Byung-Joo Park

Hepatitis E virus infections in humans and animals

Hepatitis E has traditionally been considered an endemic disease of developing countries. It generally spreads through contaminated water. However, seroprevalence studies have shown that hepatitis E virus (HEV) infections are not uncommon in industrialized countries. In addition, the number of autochthonous hepatitis E cases in these countries is increasing. Most HEV infections in developed countries can be traced to the ingestion of contaminated raw or undercooked pork meat or sausages. Several animal species, including pigs, are known reservoirs of HEV that transmit the virus to humans. HEVs are now recognized as an emerging zoonotic agent. In this review, we describe the general characteristics of HEVs isolated from humans and animals, the risk factors for human HEV infection, and the current status of human vaccine development.

Hepatitis E virus as an emerging zoonotic pathogen

Hepatitis E outbreaks are a serious public health concern in developing countries. The disease causes acute infections, primarily in young adults. The mortality rate is approximately 2%; however, it can exceed 20% in pregnant women in some regions in India. The causative agent, hepatitis E virus (HEV), has been isolated from several animal species, including pigs. HEV genotypes 3 and 4 have been isolated from both humans and animals, and are recognized as zoonotic pathogens. Seroprevalence studies in animals and humans indirectly suggest that HEV infections occur worldwide. The virus is primarily transmitted to humans via undercooked animal meats in developed countries. Moreover, transfusion- and transplantation-mediated HEV infections have recently been reported. This review summarizes the general characteristics of hepatitis E, HEV infection status in animals and humans, the zoonotic transmission modes of HEV, and HEV vaccine development status.

Prevalence and genetic features of rabbit hepatitis E virus in Korea

Hepatitis caused by hepatitis E virus (HEV) is a public health concern worldwide. HEV strains have been isolated from several animal species, some of which induce zoonosis. Recently, the isolation of HEV from rabbits was reported. Here, the partial capsid gene (320 bp) of HEV was detected in rabbit feces via reverse transcriptase‐polymerase chain reaction (RT‐PCR). Rabbit HEV was found in two of six rabbit farms and 17 of 264 rabbit fecal samples (6.4%). A phylogenetic analysis of the partial capsid gene classified the 17 HEV isolates into the putative rabbit HEV clade. A full genomic sequence, KOR‐Rb‐1, was obtained from one rabbit HEV isolate by 5′ and 3′ rapid amplification of cDNA ends‐PCR and RT‐PCR, and comprised 7275 bp excluding the 3′ poly(A) tail. It shared 77.5‐86.8%, 86.6%, and 80.2‐84.3% nucleotide identities with rabbit HEV isolates from China, the US, and France, respectively. It also shared 72.3‐73.0%, 71.4%, 76.7‐78.3%, 72.8‐73.3%, and 47.1‐47.2% nucleotide identities with representative strains of HEV‐1, HEV‐2, HEV‐3, HEV‐4, and avian HEV, respectively. A full‐genome phylogenetic analysis classified KOR‐Rb‐1 into the provisional rabbit HEV clade. This isolate could be used to study the pathogenesis and zoonotic potential of rabbit HEV.

Analysis of cytokine production in a newly developed canine tracheal epithelial cell line infected with H3N2 canine influenza virus

The Madin-Darby canine kidney (MDCK) cell line is typically used to analyze pathological features after canine influenza virus (CIV) infection. However, MDCK cells are not the ideal cell type, because they are kidney epithelial cells. Therefore, we generated an immortalized canine tracheal epithelial cell line, KU-CBE, to more reliably study immune responses to CIV infection in the respiratory tract. KU-CBE cells expressed the influenza virus receptor, α-2,3-sialic acid (SA), but not α-2,6-SA. KU-CBE and MDCK cells infected with H3N2 CIV demonstrated comparable virus growth kinetics. Gene expression levels of interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor (TNF)-α, and interferon (IFN)-β were estimated in both KU-CBE and MDCK cells infected with CIV by real-time reverse transcription polymerase chain reaction (qRT-PCR). Of these cytokines, IL-4, IL-10, TNF-α, and IFN-β mRNAs were detected in both cell lines. Gene expression of IL-4, IL-10, and TNF-α was not significantly different in the two cell lines. However, MDCK cells exhibited a significantly higher level of IFN-β mRNA than KU-CBE cells at 18 h post infection. Additionally, the protein concentrations of these four cytokines were determined by enzyme-linked immunosorbent assay (ELISA) using cell culture supernatants obtained from the two CIV-infected cell lines. MDCK cells produced significantly higher amounts of IL-4 and IFN-β than KU-CBE cells. However, KU-CBE cells produced a significantly higher amount of TNF-α than MDCK cells. These data indicated that the newly developed canine tracheal epithelial cells exhibited different cytokine production patterns compared to MDCK cells when infected with CIV. Inflammation of the respiratory tract of dogs induced by CIV infection may be attributed to the elevated expression level of TNF-α in canine tracheal epithelial cells.

Isolation of hepatitis E virus genotype 4 from patients with acute cryptogenic hepatitis in Korea

BACKGROUND Autochthonous hepatitis E occurs sporadically in developed countries. The consumption of undercooked pork containing hepatitis E virus genotype 3 (HEV-3) or 4 (HEV-4) is the major risk factor for infection. The serological diagnostic kits currently used in hospitals sometimes produce false-negative or -positive results. Therefore, detection of both HEV RNA and antibodies to the virus is required for confirmative diagnosis of hepatitis E. OBJECTIVES We aimed to detect HEV in serum samples from patients with cryptogenic hepatitis and to determine the origin of HEV. STUDY DESIGN A nested polymerase chain reaction (PCR) method was developed for detection of HEV-3 and HEV-4 in patients with hepatitis. A total of 23 serum samples, deposited in 2006-2012, from patients with acute cryptogenic hepatitis who were serologically negative for hepatitis A, B, C, and E were examined using this method. The amplified PCR products were genetically analyzed. RESULTS Four HEV-4 isolates were detected from the 23 serum samples. Phylogenetic analysis indicated that three of the four isolates were closely related to HEV-4 isolates found in pigs in Korea and in patients with hepatitis E in Japan. CONCLUSIONS The newly developed nested PCR method was useful for detection of HEV in patients with cryptogenic hepatitis. The close relationship between the human HEV-4 isolates identified in this study and swine isolates implied that zoonotic transmission of HEV might be a source of infection in patients with hepatitis.

Evidence of hepatitis E virus infection in specific pathogen-free rabbits in Korea

Rabbits are considered a new natural reservoir of hepatitis E virus (HEV). In this study, HEV infection was verified by the detection of partial genomic sequence of HEV and anti-HEV antibodies in specific pathogen-free (SPF) rabbits. HEV RNA was found in 6.4% serum and 13.5% fecal samples from 126 SPF rabbits. Anti-HEV antibodies were also detected in 4.0% of the SPF rabbits. HEV genetic sequences isolated from the rabbits were clustered into a rabbit HEV clade with other rabbit HEV isolates; they were found to be most closely related with a rabbit HEV sequence previously reported in Korea. Therefore, HEV infection should be diagnosed before conducting experiments involving SPF rabbits.

Adverse fetal outcomes in pregnant rabbits experimentally infected with rabbit hepatitis E virus

Hepatitis E virus (HEV) causes severe hepatitis in pregnant women, with associated poor fetal outcomes. To study HEV viral pathogenesis, pregnant rabbits were infected with low- and high-dose rabbit HEV at 2 weeks gestation. HEV was identified in the serum, feces, and liver tissue of infected rabbits, and dose-dependent fetal mortality rates ranging from 67% to 80% were observed. The aspartate transaminase (AST)/alanine transaminase ratio was significantly higher (P < 0.01) in high-dose infected rabbits than low-dose infected and negative control rabbits 14 days post infection (dpi). Tumor necrosis factor-α (TNF-α) was significantly higher in low-dose (P < 0.01) and high-dose infected rabbits (P < 0.001) than in negative controls 7 dpi. High-dose HEV-infected rabbits produced significantly more interferon-γ (IFN-γ; P < 0.05) than negative control rabbits at 7 and 14 dpi. High levels of AST, TNF-α, and IFN-γ may substantially influence adverse fetal outcomes in pregnant rabbits infected with high-dose HEV.

Experimental evidence of hepatitis A virus infection in pigs

Hepatitis A virus (HAV) is the leading cause of acute viral hepatitis worldwide, with HAV infection being restricted to humans and nonhuman primates. In this study, HAV infection status was serologically determined in domestic pigs and experimental infections of HAV were attempted to verify HAV infectivity in pigs. Antibodies specific to HAV or HAV‐like agents were detected in 3.5% of serum samples collected from pigs in swine farms. When the pigs were infected intravenously with 2 × 105 50% tissue culture infectious dose (TCID50) of HAV, shedding of the virus in feces, viremia, and seroconversion were detected. In pigs orally infected with the same quantity of HAV, viral shedding was detected only in feces. HAV genomic RNA was detected in the liver and bile of intravenously infected pigs, but only in the bile of orally infected pigs. In further experiments, pigs were intravenously infected with 6 × 105 TCID50 of HAV. Shedding of HAV in feces, along with viremia and seroconversion, were confirmed in infected pigs but not in sentinel pigs. HAV genomic RNA was detected in the liver, bile, spleen, lymph node, and kidney of the infected pigs. HAV antigenomic RNA was detected in the spleen of one HAV‐infected pig, suggesting HAV replication in splenic cells. Infiltration of inflammatory cells was observed in the livers of infected pigs but not in controls. This is the first experimental evidence to demonstrate that human HAV strains can infect pigs. J. Med. Virol. 88:631–638, 2016.

Detection of hepatitis E virus genotypes 3 and 4 in pig farms in Korea

Zoonotic transmission of hepatitis E virus (HEV) is mostly mediated by HEV-3 and HEV-4 genotypes, and domestic pigs are an important reservoir of these genotypes. A survey of 14 pig farms in Korea revealed HEV RNA in 30 of 148 (20.3%) fecal samples. HEV-3a and HEV-4c subtypes were identified in five pig farms (35.7%) and two pig farms (14.3%), respectively. Phylogenetic analysis indicated that the isolated HEV strains were closely related to previously reported zoonotic strains in Korea. The results of the genetic analysis partially explain the possible source of the zoonotic transmission of HEV to humans in Korea.