Evidence of Hepatitis E Virus Breaking Through the Blood–brain Barrier and Replicating in the Central Nervous System

R. Shi; M. H. Soomro; R. She; Y. Yang; T. Wang; Q. Wu; H. Li; W. Hao


J Viral Hepat. 2016;23(11):930-939. 

In This Article

Materials and Methods

Ethics Statement

All animal experiments involved in this study were approved by the Animal Care and Use Committee of China Agricultural University (CAU) (permit number: 20140115-089).

Virus, Inoculation, Animals and Sampling

The HEV strain used for inoculation was genotype 4 swine HEV, which was derived from a swine liver sample (CHN-HB-HD-L2, GenBank accession number KM024042). The HEV-positive liver homogenate with a titre of 6.57 × 108 genome equivalents (GE) per mL was prepared and stored at −86 °C prior to inoculation.

SPF (specific-pathogen free) male Mongolian gerbils with body weights around 50 g were purchased from the Department of Experimental Animal Sciences of Capital Medical University (Beijing, China). Gerbils in the experimental group were inoculated with 0.1 mL of prepared viral homogenate via intraperitoneal injection. Gerbils injected with the same dose of HEV-negative SPF swine liver homogenate served as control group. Six gerbils (labelled as A-F, respectively) were euthanized for necropsy at 0, 7, 14, 21, 28, 42 and 56 days postinoculation (dpi), respectively. Serum was collected for the evaluation of the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (T-BIL). Brain and spinal cord of the gerbils were collected and fixed in neutral 4% paraformaldehyde or 2.5% (v/v) glutaraldehyde-polyoxymethylene solution for histopathological and immunohistochemical observation or TEM analyses, respectively.

ELISA for HEV Antigen Detection

The ELISA for the detection of serum HEV antigen was performed following the manufacturer's instructions (Wantai, Beijing, China).

RNA Extraction and PCR Protocols

Total RNA was extracted from brain and spinal cord tissues with the Ultrapure RNA kit (CWBIO, Beijing, China) according to the instructions of the manufacturer and stored at −86 °C. Primers targeting at the HEV ORF2 region consist of the following sequences: forward externer primer (P1): 5'-AATTATGCYCAGTAYCGRGTTG-3', reverse externer primer (P2): 5'-CCCTTRTCYTGCTGMGCATTCTC-3', forward interner primer (P3): 5'-GTWATGCTYTGCATWCATGGCT-3', reverse interner primer: 5'-AGCCGACGAAATCAATTCTGTC-3' (P4). Reverse transcription targeting on the negative-strand and positive-strand HEV RNA was performed with Primer P1 and Primer P2, respectively, using HiFi-MMLV kit (CWBIO, Beijing, China). The PCR parameters included an initial denaturation at 95 °C for 7 min, followed by 35 cycles of 94 °C for 1 min, 42 °C for 1 min and 72 °C for 2 min, with a final extension step at 72 °C for 10 min. PCR product of 348 bp was expected after the second round. RT-nPCR-positive brain and spinal cord tissues were arranged for real-time PCR to detect the viral load. The detailed protocol is described as Yang et al. 2015.[24]

Immunohistochemical Staining for HEV ORF2, GFAP and ZO-1

Four-micrometre tissue sections of brain and spinal cord were prepared following a standard protocol. Immunohistochemical staining was performed on the paraffin slices of brain and spinal cord following the instruction, which was included in the Histostain™-Plus kit (ZSGB-BIO, Beijing, China). 3,3'-Diaminobenzidine tetrahydrochloride (DAB; ZSGB-BIO, Beijing, China) was applied to these sections for 5 min to visualize the antigen–antibody compound, after which haematoxylin was applied as the background stain. The slices were observed under the light microscope. Positive area density of GFAP and ZO-1 was analysed (Mias, CMIASWIN).

Histopathological Examinations

Optical Microscope. Four-micrometre tissue sections of brain and spinal cord were stained with haematoxylin and eosin for histological evaluation.

Transmission Electron Microscope (TEM). Brain and spinal cord samples fixed in 2.5% (v/v) glutaraldehyde-polyoxymethylene solution were washed and postfixed in 2% osmium tetroxide for 1 h at 4 °C. After dehydration in ascending grades of ethanol, the tissues were embedded in araldite CY212. Ultrathin sections (50–60 nm) were sliced and stained with alkaline and lead citrate uranyl acetate. The sections were observed under a JEM 100CX transmission electron microscope.