Assessment of the cross-protective capability of recombinant capsid proteins derived from pig, rat, and avian hepatitis E viruses (HEV) against challenge with a genotype 3 HEV in pigs

Hepatitis E virus (HEV), the causative agent of hepatitis E, is primarily transmitted via the fecal-oral route through contaminated water supplies, although many sporadic cases of hepatitis E are transmitted zoonotically via direct contact with infected animals or consumption of contaminated animal meats. Genotypes 3 and 4 HEV are zoonotic and infect humans and other animal species, whereas genotypes 1 and 2 HEV are restricted to humans. There exists a single serotype of HEV, although the cross-protective ability among the animal HEV strains is unknown. Thus, in this study we expressed and characterized N-terminal truncated ORF2 capsid antigens derived from swine, rat, and avian HEV strains and evaluated their cross-protective ability in a pig challenge model. Thirty, specific-pathogen-free, pigs were divided into 5 groups of 6 pigs each, and each group of pigs were vaccinated with 200 µg of swine HEV, rat HEV, or avian HEV ORF2 antigen or PBS buffer (2 groups) as positive and negative control groups. After a booster dose immunization at 2 weeks post-vaccination, the vaccinated animals all seroconverted to IgG anti-HEV. At 4 weeks post-vaccination, the animals were intravenously challenged with a genotype 3 mammalian HEV, and necropsied at 4 weeks post-challenge. Viremia, fecal virus shedding, and liver histological lesions were compared to assess the protective and cross-protective abilities of these antigens against HEV challenge in pigs. The results indicated that pigs vaccinated with truncated recombinant capsid antigens derived from three animal strains of HEV induced a strong IgG anti-HEV response in vaccinated pigs, but these antigens confer only partial cross-protection against a genotype 3 mammalian HEV. The results have important implications for the efficacy of current vaccines and for future vaccine development, especially against the novel zoonotic animal strains of HEV

Rescue of a genotype 4 human hepatitis E virus from cloned cDNA and characterization of intergenotypic chimeric viruses in cultured human liver cells and in pigs

Hepatitis E virus (HEV) is an important but extremely understudied human pathogen. Genotypes 1 and 2 are restricted to humans, whereas genotypes 3 and 4 are zoonotic, infecting both humans and pigs. This report describes, for the first time, the successful rescue of infectious HEV in vitro and in vivo from cloned cDNA of a genotype 4 human HEV (strain TW6196E). The complete genomic sequence of the TW6196E virus was determined and a full-length cDNA clone (pHEV-4TW) was assembled. Capped RNA transcripts from the pHEV-4TW clone were replication competent in Huh7 cells and infectious in HepG2/C3A cells. Pigs inoculated intrahepatically with capped RNA transcripts from pHEV-4TW developed an active infection, as evidenced by faecal virus shedding and seroconversion, indicating the successful rescue of infectious genotype 4 HEV and cross-species infection of pigs by a genotype 4 human HEV. To demonstrate the utility of the genotype 4 HEV infectious clone and to evaluate the potential viral determinant(s) for species tropism, four intergenotypic chimeric clones were constructed by swapping various genomic regions between genotypes 1 and 4, and genotypes 1 and 3. All four chimeric clones were replication competent in Huh7 cells, but only the two chimeras with sequences swapped between genotypes 1 and 4 human HEVs produced viruses capable of infecting HepG2/C3A cells. None of the four chimeras was able to establish a robust infection in pigs. The availability of a genotype 4 HEV infectious clone affords an opportunity to delineate the molecular mechanisms of HEV cross-species infection in the future

Hepatitis E Virus in Rabbits, Virginia, USA

We identified hepatitis E virus (HEV) in rabbits in Virginia, USA. HEV RNA was detected in 14 (16%) of 85 serum samples and 13 (15%) of 85 fecal samples. Antibodies against HEV were detected in 31 (36%) of 85 serum samples. Sequence analyses showed that HEV from rabbits is closely related to genotype 3

Rescue of infectious chimeric viruses from shuffled infectious clones and <i>in vitro</i> growth kinetics of the shuffled chimeric viruses.

<p>Immunofluorescence assay (IFA) (<b>Panel A</b>) was performed with anti-PRRSV N protein monoclonal antibody (SDOW17) to confirm that the chimeric viruses were successfully rescued in MARC-145 cells infected with the supernatant of BHK-21 cells transfected with eight individual clones generated by DNA shuffling (GP4TS14, GP4TS19, GP4TS29, MTS1, MTS5, MTS8, MTS11 and MTS57). Parental backbone strain VR2385 and mock infection were included as positive and negative controls, respectively. The growth kinetics of GP4-shuffled chimeric viruses (<b>Panel B</b>) and M-shuffled chimeric viruses (<b>Panel C</b>) in MARC-145 cells were determined by measuring the infectious viral titers (TCID₅₀/ml) at indicated time points post-infection using the microtitration infectivity assay. The experiments were done in triplicate, and the bars indicated standard errors.</p

Nucleotide sequence alignment of the five M-shuffled chimeric viruses and their parents by clustalW method.

<p>The sequence of the backbone virus VR2385 was shown on top, and only differences were indicated for other viruses.</p

Kinetics of neutralizing antibodies in serum samples collected at 28, 35 and 43 days post-inoculation (DPI) from pigs experimentally infected with chimeric viruses GP4TS14 and MTS57 or with the backbone virus VR2385, respectively.

<p>The NA titers in serum samples at 28, 35 and 43 DPI from pigs experimentally infected with chimeras GP4TS14 and MTS57 or with VR2385 were examined by an <i>in vitro</i> serum virus neutralization test against four parental virus strains VR2385, MN184B, FL-12 and NADC20, respectively (<b>A–E</b>). Each test was performed in triplicate, the average NA titers of 3 or 4 pigs in each group are shown in the figure and the bars indicate standard errors. The asterisk (*) signs indicate a significant difference between the chimeric virus and the parental strain VR2385. One asterisk (*) signs indicate <i>p</i><0.05 and two asterisk (**) signs indicate <i>p</i><0.01. The <i>p</i> values are shown above the asterisk signs.</p

Nucleotide sequence alignment of the three GP4-shuffled chimeric viruses and their parents by clustalW method.

<p>The sequence of the backbone virus VR2385 was shown on top, and only differences were indicated for other viruses.</p

Two phylogenetic trees based on the sequence of GP4 (Panel A) or M (Panel B) genes of selected type 2 PRRSV strains.

<p>The six parental viruses (VR2385 JX044140, VR2430 JX050225, MN184B DQ176020, FL-12 AY545985, JXA1 EF112445, and NADC20 JX069953) used in the DNA shuffling are indicated with boldface in the trees. The phylogenetic trees were constructed using the neighbor-joining method with bootstraps of 100 replicates. The numbers above each branch indicate the bootstrap values (percentage of consensus support in bootstrap).</p

Sequence comparison and analyses of GP4 (Panel A) and M (Panel B) amino acid sequences of chimeric viruses and their parents.

<p>Multiple sequence alignments of GP4 and M amino acid sequences of six parental virus strains and shuffled chimeric viruses were performed. The sequence of the backbone virus VR2385 is shown on top, and only differences for other strains are indicated in the alignment.</p