Chitosan Treatment of E-11 Cells Modulates Transcription of Nonspecific Immune Genes and Reduces Nodavirus Capsid Protein Gene Expression

This study explores whether crustacean products inhibit viral infections in aquaculture. Chitosan (CHT) was extracted from waste products of Parapenaeus longirostris. Biochemical composition, viscosity measurement, molecular weight, structure and cytotoxicity tests were used to characterize the extracted chitosan. Cultures of E-11 cells derived from snakehead Ophicephalus striatus were inoculated with 106.74 TCID50 of an isolate of betanodavirus genotype RGNNV (redspotted grouper nervous necrosis virus) after being treated with solutions of 0.3% CHT for 1 h at room temperature. The antiviral effect of CHT was assessed by comparing the ability of RGNVV to replicate and produce cytopathic effects on CHT-treated cell cultures. The change in RNA expression levels of the nodavirus capsid protein gene and three mediator genes in infected cells with or without CHT treatment was evaluated by qPCR. Changes in gene expression compared to control groups were monitored at 6, 24, 48 and 71 h post treatment in all target gene transcripts. The CCR3 expression in CHT treated cells showed a significant increase (p < 0.05) until day 3. On the other hand, the expression of TNF-α decreased significantly (p < 0.05) in CHT treated cells throughout the experimental period. Likewise, the expression of the IL-10 gene showed a significant downregulation in CHT treated cells at all time points (p ≤ 0.05). As further evidence of an antiviral effect, CHT treatment of cells produced a reduction in virus load as measured by a reduced expression of the viral capsid gene and the increase in RQ values from 406 ± 1.9 at hour 1 to 695 ± 3.27 at 72 h post inoculation. Statistical analysis showed that the expression of the viral capsid gene was significantly lower in cells treated with chitosan (p ≤ 0.05). These results improve our knowledge about the antiviral activity of this bioactive molecule and highlight its potential use in fish feed industry.

An acute gastroenteritis outbreak associated with breakfast contaminated with norovirus by asymptotic food handler at a kindergarten in Shenzhen, China

Background An outbreak of acute gastroenteritis occurred in a kindergarten located Shenzhen City on March 4, 2018. We were invited to investigate to the risk factors associated with this outbreak. Methods We conducted retrospective cohort-studies on three different groups of subjects in order to figure out the difference of incidence of acute gastroenteritis among subjects of different activities on March 2: group one consisted of people who attended the Lantern festival activities; group two consisted of children and employees who ate breakfast and bread provided by the kindergarten; and groups three consisted of children and employees who did not eat breakfast or bread provided by the kindergarten. Fecal, anal swabs, dishware swabs and hand swabs specimens were collected in the study. Bacteria known to cause acute gastroenteritis were cultured. Viruses associated with acute gastroenteritis were tested using real-time PCR. Capsid gene fragment of 557 bp of norovirus was amplified and sequenced. The phylogenetic tree was constructed with MEGA 7.0 using neighbor-joining method based on capsid gene fragment of norovirus. Results A total of 143 suspected cases were identified in this outbreak. Diarrhea happened more often in adults than in children while emesis and bellyache were more frequently found in children than in adults. Higher AGE incidence was observed in group 2, children and employees who had breakfast in the kindergarten on March 2, as well as in group 3, and among employees who eating bread involved in breakfast provided on March 2. Five anal swab specimens were positive for norovirus. All noroviruses belongs to group II.3 and have an identity more than 99%. Conclusion A chef, as an asymptomatic carrier with norovirus, was the infectious resource in this outbreak. He contaminated breakfast food provided on March 2. Although morning check is implemented in kindergartens of China, employees are often excluded in morning check. Our finding highlights the importance of morning check covering employees and periodical training for cooks.

Genetic Engineering of AAV Capsid Gene for Gene Therapy Application

Adeno-associated virus (AAV) is a promising vector for in vivo gene therapy because of its excellent safety profile and ability to mediate stable gene expression in human subjects. However, there are still numerous challenges that need to be resolved before this gene delivery vehicle is used in clinical applications, such as the inability of AAV to effectively target specific tissues, preexisting neutralizing antibodies in human populations, and a limited AAV packaging capacity. Over the past two decades, much genetic modification work has been performed with the AAV capsid gene, resulting in a large number of variants with modified characteristics, rendering AAV a versatile vector for more efficient gene therapy applications for different genetic diseases.

Genetic recombination in fast-spreading coxsackievirus A6 variants: a potential role in evolution and pathogenicity

Hand, foot, and mouth disease (HFMD) is a common global epidemic. From 2008 onwards, many HFMD outbreaks caused by coxsackievirus A6 (CV-A6) have been reported worldwide. Since 2013, with a dramatically increasing number of CV-A6-related HFMD cases, CV-A6 has become the predominant HFMD pathogen in mainland China. Phylogenetic analysis based on the VP1 capsid gene revealed that subtype D3 dominated the CV-A6 outbreaks. Here, we performed a large-scale (near) full-length genetic analysis of global and Chinese CV-A6 variants, including 158 newly sequenced samples collected extensively in mainland China between 2010 and 2018. During the global transmission of subtype D3 of CV-A6, the noncapsid gene continued recombining, giving rise to a series of viable recombinant hybrids designated evolutionary lineages, and each lineage displayed internal consistency in both genetic and epidemiological features. The emergence of lineage –A since 2005 has triggered CV-A6 outbreaks worldwide, with a rate of evolution estimated at 4.17 × 10−3 substitutions site-1 year−1 based on a large number of monophyletic open reading frame sequences, and created a series of lineages chronologically through varied noncapsid recombination events. In mainland China, lineage –A has generated another two novel widespread lineages (–J and –L) through recombination within the enterovirus A gene pool, with robust estimates of occurrence time. Lineage –A, –J, and –L infections presented dissimilar clinical manifestations, indicating that the conservation of the CV-A6 capsid gene resulted in high transmissibility, but the lineage-specific noncapsid gene might influence pathogenicity. Potentially important amino acid substitutions were further predicted among CV-A6 variants. The evolutionary phenomenon of noncapsid polymorphism within the same subtype observed in CV-A6 was uncommon in other leading HFMD pathogens; such frequent recombination happened in fast-spreading CV-A6, indicating that the recovery of deleterious genomes may still be ongoing within CV-A6 quasispecies. CV-A6-related HFMD outbreaks have caused a significant public health burden and pose a great threat to children’s health; therefore, further surveillance is greatly needed to understand the full genetic diversity of CV-A6 in mainland China.

Expression of Human papillomavirus type 52 L1 capsid gene in Oryza sativa involved in cytoprotective activities

Female cervical cancer is largely formed by Human papillomavirus (HPV), the second leading cause of cancer deaths in women worldwide. HPV-52 is a regionally common high-risk type of cervical cancer found mostly in Asia and reveals geographical variations, in order of importance, as types HPV-16 and -18. However, the differing propensities of HPV types in progressing to cancer, focusing on HPV-52 vaccines, are limited. Several plant-based vaccines against cancer have been developed, and the production of candidate HPV therapeutic vaccines using plant-derived expression platforms is also proven. The objectives of this study were to assess the HPV-52L1 Capsid gene by transferring HPV-52L1 Capsid cDNA into rice (Oryza sativa L.) via an Agrobacterium-mediated transformation, and accumulating HPV-52L1 Capsid proteins in a plant-based expression system to maintain and improve antigenicity. Crude protein extracts containing 5~20 μg from OsHP-52L1 transgenic lines induced cell death and significantly reduced cell proliferation in HPV-positive HeLa cervical cancer cells compared with those non-transformant (NT) rice plants. However, no significant cytotoxicity of induced human breast MDA-MB-231 cell proliferation (as negative control) was observed at any dose compared with NT groups. HeLa cells ameliorated the effects of OsHPV crude protein extracts on cell viability as the extract concentration increased, and treatment with 20 μg of the extract from OsHPV-3 significantly reduced cell viability in HeLa cells (26%) compared with the control group (57%). Our results can be used for exploring the potential of plants for increasing the immunogenicity of OsHPV-52L1 Capsid DNA vaccines, and support the development of cost-effective HPV vaccines, which is highly desirable for resource-poor countries.