in Guangxi provinces. The cattle for routine slaughter were sourced from 15 provinces of the country. Totals of 565 bovine serum samples, 100 pig serum samples, and 100 goat serum samples were collected from abattoirs in Zhongshan, China. For the bovine serum samples, 430 of them were collected between October 2022 and April 2023, 68 of them were collected in June-August 2023, and 67 of them were collected in 2021–2022 winter. The bovine serum samples were from cattle in 16 provinces of China. The 22 human sera of cattle farm workers were obtained from the Third Affiliated Hospital of Sun Yat-sen University in the Guangdong province.

PCR detection and sequencing

The viral RNA was extracted from nasal swab samples by using the commercial RNA extraction kit (Omega, E.Z.N.A.R Viral RNA Kit) and the cDNA was generated by using the reverse transcription kit (Vazyme, HiScript R III 1st Strand cDNA Synthesis Kit), according to the manufacturer’s instructions. The viral cDNA was then tested for IDV M gene by PCR with a pair of primers IDV-M-13-Forward: 5′-CAACTACTTGCTGAACTTGAGGGAT-3′ and IDV-M-746-Reverse: 5′-AAGATTAGCCATTCCACTGAC-3′. The IDV-positive samples with intense PCR bands were selected for further molecular characterization, including amplification and sequencing of all seven genomic segments of the isolates. The sequences were deposited in GenBank under accession numbers (OR685126-OR685156).

Sequence alignment and phylogenetic analysis

IDV HEF sequences of reference strains of the D/OK lineage (D/swine/Oklahoma/1334/2011), D/660 lineage (D/bovine/Oklahoma/660/2013), D/Yama2016 lineage (D/bovine/Yamagata/10710/2016), D/Yama2019 lineage (D/bovine/Yamagata/1/2019), D/CA2019 lineage (D/bovine/California/0363/2019) and representative strains of Chinese IDVs (D/bovine/Shandong/Y217/2014 and D/bovine/CHN/JY3001/2021) were downloaded from GenBank database. The comparison of HEF sequences of the new IDV isolates and the above reference IDVs was conducted using Blast tool in NCBI (http://ncbi.nlm.nih.gov). For the phylogenetic analysis, IDV genome sequences were downloaded from the GenBank database (accessed on September 15, 2023). The sequences were aligned by ClustalW in MEGA-X [46]. Partial gene sequences (less than 60% of the full-length gene sequences) were not included in the analyses. Preliminary phylogenetic trees for individual genome segments of IDV were generated using the maximum likelihood method and Tamura-Nei model with 100 bootstrap replicates in MEGA-X. According to the preliminary phylogenetic trees, sequences of D/Yama2016, D/Yama2019 and D/CA2019 lineages, and Chinese IDVs, as well as representative sequences of D/OK and D/660 lineages were selected. Phylogenetic trees for individual genome segments of IDV were further constructed with the selected sequences using the maximum likelihood method and Tamura-Nei model with 1000 bootstrap replicates in MEGA-X. Branches with bootstrap support less than 50% were omitted.

Virus isolation

The capacity of MDCK, ST or HRT cells to support IDV propagation were examined by using rescued IDVs. We observed that IDVs replicated less efficiently in certain MDCK cells provided by different collaborators in China, though they were all originally derived from ATCC. The cells in which IDVs were well propagated were selected for further use. The supernatants of IDV-positive samples were sterilized through 0.22-µm filters and then inoculated to co-cultured MDCK, ST and HRT-18G cells (50%, 25%, and 25%) on 6-well plates. The plates were incubated at 37°C for 5–7 days. If the medium become yellow, an appreciate amount of fresh medium should be added to keep the cells in good conditions. The infectivity was checked by determining the hemagglutination (HA) titre. The culture supernatants containing HA titres were passaged on MDCK cells. New IDV isolates were further confirmed by next-generation sequencing.

Electron microscopy

MDCK cells were infected with the IDV (D/bovine/CHN/JY3002/2022) at a multiplicity of infection (MOI) of 10. Sixty hours post infection, the virus supernatants were collected for the negative-staining electron microscopy, and the infected cells were fixed and then collected for the thin-section electron microscopy.

Hemagglutination inhibition (HI) assay

Briefly, sera were pretreated with receptor-destroying enzyme (RDE; Denka Seiken, Tokyo, Japan) according to the manufacturer’s protocol and then adsorbed with a 20% suspension of chicken red blood cells (RBCs) for 1 hour at 4°C. For each virus used in the HI assay, it must be standardized to contain 4 HA units/25 µL or 8 HA units/50 µL. The treated serum (1:10) was serially diluted two-fold with PBS. Then, 25 μl of standardized virus antigen were added into 25 μl of the serial two-fold dilutions of antisera (1:10–1:1280) and the mixtures were incubated for 30 min at 37°C. Finally, 50 μl of 1% RBCs were added into 50 μl of the mixture. After incubation at room temperature for 30 min, HI titres were determined as the reciprocal of the highest dilution of the serum inhibiting agglutination (showing RBC button).

Viral replication kinetics assay

Viral growth kinetics were performed on MDCK, MDBK, ST or HRT-18G cells with an inoculum at a MOI of 0.1 or 0.01. Cells were seeded in the 12-well plate with 3 × 10⁵ cells per well 16–18 hours prior to virus inoculation. Two hours after incubation at 37 °C, cells were washed twice with PBS, and 2 mL of VGM were added. Samples were collected at 0, 24, 48-, 72-, 96-, and 120-hours post-infection and titrated by TCID₅₀ assay.

Statistical analysis

The significant differences in geometric mean HI titres against different viruses and the significant differences in replication titres among the D/bovine/CHN/JY3002/2022, D/bovine/CHN/JY3097/2022 and rD/OK/11 viruses were determined by the one-way ANOVA and Tukey’s multiple comparison test in GraphPad Prism 8.0. Values of p < 0.05 were considered significant (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). Values of p < 0.1 were also marked in figures.

Results

Molecular surveillance of IDV in cattle, China, 2022–2023

During the winter of 2022 and the spring of 2023, a total of 460 nasal swab samples were collected from cattle at routine slaughter and cattle farms. The cattle were transported from 16 provinces of China (Figure 1). Nasal swab samples were tested by amplification of partial genomic sequences of the IDV M segment by RT–PCR assay (Figure S1). IDV genomic sequences were detected in 51 (11.3%) nasal swab samples from cattle in 13 of the 16 provinces (Figure 1A,B and S1). The IDV positive rates were 4.7%−31.3% among the provinces (Figure 1B).

Figure 1. Detection and isolation of IDV in cattle among provinces of China, 2022–2023. (A) The numbers of IDV-positive nasal swab samples from cattle in each province of China, 2022–2023. The colours indicate positive rates of IDV in cattle among provinces according to the legend located at the left bottom of the map. The numbers of IDV-positive nasal swab samples are given in blue colour (bold font), and the numbers of total nasal swab samples collected in cattle in individual provinces are given in yellow colour (bold font). The overall IDV RNA positive rate in cattle in China according to this study is indicated in the top of the map. (B) IDV RNA positive rates and samples in cattle in individual provinces tested. (C) Negative-staining electron microscopy showing an enveloped spherical IDV particle ((Bar = 200 nm)). (D) Thin-section electron microscopy displaying budding and release of IDV virions from the plasma membrane of infected cells (Bar = 1 µm).

Overall, provinces in Northern China appeared to have higher prevalence of IDV than that in Southern China (Figure 1A). Relatively high IDV genome detection rates (higher than 10%) were observed in Liaoning (31.3%, n = 16), Gansu (20.8%, n = 48), Hebei (19.7%, n = 61), Inner Mongoria (16.0%, n = 25), and Shandong (11.8%, n = 34) provinces in Northern China (Figure 1B). Moderate positive rates (less than 10%) of IDV genome detection were found in Ningxia (6.7%, n = 75), Shaanxi (5.6%, n = 18) and Shanxi (5.6%, n = 18) provinces in Northern China, as well as Hunan (6.7%, n = 15) and Guangxi (4.7%, n = 106) provinces in Southern China (Figure 1B). The IDV genome detection rate was not determined for the provinces with the sample size lower than 15. Among them, IDV-positive samples were found in Xinjiang and Henan provinces in Northern China, and Guizhou province in Southern China (Figure 1B). Results from this study indicated that IDV was widespread around the country.

Genetic characterization and virus isolation of IDV in China

The near full-length HEF gene segment sequences were obtained from 10 of the 51 positive samples (Figure 1B). In total, five different HEF sequences were identified from samples 42, 97, 125, 237 and 242–242 (Figure 1B), which represented five novel IDV strains designated as D/bovine/CHN/JY3002/2022 (D/JY3002), D/bovine/CHN/JY3097/2022 (D/JY3097), D/bovine/CHN/JY3125/2022 (D/JY3125), D/bovine/CHN/JY3237/2023 (D/JY3237), and D/bovine/CHN/JY3242/2023 (D/JY3242), respectively. The HEF sequences from samples 235 and 240 were identical to those from the sample 237, while the HEF sequences from samples 66, 168, and 175 matched those from the sample 97 (Figure S3). Sequencing of other IDV genomic segments was also attempted for the five new strains. IDV isolation was successfully achieved from samples 42 and 97. To intuitively display the IDV isolate, electron microscope (EM) studies were conducted. Negative-staining EM showed an enveloped spherical IDV particle approximately 100 nm in diameter (Figure 1C). Thin-section EM displayed budding and release of IDV virions from the plasma membrane of infected cells (Figure 1D). To the best of our knowledge, this is the first report on the successful isolation of live influenza D viruses (IDVs) in China.

To understand the genetic relationship of these IDV strains to previously characterized IDVs, HEF sequences of IDVs identified in this study were aligned to those of lineage-representative IDVs. Alignment results showed that HEF sequences of the D/JY3002, D/JY3097, D/JY3125 and D/JY3237 were greatly similar (nucleotide similarity >97.50%), and they all shared the highest nucleotide similarity (99.06%, 98.93%, 97.38% and 98.90%, respectively) to the HEF sequence of the D/Yama2019 lineage-representative strain (Table 1). Consistent with this, HEF sequences of D/JY3002, D/JY3097, D/JY3125, and D/JY3237 were found to phylogenetically belong to the D/Yama2019 lineage (Figure 2A). Alignment and phylogenetic analyses were also performed for other IDV genomic segments of the D/JY3002, D/JY3097, D/JY3125, and JY3237. The PB2, PB1, P3, NP, M or NS sequences of them were very similar (nucleotide similarity >98.67%) (Figure S4). Furthermore, each segment of these four strains was phylogenetically close to that of the D/Yama2019 lineage-representative strain (Figure 2B–G). These results further confirmed that IDV strains within the D/Yama2019 lineage were predominantly circulating in Chinese cattle herds.

Figure 2. Phylogenetic trees for genomic segments of IDVs. (A–G) Nucleotide sequences of each segment are aligned and analysed in MEGA-X, with 1000 bootstrap replicates. Bootstrap scores of at least 50 are shown to the left of the nodes. Scale bar represents the number of substitutions per site. In the phylogenetic tree for the HEF segment (A), each branch of the five IDV major lineages (D/OK, D/660, D/CA2019, D/Yama2016, and D/Yama2019 lineages) is noted by red, yellow, green, light blue and dark blue colour, respectively, and the five lineage-representative IDVs are bolded. IDV strains identified previously in China are highlighted with colour background. IDV strains discovered in this study are marked with red stars. In phylogenetic trees for non-HEF segments (B-G), the five lineage-representative IDVs are bolded with red, yellow, green, light blue, and dark blue colour, respectively. IDV strains identified previously in China are highlighted with black background. IDV strains discovered in this study are marked with red stars.

Table 1. Similarity of nucleotide sequences of the IDV HEF gene.

	D/JY3002	D/JY3097	D/JY3125	D/JY3237	D/JY3242	D/OK/11	D/660/13	D/Yama2016	D/Yama2019	D/CA2019	D/JY3001	D/SD2014
D/JY3002	100
D/JY3097	99.79	100
D/JY3125	97.56	97.54	100
D/JY3237	98.89	98.77	97.99	100
D/JY3242	93.75	93.75	94.52	93.65	100
D/OK/ 11	95.01	95.03	96.49	94.8	95.86	100
D/660/ 13	94.44	94.55	95.33	94.28	98.14	96.63	100
D/Yama2016	94.81	94.87	95.12	94.33	95.13	96.29	96.14	100
D/Yama2019	99.06	98.93	97.38	98.9	94.25	95.24	94.59	94.69	100
D/CA2019	93.77	93.75	94.34	93.54	94.51	95.36	95.31	94.87	94.04	100
D/JY3001	98.96	98.82	97.48	99.53	93.63	94.94	94.39	94.49	98.9	93.63	100
D/SD2014	94.55	94.55	95.91	94.33	95.39	98.78	96.14	95.85	94.79	95.02	94.49	100

Note: Similarities of HEF sequences between reference strains are indicated in blue font. The reference strains include D/swine/Oklahoma/1334/2011 (D/OK/11, D/OK lineage), D/bovine/Oklahoma/660/2013 (D/660/13, D/660 lineage), D/bovine/Yamagata/10710/2016 (D/Yama2016, D/Yama2016 lineage), D/bovine/Yamagata/1/2019 (D/Yama2019, D/Yama2019 lineage), D/bovine/California/0363/2019 (D/CA2019, D/CA2019 lineage), D/bovine/Shandong/Y217/2014 (D/SD2014, D/China sub-lineage) and D/bovine/CHN/JY3001/2021 (D/JY3001, D/Yama2019 lineage). Similarities of HEF sequences between IDV strains identified in this study are indicated in pink font. The new IDV strains are D/JY3002, D/JY3097, D/JY3125, D/JY3237 and D/JY3242. Similarities of HEF sequences between the new IDVs and above reference IDVs are indicated in orange font.

The HEF sequence of D/JY3242 was obviously different from those of D/JY3002, D/JY3097, D/JY3125 and D/JY3237 (nucleotide similarity <95.0%), which exhibited the highest nucleotide similarity (98.14%) to the HEF sequence of the D/660 lineage-representative strain (Table 1). In the phylogenetic tree, the HEF sequence of D/JY3242 belonged to the D/660 lineage (Figure 2A). This is the first evidence of the D/660 lineage-like IDV being present in Asia. In addition, alignment and phylogenetic analyses showed that the segment PB2, P3, NP, M or NS of the D/JY3242 was distant from that of the D/JY3002, D/JY3097, D/JY3125, and JY3237 (Figures 2B, D-G, and S4). Interestingly, the segment PB2, P3, M or NS of D/JY3242 was phylogenetically close to that of the D/660 lineage-representative strain (Figure 2B, D and F-G), but the segment NP of D/JY3242 was close to that of the D/OK lineage-representative strain (Figure 2E). These results suggested that D/JY3242 likely resulted from the past reassortment event(s) between circulating D/660 lineage-like and D/OK lineage-like IDVs.

Serosurvey for IDV exposure in cattle, China, 2021–2023

To determine the seroprevalence of IDV among Chinese cattle, serum samples were tested by HI against the D/JY3002. Of the 430 serum samples taken from cattle at routine slaughter between October 2022 and February 2023, 393 serum samples were positive for antibodies to IDV, resulting in an overall seroprevalence of 91.4% in cattle (Figure 3A). The overall geometric mean titre (GMT) of seropositive samples was 89.2 (HI titres ranged from 10 to 1280) (Figure 3B). Distribution of HI titres equal to or higher than 40 was 81.4% (Figure 3B). Of the 16 provinces tested, IDV antibodies were detected in cattle from all these provinces (Figure 3A). The GMT were in the range of 25.2–276.7 in seropositive samples by the provinces (13 of them displayed the GMT higher than 40 in seropositive samples) (Figure 3B). The highest GMT was observed in cattle from Liaoning province, and the lowest GMT was shown in cattle in Guizhou province (Figure 3B). There were three provinces exhibited the 100% seropositivity rate for IDV in cattle: Inner Mongoria (n = 26, GMT: 107.3), Ningxia (n = 75, GMT: 81.1) and Hebei (n = 61, GMT: 100.6) provinces in Northern China (Figure 3). The lowest seropositivity rate (30.8%) for IDV in cattle was observed in Guangdong (n = 26, GMT: 33.6) province in Southern China (Figure 3). The bias in seropositivity rates of IDV might occur when the sample size in some of the provinces was fairly small, so the IDV seropositive rate was analysed only for the provinces with the sample size greater than 15. Overall, our results still clearly demonstrated that IDV circulated with high frequency in cattle across a number of provinces in China during the 2022–2023 winter season.

Figure 3. Serosurveillance results for IDV in cattle among provinces of China, 2022–2023. (A) The numbers of serum samples positive for IDV antibodies from cattle in each province of China, 2022–2023. The colours indicate seropositive rates of IDV in cattle among provinces according to the legend located at the left bottom of the map. The numbers of serum samples positive for IDV antibodies are given in red colour (bold font), and the numbers of total serum samples collected in cattle in individual provinces are given in yellow colour (bold font). The overall IDV seropositive rate in cattle in China according to this study is indicated in the top of the map. (B) The HI titre of each serum sample obtained from cattle across provinces. The colours indicate different levels (less than 10, 10–20, 20–40, 40–160, and 160–1280) of HI titres. The error bar in red colour represents the geometric mean HI titre. Serum samples with HI titres equal to or higher than 10 are counted as positive. Data on the right y-axis represent the distribution of serum samples with HI titres less than 10, equal to or higher than 10 but less than 40, and equal to or higher than 40.

We continued to examine the IDV exposure in Chinese cattle in the following summer of 2023. No seropositive samples were found in cattle from Guangdong (n = 15) province, but all the serum samples collected from cattle in Hebei (n = 3), Shanxi (n = 2), Inner Mongoria (n = 9), Ningxia (n = 33), Gansu (n = 1), Hunan (n = 3) and Guangxi (n = 2) provinces were positive for IDV-specific antibodies (Figure 4A). In Inner Mongoria and Ningxia provinces, containing a sufficient number of bovine serum samples, though geometric mean titres in sera from the two provinces were lower in summer (68.6 and 74.4) than those in winter (133.8 and 81.1), their IDV seropositivity rate was 100% in the two seasons (Figures 3 and 4A). In addition, the results of a retrospective serosurvey, which was conducted in cattle from Hebei (n = 22), Liaoning (n = 18), and Guangdong (n = 27) provinces in 2021–2022 winter, revealed IDV seropositivity rates of 95.5%, 83.3%, and 25.9%, and geometric mean titres of 33.9 (HI titre range 10–320), 27.6 (HI titre range 10–80), 29.7 (HI titre range 10–80) in cattle from the three provinces, respectively (Figure 4B). The overall IDV seroprevalence and GMT in cattle from Hebei, Liaoning and Guangdong provinces were lower in 2021–2022 winter than those in 2022–2023 winter (Figures 3 and 4B). Taken together, these results further confirmed that IDV exposure in cattle were frequent in a number of provinces in China during 2021–2023.

Figure 4. Serosurveillance results for IDV in cattle (in the summer season or the earlier year), pigs, goats, and farm workers. (A,B) The HI titre of each serum sample obtained from cattle across provinces in the summer of 2023 (A) or the winter during 2021 and 2022 (B). The colours indicate different levels (less than 10, 10–20, 20–40, 40–160, and 160–1280) of HI titres. The error bar in red colour represents the geometric mean HI titre. Serum samples with HI titres equal to or higher than 10 are counted as positive. (C,D) The HI antibodies against the D/JY3002 (C) or the rC/Vic/12 (D) in serum samples taken from pigs, goats, and farm workers. According to the legend with blue gradient located to the right of the panel, rectangular boxes filled with blue colours indicate HI antibody titres against the D/JY3002 or the rC/Vic/12. The blank rectangular boxes represent serum samples without HI antibody titres against the D/JY3002 or the rC/Vic/12.

Serosurvey for IDV exposure in pigs, goats, and farm workers, China, 2022–2023

To investigate IDV exposure in other animal species, we collected 100 serum samples from pigs and 100 serum samples from goats at routine slaughter in the Guangdong province in February–April 2023. Our data showed that three serum samples (3%) from pigs and 1 serum sample (1%) from goat were tested positive for IDV specific antibodies (Figure 4C,D), while the titres were very low (HI antibody titres: 10–20) (Figure 4C). These results indicated a very low seroprevalence of IDV in pigs and goats in the Guangdong province.

To determine if farm workers are susceptible to IDV, we measured 22 serum samples for the presence of IDV-specific antibodies. Our results revealed five serum samples showing low levels of HI antibodies against the IDV (HI titre range: 10–20), while these serum samples also reacted with the influenza C virus (ICV) (HI titre range: 20–40) (Figure 4C,D). Moreover, 21 out of the 22 human serum samples were positive for antibodies to the ICV, with HI titres ranged between 10 and 80 (Figure 4D). Generally, no antigenic cross-reactivity has been found between the swine or bovine IDV and the human ICV [6]. In light of this, a large sample size of ICV-preabsorbed sera should be used to further examine IDV exposure among farm workers in China.

Replication fitness and antigenicity of the Chinese IDV strain

To understand the replication fitness of Chinese IDV strains (D/JY3002 and D/JY3097), we compared their replication kinetics to that of the D/OK lineage-representative strain rD/OK/11 in Madin-Darby Canine Kidney (MDCK) cells, Swine Testis (ST) cells, Madin-Darby Bovine Kidney (MDBK) cells, and Human Rectal Tumour (HRT-18G) cells at MOIs of 0.01 and 0.1. Our data showed that there were no statistically significant differences in the replication efficiency between the D/JY3002 and D/JY3097 viruses, while they both replicated to significantly higher titres than the rD/OK/11 virus in all these cell lines (Figure 5A–D). They grew robustly in MDCK, MDBK and HRT-18G cell lines, with the peak viral titres of 1 × 10⁸ TCID₅₀/ml^-1 × 10⁹ TCID₅₀/ml at 3 or 4 days after infection (Figure 5A,B and D). Interestingly, they replicated less robustly in the ST cell line, in which the peak viral titre was 2 log₁₀TCID₅₀/ml lower than that in MDCK, MDBK and HRT-18G cell lines (Figure 5A–D). The differences of peak viral titres between the D/JY3002 or D/JY3097 and rD/OK/11 viruses were 3–5 log₁₀(TCID₅₀/ml) in MDCK and MDBK cells (Figure 5A,B), 1–3 log₁₀(TCID₅₀/ml) in ST cells (Figure 5C), and 3–4 log₁₀(TCID₅₀/ml) in HRT-18G cells (Figure 5D). These data indicated that the Chinese IDV isolates possessed robust replication capacity in multiple cell lines.

Figure 5. Replication kinetics and antigenicity of the Chinese IDV strain. (A-D) Replication kinetics of the D/JY3002, D/JY3097, and rC/Vic/12 in different cell lines. MDCK (A), MDBK (B), ST (C) and HRT-18G (D) cell lines are inoculated with 0.01 or 0.1 MOI of the D/JY3002 or the rC/Vic/12. Aliquots of the tissue culture supernatant are taken at periodic intervals and virus titres are determined by TCID₅₀ per mL in MDCK cells. (E) Viral titres of the D/JY3002, the rD/OK/11, the rD/OK^660-HEF, and the rC/Vic/12. (F-H) The comparison of HI antibody titres of cattle serum samples against the D/JY3002, the rD/OK/11, and the rD/OK^660-HEF.

To examine if the bovine sera with HI antibodies against the D/JY3002 virus could react to other lineage-representative IDVs, we conducted HI tests involving the recombinant D/OK lineage-representative virus rD/OK/11 and a chimeric virus rD/OK^660-HEF that the HEF was from the D/660 lineage-representative strain D/660/13 and the other six segments were from the D/OK/11 virus. In the HI tests, we included 33 serum samples showing diverse HI titres against the D/JY3002. The results showed that all of them cross-reacted to the rD/OK/11, and 31 of them cross-reacted to the rD/OK^660-HEF (Figure 5F–H). Among 22 serum samples with geometric mean titres less than 320 against the D/JY3002, half reacted more strongly with the rD/OK/11 than with the D/JY3002 or the rD/OK^660-HEF (Figure 5F,G), suggesting that IDV exposed in these cattle may have D/OK-lineage antigenicity. Among the other 11 serum samples showing geometric mean titres higher than 320 against the D/JY3002, 9 of them reacted more strongly with the D/JY3002 than with the rD/OK/11 or the rD/OK^660-HEF (Figure 5H). Similarly, geometric mean titres of serum samples 241 and 246 were also significantly higher against the D/JY3002 than against the rD/OK/11 or the rD/OK^660-HEF (Figure 5G). These results implied that the IDV exposed in the corresponding cattle displayed D/Yama2019-lineage antigenicity. Taken together, our data indicated that antigenic cross-reactivity was commonly exited among the three lineage-representative IDVs and IDV exposed in Chinese cattle might have D/OK – or D/Yama2019-lineage antigenicity, which warrants further investigation by using IDV lineage-specific anti-sera.

Discussion

In 2014, China reported its first detection of IDV in cattle [42]. Subsequently, several studies demonstrated that IDVs existed not only in bovines but also in pigs and goats in China [37,41,43]. Despite continuous documentation of IDV cases in Chinese herds, there have been no reports of isolation of live IDV in the country, which impedes our understanding of the infectivity, pathogenicity, and transmissibility of IDV in China. In this study, we successfully isolated live IDVs from cattle. Moreover, we expanded IDV surveillance in China from the regional to national level towards further studying of the national distribution, genetic diversity, and natural exposure of IDVs in China.

In the earliest survey in China, IDV RNA was only detected in 0.66% (3/453) of cattle in Shandong province between April and May of 2014 [42]. In 2016, the survey conducted in Guangdong province revealed that IDV RNA positive rate in cattle was 7.9% (40/504) [4,43]. In a small-scale survey during the winter and spring of 2021–2022, we found an overall IDV RNA positive rate of 4% (10/250) in cattle from different provinces of China [37]. In the present study, IDV RNA was detected in 11.1% (51/460) of cattle in 13 of the 16 provinces tested, and the positive rates among the provinces varied from 4.7% to 31.3% (Figures 1A,B and S1). The findings, combined with those from previous survey studies, demonstrate that IDV is widely circulating in cattle in China.

To date, no comprehensive serosurvey has been done to assess IDV exposure in farm animals in China. This first IDV serosurveillance in China during the winter and spring of 2022–2023, showed that the overall seroprevalence of IDV in cattle was 91.4% (393/430), with the overall GMT of 89.2 (HI titres ranged from 10 to 1280) in seropositive samples (Figure 3A,B). Notably, all the 16 provinces from which cattle originated had IDV-seropositive animals (Figure 3A). There were three provinces exhibited the 100% seropositivity rate for IDV in cattle (Figure 3). Even if in the summer season of 2023, Ningxia province (n = 33) displayed the 100% IDV-seropositivity rate in cattle (Figure 4A). In addition, high IDV-seropositivity rates in cattle were also observed in Hebei (95.5%, n = 22) and Liaoning (83.3%, n = 18) provinces in 2021–2022 winter, according to the retrospective serosurvey (Figure 4B). A solid study has revealed limited protection from preexisting immunity against IDVs in cattle herds [47], which may explain the recurrent and escalating nature of IDV exposure within Chinese cattle populations. High IDV seroprevalence in cattle were also reported in the United States (77.5%) [48], Luxembourg (80.2%) [49], Ireland (94.6%) [50] and Italy (95.6%) [51]. In other Asian countries, the seroprevalence of IDV in cattle was 54.6% (405/742) in Korea and 30.5% (386/1267) in Japan [9,52]. In terms of our serosurvey study, the findings are important as they establish that IDV exposure in the Chinese cattle population is far more frequent than expected. High seroprevalence levels of IDV in cattle is consistent with the finding that cattle are primary reservoir for IDV.

A variety of non-bovine hosts, such as pigs, goats, sheep, camels, and equids, have been shown to be susceptible to IDV [6,49,50,53–55]. In China, IDV RNA has been also detected in pigs and goats [41,43]. Although high levels of IDV RNA were found in pigs (18.4%, n = 114) and goats (20.3%, n = 138) in Guangdong province in 2016 [4,43], our serological surveys revealed low titres and low percentages of IDV-seropositive pigs (3%, n = 100, GMT: 12.6) as well as goats (1%, n = 100, GMT: 10) in 2023 (Figure 4C). Part of these findings are in line with the low IDV RNA positive rate reported in pigs from multiple provinces in Eastern China [41]. In addition, a high seroprevalence (91%, n = 35) of IDV has been detected among human populations that have been in close contact with cattle [15] in U.S., suggesting that the virus might be transmitted from animal to human. In our study, we tested 22 serum samples from cattle farm workers in Guangdong. There were five serum samples (22.7%) showing low levels of HI antibodies against IDV (HI titre range 10–20) (Figure 4C), but they also reacted with human ICV (HI titre range 20–40) (Figure 4D). Considering that the seropositivity rate for IDV in cattle in Guangdong province was low, future studies on IDV exposure to the occupational population in China should be conducted in regions where IDV is highly prevalent.

On the basis of the HEF gene, five IDV phylogenetic lineages have been identified to date. Chinese IDV strains reported before 2021 formed a distinct sub-clade within the D/OK lineage [41]. However, according to this study (Figure 2A) and our previous work [37], the IDV strains reported in China after 2021 mainly fall within the D/Yama2019 lineage. It appears that the D/Yama2019-like IDVs are the currently dominant strains in Asia, with widespread distribution in Japan, China, and Korea [9,36,37]. The D/660 lineage is mostly found in North America and Europe [4]. Here, we discovered the D/660 lineage-like IDV in Chinese cattle (Figure 2A). As far as we know, this is the first time the D/660 lineage-like IDV has been detected in Asia. Importantly, this D/660 lineage-like IDV is a reassortant virus carrying the NP gene from the D/OK lineage-like IDV and remain genes from the D/660 lineage-like IDV (Figure 2). As a result, more studies are needed to understand the genetic diversity and reassortment of Chinese IDVs.

It has been proved that IDV has a broad cellular tropism [1]. Both the D/OK and D/660 lineage-representative IDVs can replicate efficiently in diverse cell lines derived from human, swine, bovine, and canine [1,44]. As for the Chinese IDV strains D/JY3002 and D/JY3097, we compared their replication kinetics to that of the D/OK lineage-representative strain rD/OK/11 in HRT-18G, ST, MDBK and MDCK cells at different MOIs. According to the results, they replicated more robustly than the rD/OK/11 virus in all these cell lines (Figure 5A–D). It is interesting that the Chinese IDV isolates replicated the least efficiently in the swine cell line (Figure 5C). In this case, further investigation is warranted to determine if the bovine IDV prefers bovine cells for replication over swine cells.

The antigenic cross-reactivity is common between IDV lineages [35,36,38]. Although the IDV lineage-representative antigens cross-reacted to each other, each IDV lineage-representative antigen has its own specific antigenicity. In this study, we investigated how the bovine sera with HI antibodies against the D/JY3002 virus reacted to other lineage-representative IDVs. Since genomic RNAs of IDVs in D/OK, D/660, and D/Yama2019 lineages were reported in China by this (Figure 2) and other studies [37,41–43], we intended to identify the major lineage-specific antigenicity of IDVs exposed in Chinese cattle. According to our results, IDV exposed in Chinese cattle might have D/OK – or D/Yama2019-lineage antigenicity (Figure 5F,G). However, it still warrants further investigation by including IDV lineage-specific anti-sera.

Ethics statement

Ethical review and approval were not required for this investigation because the nasal swab and serum samples from farm animals were taken by veterinarians, and the human serum samples were obtained from the Third Affiliated Hospital of Sun Yat-sen University in the Guangdong province.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This study was supported by the following grants: the NSFC grant (Nos. 32202795 and 32102750), the special funds (Nos. R2021YJ-QG008 and R2021QD-034) for Talent Introduction Program and the Project of Collaborative Innovation Center Fund (Nos. XTXM202202 and XT202207) from Guangdong Academy of Agricultural Sciences, the Project of Innovation Fund (No. 202201) from Agro-biological Gene Research Center, Guangdong Academy of Agricultural Sciences, and the grants from the open competition program of top 10 critical priorities of Agricultural Science and Technology Innovation for the 14th Five-Year Plan of Guangdong Province (2022SDZG02).