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. 2018 Oct 12;92(21):e01208-18.
doi: 10.1128/JVI.01208-18. Print 2018 Nov 1.

Infection Dynamics of Hepatitis E Virus in Wild-Type and Immunoglobulin Heavy Chain Knockout J H -/- Gnotobiotic Piglets

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Free PMC article

Infection Dynamics of Hepatitis E Virus in Wild-Type and Immunoglobulin Heavy Chain Knockout J H -/- Gnotobiotic Piglets

Danielle M Yugo et al. J Virol. .
Free PMC article

Abstract

Hepatitis E virus (HEV), the causative agent of hepatitis E, is an important but incompletely understood pathogen causing high mortality during pregnancy and leading to chronic hepatitis in immunocompromised individuals. The underlying mechanisms leading to hepatic damage remain unknown; however, the humoral immune response is implicated. In this study, immunoglobulin (Ig) heavy chain JH -/- knockout gnotobiotic pigs were generated using CRISPR/Cas9 technology to deplete the B-lymphocyte population, resulting in an inability to generate a humoral immune response to genotype 3 HEV infection. Compared to wild-type gnotobiotic piglets, the frequencies of B lymphocytes in the Ig heavy chain JH -/- knockouts were significantly lower, despite similar levels of other innate and adaptive T-lymphocyte cell populations. The dynamic of acute HEV infection was subsequently determined in heavy chain JH -/- knockout and wild-type gnotobiotic pigs. The data showed that wild-type piglets had higher viral RNA loads in feces and sera compared to the JH -/- knockout pigs, suggesting that the Ig heavy chain JH -/- knockout in pigs actually decreased the level of HEV replication. Both HEV-infected wild-type and JH -/- knockout gnotobiotic piglets developed more pronounced lymphoplasmacytic hepatitis and hepatocellular necrosis lesions than other studies with conventional pigs. The HEV-infected JH -/- knockout pigs also had significantly enlarged livers both grossly and as a ratio of liver/body weight compared to phosphate-buffered saline-inoculated groups. This novel gnotobiotic pig model will aid in future studies into HEV pathogenicity, an aspect which has thus far been difficult to reproduce in the available animal model systems.IMPORTANCE According to the World Health Organization, approximately 20 million HEV infections occur annually, resulting in 3.3 million cases of hepatitis E and >44,000 deaths. The lack of an efficient animal model that can mimic the full-spectrum of infection outcomes hinders our ability to delineate the mechanism of HEV pathogenesis. Here, we successfully generated immunoglobulin heavy chain JH -/- knockout gnotobiotic pigs using CRISPR/Cas9 technology, established a novel JH -/- knockout and wild-type gnotobiotic pig model for HEV, and systematically determined the dynamic of acute HEV infection in gnotobiotic pigs. It was demonstrated that knockout of the Ig heavy chain in pigs decreased the level of HEV replication. Infected wild-type and JH -/- knockout gnotobiotic piglets developed more pronounced HEV-specific lesions than other studies using conventional pigs, and the infected JH -/- knockout pigs had significantly enlarged livers. The availability of this novel model will facilitate future studies of HEV pathogenicity.

Keywords: B cell depletion; HEV; Ig heavy chain knockout; gnotobiotic pig; hepatitis E virus.

Figures

FIG 1
FIG 1
Ig heavy chain targeting CRISPR sequences and representative types of Ig heavy chain mutations in this study. (A) Disruption of Ig heavy chain in pigs by CRISPR/Cas9 with a total of four target sites designed using a web-based program. Boldface letters indicate the protospacer adjacent motif (PAM) sequence of target sites. Black arrows indicate primers used to amplify the target region. (B) Types of genetic mutations of the Ig heavy chain in single blastocyst. The sequencing results indicated either homozygous or biallelic mutation of Ig heavy chain in single embryos with no wild-type alleles observed. (C) Representative genotype of Ig heavy chain knockout pigs. Arrows indicate sites of mutations.
FIG 2
FIG 2
B-lymphocyte cell counts in peripheral blood samples of wild-type and immunoglobulin JH knockout gnotobiotic piglets experimentally infected with HEV. PBMCs were collected from each piglet at 28 dpi at necropsy. Cells were gated based on CD3 and quantified for the intracellular marker CD79a+ as a measure of the total B-cell population in the peripheral blood. Asterisks indicate statistical significance between designated groups determined by Tukey's t test with a P value of <0.001.
FIG 3
FIG 3
Quantification of HEV RNA loads in serum, fecal, and bile samples of wild-type and JH knockout gnotobiotic piglets experimentally infected with HEV. Viral RNAs were extracted from fecal samples three times weekly (A), intestinal contents and bile at 28 dpi (B), as a scatterplot of fecal viral RNA, with each symbol indicating the value for an individual piglet (C), and serum samples at 0, 7, 14, 21, and 28 dpi (D) for the quantification of HEV RNA copy numbers by qRT-PCR. The data are expressed as means ± the SEM. The detection limit is 10 viral genomic copies, which corresponds to 400 copies per 1-ml sample or per gram of tissue. Titers below the reported detection limit were considered negative. Asterisks indicate statistical significance between designated groups as determined by two-way ANOVA and a P value of <0.05.
FIG 4
FIG 4
Circulating levels of liver enzymes in serum samples collected weekly from HEV-infected wild-type and HEV-infected JH−/− knockout gnotobiotic piglets. Serum was collected weekly from each piglet beginning with 0 wpi and continuing until necropsy at 4 wpi. Liver enzymes, including AST (A), total bilirubin (B), alkaline phosphatase (C), and GGT (D), were measured at the Iowa State University Veterinary Diagnostic Lab. Normal limits were provided with the analysis based on the laboratory's standards for each specific test, which are indicated on each scatterplot graph as a dotted line. Each sample indicates an individual piglet for each time point and are expressed as means ± the SEM. Analysis was completed using two-way ANOVA (*, P < 0.05).
FIG 5
FIG 5
Histopathological lesions in wild-type and JH knockout gnotobiotic piglets experimentally infected with HEV. (A) Histopathological lesions in the liver, including lymphoplasmacytic hepatitis and hepatocellular necrosis. Lymphoplasmacytic hepatitis was scored as follows: 0, no inflammation; 1, 1 to 2 focal lymphoplasmacytic infiltrates/10 hepatic lobules; 2, 3 to 5 focal infiltrates/10 hepatic lobules; 3, 6 to 10 focal infiltrates/10 hepatic lobules; and 4, >10 focal infiltrates/10 hepatic lobules. Hepatocellular necrosis was characterized by the presence of individual hepatocytes with an eosinophilic cytoplasm with or without fragmented or absent nuclei. (B) Liver/body weight ratio. The liver weights were evaluated as a ratio of the overall body weight. Asterisks indicate statistical significance as determined by two-way ANOVA (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
FIG 6
FIG 6
Frequencies of IFN-γ and IL-4 intracellular cytokines in mononuclear cells isolated from spleen and mesenteric lymph node tissues and PBMCs from the peripheral blood of wild-type and JH knockout piglets experimentally infected with HEV. PBMCs and MNCs were collected from each piglet at 28 dpi at necropsy. Cells were gated on CD3 and stained for extracellular and intracellular markers CD4+ and IFN-γ and IL-4, respectively, after stimulation with HEV-specific capsid protein. The mean frequencies of IFN-γ (A), IL-4 (B), and CD4+ (C) are indicated and expressed as means ± the SEM. All frequencies were determined by flow cytometry with 100,000 events per sample (*, P < 0.05).
FIG 7
FIG 7
Frequencies of TGF-β and IL-10 intracellular cytokines in mononuclear cells isolated from spleen and mesenteric lymph node tissues and PBMCs from the peripheral blood of wild-type and JH knockout piglets experimentally infected with HEV. PBMCs and MNCs were collected from each piglet at 28 dpi at necropsy. Cells were gated on CD3 and stained for extracellular and intracellular markers CD4+, CD25+, Foxp3, and TGF-β and IL-10, respectively, after stimulation with HEV-specific capsid protein. The mean frequencies of CD25+ Foxp3+ (A), CD25 Foxp3+ (B), TGF-β (C), and IL-10 (D) are indicated and expressed as means ± the SEM. All frequencies were determined by flow cytometry with 100,000 events per sample.

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