Development and evaluation of a time-saving RT-qRPA method for the detection of genotype 4 HEV presence in raw pork liver.

Hepatitis E virus (HEV) is a zoonotic pathogen spreading worldwide. Pig was known as its first and main animal reservoir. In China, pork consumption is very large and the risk of potential HEV contamination should not be underestimated. The present study aims to develop a quantitative real-time reverse transcription combining recombinase polymerase amplification assay (RT-qRPA) for the rapid detection of HEV RNA presence in raw pork liver on the Jinzhou markets in China. Methods: the specific primers and probes for RT-qRPA assay were designed targeting the ORF2/3 conserved region in genotype 4 swine HEV isolate (accession no. DQ279091.2) according to the TwistDx manual instructions. The specificity, sensitivity and reproducibility evaluations of the RT-qRPA method were subsequently conducted in assessing agreement with the standard RT-qPCR method. Results: the qRPA method step exhibited the obvious time-saving advantage which worked under the isothermal condition at 39 °C within about 30 min to complete the run while the compared standard qPCR method in the same cycle took almost 60 min to do. Both methods could exclusively detect the HEV genome equivalents from the quantified HEV-VLPs spiked samples. And both methods shared the same limit of detection (LOD) that was estimated at 1.25 × 103 genome equivalents copies/g spiked sample by the probit analysis. The recovery rate of HEV-VLPs reached a range of 9.56-14.65% by the RT-qRPA method which was higher than that of 1.34-2.34% by the standard RT-qPCR method. The detected HEV RNA positive rate in the field was 1.8% (1 out of 55) by both methods under Cohen's kappa statistic accessing with perfect agreement (κ = 1.00, p < 0.0005). The viral load in positive sample detected by the RT-qRPA method was estimated at 2.2125 × 105 genome copies/g pork liver sample. Conclusions, the present reported RT-qRPA method mainly targeting genotype 4 HEV is a rapid and reliable method. Its time-saving quality offers a promising for the development of a portable tool used in the routine monitoring of HEV contamination in the field.

Isolation and identification of porcine epidemic diarrhea virus strain LN-2015-1 and its S gene mutation analysis / 中国兽医学报

In order to understand the porcine epidemic diarrhea virus (PEDV) origin and variant characteristics in Liaoning province,diagnosed by PCR,separated by Vero cell,and identified by cell pathological observation,RT-PCR and S gene sequence analysis,1 PEDV strains (LN-2015-1) was successfully isolated from a pig farm of Liaoning province.Analysis of S gene sequence showed that compared withCV777 strain,there were the longest 9 bp insertion,6 bp deletion and 13 bp continuous mutation in addition to point mutation.There also were the longest 3 AA insert,2 AA deletion,and 3 AA or more continuous mutation.The epitope analysis showed that there were 16AA mutations in the 5 epitope regions.Homology analysis show that it had the highest sequence similarity of 99.2％ with HB-HA2015 strain,higher sequence similarity of 98.5％-98.8％ with the domestic and foreign representative strains isolated since 2010,and lower sequence similarity of 93.2％-95.6％ with the traditional strain isolated before 2010;the phylogenetic analysis showed that LN-2015-1 was clustered into the same group with home and abroad variation strain in recent years,and formed a small subgroup with HB-HA2015 at the same time.The evolutionary distance was far from the traditional strains.

Effects of temperature and pH on the growth of H1N1 subtype of influenza A virus by surface-enhanced Raman spectroscopy / 生物工程学报

Surface enhanced Raman spectroscopy technology (SERS), using gold nanoparticles as a base, was developed for rapid and sensitive detection of virus strains. SERS can be used as a rapid and reliable method to distinguish the titers of viral replication. In the present study, we characterized H1N1 subtypes of influenza A virus strains in different conditions of pH or temperatures, while we analyzed data from SERS technology using gold nanoparticles as a base and cell cultures were employed to further confirm the data from virus strains. Origin8.0 was used to collect Raman spectra, smooth and homogenize data, and to contrast spectra. Our results indicated that the peaks of different virus strains in optimal environmental conditions (T=37 ℃/pH=7.2) reached ≥3 000. This criterion was verified by subsequent virological method. The present data indicate that the established SERS protocol can be used as a rapid and reliable method to distinguish the replication rate of virus, which can be further used in clinical samples.

Design and evaluation of a recombinant multi-epitope-based ELISA for the serological surveillance of HEV infection in northern China.

In this study, seven recombinant epitope peptides from within the ORF2 protein of the local genotype 4 swine hepatitis E virus (HEV) DQ strain were designed and analyzed. Then, a new multi-epitope-based ELISA was established. In comparison with a commercial kit, this test exhibited good specificity and sensitivity for anti-HEV genotype 4. Subsequently, this test was applied for analyzing serum samples from either swine herds or human populations in northern China. The overall seroprevalence rate of anti-HEV IgG reached up to 40.4% for swine and 8.1% for humans. A statistical difference was observed for humans in rural and urban areas, with a higher prevalence for people living in rural than urban areas. Moreover, sequencing confirmed that all RNA-positive samples belonged to genotype 4.