In silico and in vitro analysis of a rational mutation in gIII signal peptide and its effects on periplasmic expression of rhGH in E. coli.

The secretion efficiency of a heterologous protein in E. coli is mainly dictated by the N-terminal signal peptide fused to the desired protein. In this study, we aimed to select and introduce mutations into the - 1, - 2 and - 3 positions of the gIII signal peptide (originated from filamentous phage fd Gene III) fused to the N-terminus of the human growth hormone (hGH), and study its effect on the secretion efficiency of the recombinant hGH into the periplasmic space of E. coli Top10. Bioinformatics software such as SignalP-5.0 and PrediSi were employed to predict the effects of the mutations on the secretion efficiency of the recombinant hGH. Site-directed mutagenesis was applied to introduce the desired mutations into the C-terminus of the gIII signal peptide. The periplasmic expression and the secretion efficiency of the recombinant hGH using the native and mutant gIII signal peptides were compared in E. coli Top10 under the control of araBAD promoter. Our results from bioinformatics analysis indicated that the mutant gIII signal peptide was more potent than the native one for secretion of the recombinant hGH in E. coli. While our experimental results revealed that the mutation had no effect on hGH secretion. This result points to the importance of experimental validation of bioinformatics predictions.

Distribution of Japanese Encephalitis Virus, Japan and Southeast Asia, 2016-2018.

During 2016-2018, we conducted surveillance for Japanese encephalitis virus (JEV) in mosquitoes and pigs in Japan, Thailand, the Philippines, and Indonesia. Phylogenetic analyses demonstrated that our isolates (genotypes Ia, Ib, III, IV) were related to JEV isolates obtained from the same regions many years ago. Indigenous JEV strains persist in Asia.

Virulence of Japanese Encephalitis Virus Genotypes I and III, Taiwan.

The virulence of genotype I (GI) Japanese encephalitis virus (JEV) is under debate. We investigated differences in the virulence of GI and GIII JEV by calculating asymptomatic ratios based on serologic studies during GI- and GIII-JEV endemic periods. The results suggested equal virulence of GI and GIII JEV among humans.

First Detection and Genomic Characterization of Bovine Norovirus from Yak.

Yak are a unique free-grazing bovine species in high-altitude areas. The objective of this study was to investigate the presence and molecular characteristics of BNoV in yak. A total of 205 diarrheal samples of yak (aged ≤ 3 months) were collected from 10 farms in Sichuan Province, China, from May 2018 to October 2020, and four samples were detected as BNoV-positive with RT-PCR. Moreover, a nearly full-length genome of SMU-YAK-J1 containing three complete ORFs was successfully sequenced. Sequence analysis with only nine genome sequences of the GIII genogroup showed that SMU-YAK-J1 was most closely related with GIII.P2 GIII.4, sharing 90.9% gnomic nucleotide identity, but only shared 71.6-85.9% with other genotypes, which confirmed that SMU-YAK-J1 belongs to genotype GIII.P2 GIII.4. However, compared with the sole genome of GIII.4 in GenBank, the BNoV in this study also exhibited many unique amino acid changes among all the three ORFs, which may represent the unique genetic evolution of BNoV in yak. This study first determined the presence of BNoV in yak, contributing to a better understanding of the prevalence and genetic evolution of BNoV.

Evolutionary history and spatiotemporal dynamic of GIII norovirus: From emergence to classification in four genotypes.

Noroviruses belong to a genetically diverse group of viruses infecting a wide range of mammalian host species, and those detected in cattle and sheep are classified within genogroup III (GIII). The current classification of norovirus in genogroups and genotypes is based on phylogenetic clustering and average distances within and between these phylogenetic clusters; however, the classification studies have been focused mainly on human norovirus, being GIII norovirus relegated. Due to the increasing number of studies on GIII norovirus, the need of an updated and extensive classification is evident. The aim of this study was to update the classification of norovirus within GIII, to describe the emergence of a circulating recombinant strain, and to reconstruct the evolutionary history of this genogroup. Two P-types (GIII.P1-2) and four genotypes (GIII.1-4) were described. For the genogroup GIII, the evolutionary rate estimated was 2.78E-3 s/s/y (95%HPD, 1.79E-3 s/s/y-3.78E-3 s/s/y), and the tMRCA was estimated around 1500 (95%HPD, 1247-1688). Despite the long history of this genogroup, the genotypes detected at present emerged in the last 100 years. Interestingly, most of the recombinant GIII.2P[1] strains detected worldwide were originated from a single recombination event and this recombinant strain was later dispersed through the world. Finally, our results indicate that a scenario of genotypes replacement through the time is highly probable.

High Genetic Diversity of Porcine Sapovirus From Diarrheic Piglets in Yunnan Province, China.

As one of the most important enteric viruses, sapovirus (SaV) can infect humans and a variety of animals. Until now, 19 SaV genogroups have been identified, among which 4 from human (GI, GII, GIV, and GV) and 8 from swine (GIII, GV-GXI). Porcine sapovirus (PoSaV) GIII has been prevalent in China; however, the status of PoSaV infection in Yunnan province remains unknown. In this study, 202 fecal samples were collected from piglets associated with outbreaks of acute diarrhea in Yunnan between January and May 2020. PoSaV detection revealed that the total PoSaV infection rate in Yunnan was 35.2%, with 21 PoSaV strains determined and phylogenetically analyzed. The phylogenetic tree analyses demonstrated that twenty PoSaV strains belonged to GIII and fell into five genotypes, whereas one PoSaV strain (YNQB) belonged to GV. Sequence alignments revealed deletions in VP2 region in 10 of the 20 GIII strains, as well as deletions and insertions in VP1 region of the GV strain (YNQB). Furthermore, genomic recombination analyses showed that two GIII strains (YNAN and YNJD) were recombinants, closely related to reference sequences MK965898 and LC215880, MK965898 and FJ387164, respectively. In summary, PoSaV-GIII strains were identified in Yunnan in 2020, and for the first time, a PoSaV-GV strain was identified from China, whereas the comprehensive analyses illustrated high genetic diversity of Yunnan PoSaV strains. This study may shed new light on the current PoSaV infections in Yunnan and pave the way toward further control of the PoSaV infections in China.

Acid and Alkaline Phosphatases Specific activities and Expression as Biomarkers in Brain Tumor Grading and Correlation of Anaplastic Oligodendroglioma G-III to Meningiomas G-I among Brain Tumors and their primary culture.

BACKGROUND: Acid Phosphatase (ACP) and Alkaline Phosphatases (ALP) are hydrolases that remove phosphate groups from protein and nucleic acid respectively for regulation of cell function from ACP as lysosomal defence function and ALP membrane-bound as a barrier of the cell. The ACP and ALP-specific activities of Meningiomas (n = 75) and gliomas (n = 81) were compared among brain tumors, normal brain, and derived primary cell culture. METHODS: Total Protein and Phosphatases assays estimated by Spectrophotometer and Native PAGE Gel Electrophoresis. Brain tumor and primary explant lysosome studies were performed with an electron microscope. RESULTS: Average ACP specific activity exhibited 9.32617 ± 4.1144 for meningiomas (n = 55) and 5.91 ± 5.8305 for gliomas (n = 60) respectively as compared to normal brain 7.104 ± 1.33 (n = 120) nm/min/mg of protein. Average ALP exhibited 37.1862 ± 39.91 (n = 36) for meningiomas and 5.91 ± 5.83 (n = 60) for gliomas respectively as compared to normal brain (n = 117) 2.463 ± 1.01 nm/min/mg of protein. ACP and ALP exhibited higher activities for meningiomas but not for gliomas as compared to normal brain, in contrast, both expressed more activities in the majority of glioma cell lines and lower in meningioma cell lines. Interestingly gliomas exhibited similar average specific activities for ACP and ALP. While GBM IV exhibits lower ALP activities due to cell migration and higher ACP activity correlate too many storage lysosomes from Electron microscopic observation as compared to meningiomas. CONCLUSIONS: Higher ALP activities can be surrogate markers from meningiomas G-I, G-II to G-III respectively. However meningiomas G-III are similar to gliomas excluding Anaplastic Oligodendroglioma G- III which is similar to Meningiomas G-I even for cells growth patterns. Therefore, an ALP level in meningiomas indicates complementary diagnosis as antibody-ALP conjugates with anticancer drugs for efficiency in targeting brain tumor reduction.

Prokaryotic High-Level Expression System in Producing Adhesin Recombinant Protein E of Nontypeable Haemophilus influenzae.

BACKGROUND: Adhesion protein E (PE) of Haemophilus influenzae is a 16 - 18 kDa protein with 160 amino acids which causes adhesion to epithelial cells and acts as a major factor in pathogenesis. OBJECTIVES: In this study, we performed cloning, expression and purification of PE as a candidate antigen for vaccine design upon further study. MATERIALS AND METHODS: At first, the pe gene of NTHi ATCC 49766 strain (483 bp) was amplified by PCR. Then, to sequence the resulted amplicon, it was cloned into TA vector (pTZ57R/T). In the next step, the sequenced gene was sub-cloned in pBAD/gIII A vector and transformed into competent Escherichia coli TOP10. For overexpression, the recombinant bacteria were grown in broth medium containing arabinose and the recombinant protein was purified using metal affinity chromatography (Ni-nitrilotriacetic acid) (Ni-NTA agarose). Finally, the protein was detected using sodium dodecyl sulfate polyacrylamide gel electrophores (SDS-PAG) and confirmed by western blotting. RESULTS: The cloned gene was confirmed by PCR, restriction digestion and sequencing. The sequenced gene was searched for homology in GenBank and 99% similarity was found to the already deposited genes in GenBank. Then we obtained PE using Ni-NTA agarose with up to 7 mg/mL concentration. CONCLUSIONS: The pe gene was successfully cloned and confirmed by sequencing. Finally, PE was obtained with high concentration. Due to high homology and similarity among the pe gene from NTHi ATCC 49766 and other NTHi strains in GenBank, we believe that the protein is a universal antigen to be used as a vaccine design candidate and further studies to evaluate its immunogenicity is underway.

Cross-protection elicited by primary and booster vaccinations against Japanese encephalitis: a two-year follow-up study.

BACKGROUND: The inactivated Vero cell-derived vaccine (JE-VC, IXIARO) has replaced the traditional mouse brain-derived preparations (JE-MB) in travelers' vaccinations against Japanese encephalitis. We showed recently that a single JE-VC dose efficiently boosts immunity in JE-MB-primed vaccinees, and that JE-VC elicits cross-protective immunity against non-vaccine genotypes, including the emerging genotype I. While these studies only provided short-term data, the present investigation evaluates the longevity of seroprotection in the same volunteers. METHODS: The study comprised 48 travelers who had received (1) JE-VC primary series, (2) JE-MB primary series followed by a single JE-VC booster dose, or (3) JE-MB primary series and a single JE-MB booster dose. Serum samples were collected two years after the last vaccine dose, and evaluated with the plaque-reduction neutralization test against seven Japanese encephalitis virus strains representing genotypes I-IV. PRNT50 titers ≥ 10 were considered protective. RESULTS: Two years after the primary series with JE-VC, 87-93% of the vaccinees proved to be cross-protected against test strains representing genotypes II-IV and 73% against those of genotype I. After a single homologous or heterologous booster dose to JE-MB-primed subjects, the two-year seroprotection rates against genotype I-IV strains were 89-100%. CONCLUSIONS: After JE-VC primary series, seroprotection appeared to wane first against genotype I. The first booster should not be delayed beyond two years. In JE-MB-primed subjects, a single JE-VC booster provided cross-protective immunity against genotype I-IV strains in almost all vaccinees, suggesting an interval of two years or even longer for the second booster. These data further support the use of a single JE-VC dose for boosting JE-MB immunity.