key: cord-0798720-g8d00m6j
authors: Li, Hai‐Yan; Li, Bing‐Xiao; Liang, Qing‐Qing; Jin, Xiao‐Hui; Tang, Lei; Ding, Qing‐Wen; Wang, Zhi‐Xiang; Wei, Zhan‐Yong
title: Porcine deltacoronavirus infection alters bacterial communities in the colon and feces of neonatal piglets
date: 2020-04-01
journal: Microbiologyopen
DOI: 10.1002/mbo3.1036
sha: 4fc1d8b553b16075958d57a3375cf4790c5a4dfd
doc_id: 798720
cord_uid: g8d00m6j

Porcine deltacoronavirus (PDCoV) is a novel enteropathogenic coronavirus that causes watery diarrhea in piglets. Little is known regarding the alteration of the gut microbiota in PDCoV‐induced diarrhea piglets. In this study, 5‐day‐old piglets were experimentally infected with PDCoV strain CH‐01, and all piglets developed typical clinical disease, characterized by acute and severe watery diarrhea. Histologic lesions were limited to the villous epithelium of the duodenum and ileum. Gut microbiota profiles in the colon and feces of piglets inoculated with PDCoV were investigated using 16S rRNA sequencing. The results showed that PDCoV infection reduced bacterial diversity and significantly altered the composition of the microbiota from the phylum to the genus level in the colon and feces of piglets. Firmicutes (phylum), Lactobacillaceae (family), and Lactobacillus (genus) were significantly increased (p < .01), while the abundance of Bacteroidetes (phylum) was markedly reduced in the colon and feces of the PDCoV‐infected piglets (p < .01) when compared to those of the healthy piglets. Furthermore, microbial function prediction indicated that the changes in the intestinal flora also affected the nucleotide transport and metabolism, defense, translation, and transcription function of the intestinal microbiota. The current study provides new insight into the pathology and physiology of PDCoV.

Porcine deltacoronavirus (PDCoV), a member of the genus Coronavirus of the family Coronaviridae, causes acute diarrhea/ vomiting and dehydration accompanied by severe atrophic enteritis in neonatal piglets Hu, Jung, Vlasova, & Saif, 2016) . The clinical symptoms of PDCoV resemble those of other porcine enteric pathogens, such as porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) (Zhang, 2016) . To date, PDCoV has widely spread around the world, which has caused serious economic losses for the pig farming industries.

However, there are no effective reagents or vaccines to control PDCoV infection.

In pigs, several studies have reported that there exists a close relationship between diarrhea-relating coronaviruses and intestinal microbiota (Huang et al., 2018; Liu et al., 2015; Song et al., 2017; Tan et al., 2019) . It has been reported that PEDV infection induced gut microbial dysbiosis, leading to a reduction in beneficial bacteria and an increase in pathogenic bacteria. The proportions of the phyla Fusobacteria, Proteobacteria, and Verrucomicrobia in PEDV-infected piglets were higher than that of the healthy piglets (Huang et al., 2018; Liu et al., 2015; Song et al., 2017; Tan et al., 2019) . TGEV can cause severe vomiting and diarrhea in pigs. Recently, a report showed that TGEV infection led to higher levels of Enterobacteriaceae and lower levels of Lactobacillus in intestinal mucosal when compared to the healthy pigs (Xia, Yang, Wang, Jing, & Yang, 2018) . However, little is known regarding the composition of the gut microbiota in PDCoV-infected piglets. Therefore, in the current study, the bacterial community profiles in the colon and feces of piglets inoculated with PDCoV were investigated using high-throughput sequencing of 16S rRNA gene amplicons, and our results will provide new information on the understanding of gut microbiota associated with PDCoV infection.

The LLC porcine kidney (LLC-PK) cell line was used to serially propagate PDCoV. Minimum essential medium (MEM) (NJJCBIO) supplemented with 5% fetal bovine serum (FBS, Gibco) was used for cell growth. The virulent PDCoV CH-01 strain (GenBank accession number: KX443143) was isolated by our laboratory from the intestinal contents of a piglet with diarrhea on a farm in Henan Province, China. Experimental infection with this strain can cause severe diarrhea in piglets. Passage 5 (P5) of PDCoV CH-01 was used in the current study. The cell-cultured PDCoV CH-01 P5 was detected using real-time quantitative RT-PCR (qRT-PCR) and titrated by plaque assay, as reported previously . The titer of PDCoV CH-01 P5 was 9.02 log 10 genome equivalents (GE)/ml and 10 7 PFU/ ml, respectively. The propagated PDCoV was confirmed to be negative for TGEV, PEDV, and Rotaviruses using RT-PCR, as reported previously (Jung et al., 2014) .

Six three-day-old healthy Duroc × Landrace × Yorkshire piglets with similar weights (about 2.2 kg/head) were purchased from a pig farm in Henan Province, China. The piglets were divided randomly into two groups: the control group (n = 3) and the virus-inoculated group (n = 3). Piglets were housed in two separate rooms (temperature, 25 ± 2°C) and artificially fed with milk powder every 3 hr throughout the experiment to meet or exceed the requirements of the national research council (Austin & Ruthie, 2012) . Blood and rectal swabs were collected from all the piglets before viral inoculation for the detection of PDCoV, TGEV, PEDV, and other diarrhea-related viruses by

Two days later (at 5-days old), the piglets in the viral group were inoculated orally with PDCoV CH-01 (10 ml/piglet). The piglets in the control group were inoculated orally with the same volume of MEM.

After infection, clinical signs and diarrhea occurrence in the piglets were observed daily. Diarrhea was assessed by scoring fecal consistency as follows: 0 = solid; 1 = pasty; 2 = semiliquid; and 3 = liquid.

Piglets with scores of 2 or more were considered diarrheic (Lin et al., 2015) .

To evaluate viral shedding, fecal samples were collected daily from each piglet with sterile cotton swabs. Piglets in both groups were euthanized at 4 days postinoculation (dpi), and different intestinal segments from duodenum, jejunum, ileum, cecum, colon, and rectum tissues were collected, to examine the viral distribution and pathological features in those tissues. The colonic contents were collected and used for microbial DNA extraction.

All the collected fecal samples were diluted fivefold with MEM.

About 1 g of tissue samples was collected, ground, and diluted in 5 ml of MEM. The samples were centrifuged at 1,847 g at 4°C for 20 min, and the supernatants were used for viral RNA extraction. Viral RNA was extracted using the TRIzol method (Solarbio) according to the manufacturer's instructions. The viral RNA was further reverse transcribed into cDNA using a Vazyme Reverse Transcription kit.

Viral RNA titers were determined using qRT-PCR as reported previously . qRT-PCR was conducted using the Premix Ex Taq (Probe qPCR) kit (TaKaRa). Amplification reactions were performed on a real-time thermocycler (CFX96TM Optics Module, BIO-RAD), and the result data were analyzed using the system software. The detection limit of the qRT-PCR was 4.6 log10 GE/ ml of PDCoV in fecal and tissue samples.

The fixed intestinal tissues were dehydrated, embedded in paraffin, sectioned, mounted on slides, and stained with hematoxylin and eosin (H&E). Slides were examined by conventional light microscopy.

The villus height (VH) and crypt depth (CD) of duodenum and ileum were measured using a computerized image system following previously described (Madson et al., 2014) , and the ratio of VH/CD was calculated. Paraffin slides were processed according to previous studies Ma et al., 2015) , and the anti-PDCoV-N protein was used as a primary antibody in this study. Finally, the slides were observed under a light microscope.

Total DNA was extracted from 500 mg of colonic contents and (5′-GGACTACHVGGGTWTCTAAT-3′) primers specific for the V3-V4 regions (Mori et al., 2013) . The PCR program was as follows:

95°C for 3 min, 27 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 45 s, and finally, extension at 72°C for 10 min. After PCR products extracted, purified, and quantified, equimolar concentrations were pooled and sequenced on an Illumina MiSeq instrument using paired-end sequencing (2 × 300) (Illumina) according to standard protocols, by Majorbio Bio-Pharm Technology Co. Ltd.

Raw DNA fragments were quality-filtered by Trimmomatic and merged by FLASH software (Magoč & Salzberg, 2011) . The highquality sequences were assigned to samples according to barcodes.

The high-quality reads were clustered into operational taxonomic units (OTUs) using Mothur. OTUs with a 97% similarity was used for 

The differences between groups were analyzed using a one-way analysis of variance (ANOVA) followed by Waller-Duncan's multiple comparison test and an unpaired t test. The results were expressed as the mean ± standard deviation (SD). Differences between samples were considered significant at p < .05. Statistical significance is indicated in the figures as follows: *p < .05, **p < .01.

To reproduce the disease with our newly isolated strain of PDCoV CH-01 in 5-day-old piglets, we orally inoculated piglets with the virus at a dose of 1 × 10 8 PFU/head ( Figure 1a ). PDCoV RNA was detected in the fecal swabs by qRT-PCR from 1 to 4 dpi. Peak viral RNA shedding was observed at 3 dpi ( Figure 1b ). As expected, all inoculated piglets showed typical clinical syndrome, characterized by acute and severe watery diarrhea and vomiting, while the control piglets were normal ( Figure 1c ).

ined at necropsies (4 dpi) by qRT-PCR assay. PDCoV was detected from all intestinal segments at 4 dpi. The high titers of viral RNA copies of PDCoV were detected in small intestines, especially in the duodenum and ileum (Figure 1d ). All fecal and tissue samples from the control piglets were negative for PDCoV.

Macroscopic examination showed that PDCoV-inoculated piglets 

The pyrosequencing studies provided 637,151 usable sequences According to the Sobs, Chao, Shannon, and Simpson indexes analysis, the richness and diversity of gut microbial in the VI and VF groups was significantly reduced when compared to that of the CI and CF groups, respectively (p < .05) ( Table 2) , but there was no significant difference within the VI and VF, the CF and CI groups (p > .05). To better understand the shared richness among the four groups, a Venn diagram displaying the overlaps between groups was developed. The result showed that only 68 of the total richness of 338 OTUs were shared among all the samples (Figure 3) , while the number of OTUs in VI (159) and VF (132) was lower than that of CI (244) and CF (293), respectively. These data suggest that PDCoV infection decreased the relative abundance of OTUS at 4 dpi. To measure the extent of the distinction between microbial communities, beta diversity was calculated using Bray-Curtis, and PCoA was performed. The fecal and colonic microbiota from piglets with PDCoV inoculation and the control could be divided into two different clusters, respectively, according to the community composition using Bray-Curtis metrics (Figure 4 ).

The microbiota composition was assessed in the colon and feces of piglets at 4 dpi. The intestinal microbiota was affected by PDCoV The analysis of the predominant abundant showed that the relative abundance of Lactobacillaceae and Veillonellaceae was higher in the PDCoV-inoculated groups (for Lactobacillaceae, 5.53% in the CF group vs. 60.65% in VF group, p < .01, for Veillonellaceae, 0.55% in the CF vs. 30% in the VF, p < .05), whereas the proportions of Becteroidaceae, Lachnospiraceae, and Ruminococcaceae decreased sharply in the VF group when compared to the CF group (p < .01) (Figure 5d) . Similarly, the abundance of Lachnospiraceae and Fusobacteriaceae was decreased in the VI group compared with that in the CI group (p < .01). Note: The results were presented as the mean ± standard deviation (SD); n = 3 for each treatment, five villus, and five crypts were counted per piglets. p-value < .05 was defined as statistically significant.

Abbreviations: MEM, minimum essential medium; SD, standard deviation.

At the genus level, PDCoV infection significantly increased the relative abundant of Lactobacillus (7.82% in the CI group vs. 86.22% in the VI group, p < .01 and 5.53% in the CF group vs. 60.65% in the VF group, p < .01), whereas relative abundance of Lachnoclostridium was lower in the VI (p < .01) and VF groups (p < .05) compared with that in the CI and CF groups, respectively. Also, in the VF group, the abundance of Veillonella was significantly increased (p < .05) and the abundance of Bacteroides and Holdemanella was markedly decreased compared with that of the CF group (p < .01). In the VI group, the abundance of Streptococcus and

Holdemanella was observed to decline notably (p < .05) (Figure 5e-f ).

The relative functional abundance in the samples was inferred by PICRUSt ( Figure 6 ). The COG functions mainly related to metabolism, genetic information processing, cellular processing, etc. After

PDCoV infection, the abundance of bacteria associated with defense (p < .05), translation (p < .05), and signal transduction mechanisms (p < .01) was significantly decreased, while the abundance of bacteria associated with nucleotide metabolism, translation, and function unknown was increased in the VI and VF groups compared with that in the CI and CF groups, respectively (p < .01). Moreover, PDCoV infection also caused a significant increase in the relative abundance of bacteria associated with coenzyme (p < .05) and amino acid metabolism (p < .01) in the VF group. These results suggest that the PDCoV infection might induce the change in the abundances of a bacterial functional gene.

Porcine deltacoronavirus could infect farmed pigs with watery diarrhea at various ages (Li et al., 2019 Note: The results were presented as the mean ± standard deviation (SD); n = 3 for each treatment.

In the same row, values with the same superscript letter are not significantly different (p > .05); those with different superscript letters differ significantly (p < .05).

Abbreviations: CF, feces of the control group; CI, intestinal content of the control group; VF, feces of the virus-inoculated group; VI, intestinal content of the virus-inoculated group.

The alpha diversity indexes of the colonic and fecal microflora in piglets (n = 3) Histopathological examination showed there is some structural damage in the duodenum and ileum, but the severity of the intestinal tissue damage differed from the previous studies (Dong et al., 2016; Hu et al., 2016; Ma et al., 2015; Zhang et al., 2019) . We deduced that these differences may be induced by the different PDCoV strains, virus administration dose, the duration of virus shedding, or the immune responses induced by PDCoV.

Gut microflora plays an important role in the immune response and pathogenesis of gastrointestinal tract infections. Nursing pigs are most susceptible to PDCoV, and infected piglets display acute watery diarrhea/vomiting and dehydration Hu et al., 2016) .

Diarrhea is associated with dysbiosis of the intestinal microbiota (Pop et al., 2014) . In this study, PDCoV infection induced a significant decrease in microbial diversity in the colon and feces of the 5-day-old piglets, which was similar to the result in a previous PEDV-related report (Huang et al., 2018) . Also, the microbial composition was altered in the colon and feces of the piglets infected with PDCoV. The diversity of gut microbiota is correlated with the host disease states (Ianiro, Tilg, & Gasbarrini, 2016; Ley, Turnbaugh, Klein, & Gordon, 2006) .

Bacteroidetes and Firmicutes are usually dominant in the healthy host, and the Firmicutes/Bacteroidetes ratio may reflect the eubiosis or dysbiosis of the gastrointestinal tract (Ling et al., 2014) . In this study, the increased ratio of Firmicutes/Bacteroidetes could be regarded as an important marker for intestinal dysbiosis. This change is often associated with the susceptibility to disease (Ley et al., 2006) . With the development of diarrhea, the greater flux of oxidative metabolites and oxygen spread into the intestinal lumen, which can induce a significant decrease in the abundance of strict anaerobes and a relative increase in the abundance of facultative anaerobic bacteria (Albenberg et al., 2014; Lupp et al., 2007) .

This may be a reason why the ratio of Firmicutes/Bacteroidetes The intestinal microbiota acts as a "metabolic organ" that interacts with the host and performs many essential functions to maintain host health status (Huang et al., 2018) . Previous research has shown F I G U R E 6 Functional predictions of the bacterial communities by PICRUSt.

The abscissa of functional prediction means the relative abundance proportions of the microbial function information. Statistical analysis was performed by Student's t test for multiple testing. Asterisks indicate a difference from the corresponding control group: (*p < .05, **p < .01). CF, control feces group; CI, control intestinal content group; VF, virus feces group; VI, virus intestinal content group that the diversity of microbiota confers functional redundancy, which aids the development of a functional immune system and immune regulation at the intestinal surface and protects against pathogens (Ivanov & Littman, 2011; Schippa & Conte, 2014) . In this study, the changes in the colonic and fecal microbiota altered the function of the microbial flora after PDCoV infection. Notably, the abundance of Bacteroidetes, a known carbohydrate producer (Duerkop, Vaishnava, & Hooper, 2009) , was decreased by PDCoV infection, which might partially explain the reason for the low functional abundance of carbohydrate transport and metabolism in the VI and VF groups.

The microbiota composition in the colon and feces was altered dur- of Henan Agricultural University.

The authors thank Dr. Hui Hu from Henan Agricultural University for designing the study and revising the manuscript. This work was supported by the National Key R&D Program (2018YFD0500102 and 2016YFD0500102) and the National Natural Science Foundation of China (U1704231).

None declared. funding acquisition (lead); supervision (lead); writing -review and editing (lead). Zhan-Yong Wei: Conceptualization (lead); funding acquisition (lead); supervision (lead); writing -review and editing (lead).

All data are provided in full in the results section of this paper apart from the raw pyrosequencing data which is available at https:// www.ncbi.nlm.nih.gov/biopr oject /PRJNA 549812.

Zhan-Yong Wei https://orcid.org/0000-0001-5731-3145

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