Browsing by Author "Cao, Dianjun"
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- Comparison of Real-Time Reverse Transcriptase PCR Assays for Detection of Swine Hepatitis E Virus in Fecal SamplesGerber, Priscilla F.; Xiao, Chao-Ting; Cao, Dianjun; Meng, Xiang-Jin; Opriessnig, Tanja (American Society for Microbiology, 2014-01-15)Hepatitis E virus (HEV) is a major cause of acute viral hepatitis in people in many developing countries and is also endemic in many industrialized countries. Mammalian HEV (mHEV) isolates can be divided into at least four recognized major genotypes. Several nucleic acid amplification techniques have been developed for mHEV detection, with great differences in sensitivity. The aim of this study was to compare the performances of two singleplex real-time reverse transcriptase (RT) PCR assays for broad detection of all four mHEV genotypes (assays A and B) and two duplex real-time RT-PCR assays for detection and differentiation of mHEV genotypes 3 and 4 (assays C and D). RNAs extracted from 28 fecal samples from pigs experimentally inoculated with HEV genotype 3 and 186 fecal samples from commercial pigs with unknown HEV exposure were tested by all four assays. In experimental samples, HEV RNA was detected in 96.4% (assay A), 39.2% (assay B), 14.2% (assay C), and 0% (assay D) of the samples. In field samples with unknown HEV exposure, HEV RNA was detected in 67.2% (assay A), 36.4% (assay B), 1.1% (assay C), and 0.5% (assay D) of the samples. The assays showed overall poor agreement (kappa = 0.19 to 0.03), with differences in detection rates between assays (P < 0.01). Assays A and B, which broadly detect HEV genotypes 1 to 4, had significantly higher detection rates for HEV RNA than the duplex assays C and D, which were both designed to detect and differentiate between HEV genotypes 3 and 4.
- Development and evaluation of two subunit vaccine candidates containing antigens of hepatitis E virus, rotavirus, and astrovirusXia, Ming; Wei, Chao; Wang, Leyi; Cao, Dianjun; Meng, Xiang-Jin; Jiang, Xi; Tan, Ming (Springer Nature, 2016-05-19)Hepatitis E virus (HEV), rotavirus (RV), and astrovirus (AstV) are important pathogens that transmit through a common fecal-oral route, causing hepatitis (HEV) and gastroenteritis (RV and AstV) respectively in humans. In this study, we developed and evaluated two subunit vaccine candidates that consisted of the same protruding or spike protein antigens of the three viruses in two formats, a fusion of the three antigens into one molecule (fused vaccine) vs. a mixture of the three free antigens together (mixed vaccine). Both vaccines were easily made via E. coli expression system. Mouse immunization experiments showed that the fused vaccine elicited significantly higher antibody responses against the three viral antigens than those induced by the mixed vaccine. In addition, the mouse post-immune antisera of the fused vaccine revealed significantly higher neutralizing titers against HEV infection in cell culture, as well as significantly higher 50% blocking titers (BT50) against RV VP8-HBGA receptor interactions than those of the post-immune antisera after immunization of the mixed vaccine. Thus, the fused vaccine is a promising trivalent vaccine candidate against HEV, RV, and AstV, which is worth for further development.
- Increased frequency of porcine epidemic diarrhea virus shedding and lesions in suckling pigs compared to nursery pigs and protective immunity in nursery pigs after homologous re-challengeGerber, Priscilla F.; Xiao, Chao-Ting; Lager, Kelly; Crawford, Kimberly; Kulshreshtha, Vikas; Cao, Dianjun; Meng, Xiang-Jin; Opriessnig, Tanja (2016-11-21)Porcine epidemic diarrhea virus (PEDV) causes enteric disease in pigs and spreads rapidly after entering naïve pig populations. The objectives were to (1) compare the disease course following inoculation with PEDV isolate US/Colorado/2013 in naïve 10 day and 8 week-old pigs, and (2) contrast the naïve response to homologous challenge in 8 week-old pigs. Pigs were randomly assigned into group 1 (n = 40, no PEDV exposure), group 2 (n = 43, PEDV inoculation at 10 days of age) and group 3 (n = 48, PEDV inoculation at 8 weeks of age). Thirty-three group 2 pigs received a homologous challenge at 8 weeks of age. Following primary or secondary inoculation, 3–10 pigs were euthanized at days post-inoculation (dpi) 1, 2, 3, 7 or 14. Clinical signs were more pronounced in 10 day-old pigs compared to 8 week-old pigs at dpi 2 and 3, a higher number of 10 day-old pigs shed PEDV RNA in feces compared to 8 week-old pigs. Typical severe atrophic enteritis of PEDV infection was observed at dpi 3 in both age groups, and at dpi 4 and 14 fecal shedding patterns were also similar. While both age groups had seroconverted to PEDV by dpi 14, IgG levels were higher in 8 week-old pigs. PEDV IgA antibodies were detected in feces of approximately 50% of the pigs at dpi 44. In homologous challenged pigs, no clinical signs or lesions were found, and PEDV fecal shedding was restricted to less than 10% of the pigs indicating the existence of homologous protection 44 days after initial PEDV exposure.
- Molecular epidemiology of hepatitis E virus infections in Shanghai, ChinaZhu, Yumin; Si, Fusheng; Cao, Dianjun; Yu, Xiaoming; Yu, Ruisong; Dong, Shijuan; Huang, Fenfen; Zhang, Yuanshu; Li, Zhen (2011-12-15)Background Hepatitis E virus (HEV) causes acute or fulminant hepatitis in humans and is an important public health concern in many developing countries. China has a high incidence of HEV epidemics, with at least three genotypes (1, 3 and 4) and nine subtypes (1b, 1c, 3b, 4a, 4b, 4d, 4g, 4h and 4i) so far identified. Since genotype 3 and the newly identified subtype 4i have been exclusively limited geographically to Shanghai and its neighboring provinces, the epidemiology of HEV infections within the municipality, a major industrial and commercial center, deserves closer attention. Findings A total of 65 sequences, 60 located within the HEV SH-SW-zs1 genome [GenBank:EF570133], together with five full-length swine and human HEV genomic sequences, all emanating from Shanghai, were retrieved from GenBank. Consistent with the primary role of genotype 4 in China overall, analysis of the sequences revealed this to have been the dominant genotype (58/65) in Shanghai. Six HEV subtypes (3b, 4a, 4b, 4d, 4h and 4i) were also represented. However, although subtype 4a is the dominant subtype throughout China, subtype 4i (29/65) was the most prevalent subtype among the Shanghai sequences, followed by subtypes 4d (10/65) and 4h (9/65). Subtypes 4h, 4i and 4d were found in both swine and humans, whereas 4b was found only in swine and subtype 4a only in humans. Conclusions Six different swine and human HEV subtypes have so far been documented in Shanghai. More molecular epidemiological investigations of HEV in swine, and particularly among the human population, should be undertaken.
- A Novel Pathogenic Mammalian Orthoreovirus from Diarrheic Pigs and Swine Blood Meal in the United StatesNarayanappa, Athmaram Thimmasandra; Sooryanarain, Harini; Deventhiran, Jagadeeswaran; Cao, Dianjun; Venkatachalam, Backiyalakshmi Ammayappan; Kambiranda, Devaiah; LeRoith, Tanya; Heffron, C. Lynn; Lindstrom, Nicole; Hall, Karen; Jobst, Peter; Sexton, Cary; Meng, Xiang-Jin; Elankumaran, Subbiah (American Society for Microbiology, 2015-05)Since May 2013, outbreaks of porcine epidemic diarrhea have devastated the U.S. swine industry, causing immense economic losses. Two different swine enteric coronaviruses (porcine epidemic diarrhea virus and Delta coronavirus) have been isolated from the affected swine population. The disease has been reported from at least 32 states of the United States and other countries, including Mexico, Peru, Dominican Republic, Canada, Columbia, Ecuador, and Ukraine, with repeated outbreaks in previously infected herds. Here we report the isolation and characterization of a novel mammalian orthoreovirus 3 (MRV3) from diarrheic feces of piglets from these outbreaks in three states and ring-dried swine blood meal from multiple sources. MRV3 could not be isolated from healthy or pigs that had recovered from epidemic diarrhea from four states. Several MRV3 isolates were obtained from chloroform-extracted pig feces or blood meal in cell cultures or developing chicken embryos. Biological characterization of two representative isolates revealed trypsin resistance and thermostability at 90 degrees C. NextGen sequencing of ultrapurified viruses indicated a strong homology of the S1 segment to mammalian and bat MRV3. Neonatal piglets experimentally infected with these viruses or a chloroform extract of swine blood meal developed severe diarrhea and acute gastroenteritis with 100% mortality within 3 days postinfection. Therefore, the novel porcine MRV3 may contribute to enteric disease along with other swine enteric viruses. The role of MRV3 in the current outbreaks of porcine epidemic diarrhea in the United States remains to be determined, but the pathogenic nature of the virus warrants further investigations on its epidemiology and prevalence. IMPORTANCE Porcine orthoreoviruses causing diarrhea have been reported in China and Korea but not in the United States. We have isolated and characterized two pathogenic reassortant MRV3 isolates from swine fecal samples from porcine epidemic diarrhea outbreaks and ring-dried swine blood meal in the United States. These fecal and blood meal isolates or a chloroform extract of blood meal induced severe diarrhea and mortality in experimentally infected neonatal pigs. Genetic and phylogenetic analyses of two MRV3 isolates revealed that they are identical but differed significantly from nonpathogenic mammalian orthoreoviruses circulating in the United States. The present study provides a platform for immediate development of suitable vaccines and diagnostics to prevent and control porcine orthoreovirus diarrhea.
- Substitution of amino acid residue V1213 in the helicase domain of the genotype 3 hepatitis E virus reduces virus replicationCao, Dianjun; Ni, Yan-Yan; Meng, Xiang-Jin (2018-02-08)Background Genotype 3 hepatitis E virus (HEV) infection is generally associated with mild disease. However, recently eight genotype 3 HEV isolates were identified from patients with severe hepatitis. Importantly, three mutations (S605P, I978V and V1213A) in these genotype 3 isolates were found to be typical of genotype 4 HEV, which is sometime associated with more severe hepatitis. Therefore in this study we seek to determine if these unique mutations contribute to enhanced virus replication and thus potentially severe disease. Methods In the lack of an efficient cell culture system to study the effect of mutations on HEV replication, we developed a genotype 3 HEV replicon with Renilla luciferase (Rluc) as reporter and subsequently used it to construct numerous mutants, including swMu-1 (V1213A), swMu-2 (Q1246H), swMu-3 (V1213A and Q1246H), swMu-4 (S605P and I978V), and swMu-5 (V1213A, S605P and I978V). RNA transcripts from mutant replicons were transfected into Huh7 S10–3 liver cells to measure the effect of mutations on HEV replication efficiency. Results The results showed that the V1213A mutant had the highest reduction in HEV replication efficiency than other mutants. The V1213A and S605P + I978V mutations have a cumulative, if not synergistic, effect on HEV replication. The Q1246H mutant decreased HEV replication compared to the wild-type HEV Rluc replicon but replicated better than the V1213A mutant. The amino acid residue V1213 favors the replication of both genotypes 3 and 4 HEV strains, but not genotype 1 HEV. Conclusion The results suggested that the V1213A mutation reduced HEV replication, but is likely not associated with the reported severe hepatitis caused by genotype 3 HEV isolates containing this mutation.