The untranslated regions of EV-A71 contribute to its pathogenicity and virulence.

Enterovirus A71 (EV-A71) is known for its manifestation as hand foot and mouth disease (HFMD), which has caused countless large-scale epidemic outbreaks throughout the world. However, the molecular pathogenesis of EV-A71 infection is still elusive. Previous studies found that the biological characteristics of a mild EV-A71 strain (SDLY1) and a severe EV-A71 strain (SDLY107) are significantly different, and sequence analysis showed that there are several differences in nucleotide sites of UTRs (88 nt, 123 nt, 143 nt, 154 nt, 187 nt, 241 nt, 243 nt, 253 nt, 291 nt, 438 nt, 440 nt, 571 nt, 579 nt, 602 nt, 658 nt, 664 nt, 690 nt, 696 nt, 7328 nt, 7335 nt, 7367 nt, and 7395 nt). The aim of this study was to determine whether these amino sites in UTRs are associated with the pathogenesis of EV-A71 and are responsible for different clinical manifestations. Based on the reverse genetics technology, we rescued two chimeric viruses SDLY107(1-5'UTR) and SDLY107(1-3'UTR) by replacing 5'UTR/3'UTR gene fragments of an infectious cDNA clone. Replication kinetics and cytotoxicity assays showed that the virulence of the two chimeric strains significantly changed in vitro. The viral loads of the two chimeric strains in infected ICR mice were reduced and pathological damage in the brains, lungs, intestinal tissues, and muscles were lightened. Our findings suggest that some nucleotide sites in UTRs may have a function in the pathogenicity and virulence of EV-A71.

Novel Bartonella Species in Insectivorous Bats, Northern China.

Bartonella species are emerging human pathogens. Bats are known to carry diverse Bartonella species, some of which are capable of infecting humans. However, as the second largest mammalian group by a number of species, the role of bats as the reservoirs of Bartonella species is not fully explored, in term of their species diversity and worldwide distribution. China, especially Northern China, harbors a number of endemic insectivorous bat species; however, to our knowledge, there are not yet studies about Bartonella in bats in China. The aim of the study was to investigate the prevalence and genetic diversity of Bartonella species in bats in Northern China. Bartonella species were detected by PCR amplification of gltA gene in 25.2% (27/107) bats in Mengyin County, Shandong Province of China, including 1/3 Rhinolophus ferrumequinum, 2/10 Rhinolophus pusillus, 9/16 Myotis fimbriatus, 1/5 Myotis ricketti, 14/58 Myotis pequinius. Phylogenetic analysis showed that Bartonella species detected in bats in this study clustered into ten groups, and some might be novel Bartonella species. An association between Bartonella species and bat species was demonstrated and co-infection with different Bartonella species in a single bat was also observed. Our findings expanded our knowledge on the genetic diversity of Bartonella in bats, and shed light on the ecology of bat-borne Bartonella species.

Genetic diversity and evolutionary dynamics of Ebola virus in Sierra Leone.

A novel Ebola virus (EBOV) first identified in March 2014 has infected more than 25,000 people in West Africa, resulting in more than 10,000 deaths. Preliminary analyses of genome sequences of 81 EBOV collected from March to June 2014 from Guinea and Sierra Leone suggest that the 2014 EBOV originated from an independent transmission event from its natural reservoir followed by sustained human-to-human infections. It has been reported that the EBOV genome variation might have an effect on the efficacy of sequence-based virus detection and candidate therapeutics. However, only limited viral information has been available since July 2014, when the outbreak entered a rapid growth phase. Here we describe 175 full-length EBOV genome sequences from five severely stricken districts in Sierra Leone from 28 September to 11 November 2014. We found that the 2014 EBOV has become more phylogenetically and genetically diverse from July to November 2014, characterized by the emergence of multiple novel lineages. The substitution rate for the 2014 EBOV was estimated to be 1.23 × 10(-3) substitutions per site per year (95% highest posterior density interval, 1.04 × 10(-3) to 1.41 × 10(-3) substitutions per site per year), approximating to that observed between previous EBOV outbreaks. The sharp increase in genetic diversity of the 2014 EBOV warrants extensive EBOV surveillance in Sierra Leone, Guinea and Liberia to better understand the viral evolution and transmission dynamics of the ongoing outbreak. These data will facilitate the international efforts to develop vaccines and therapeutics.

An inactivated recombinant rabies CVS-11 virus expressing two copies of the glycoprotein elicits a higher level of neutralizing antibodies and provides better protection in mice.

The rabies virus (RABV) G protein is the primary contributor to the pathogenicity and protective immunity of RABV. In this study, we generated a recombinant rCVS-11-G strain containing two copies of the G protein derived from the pathogenic wild-type (wt) CVS-11 strain and based on its infectious clone. Compared with the wtCVS-11 strain, the rCVS-11-G strain possessed a larger virion and 1.4-fold more G protein, but it exhibited a similar growth property to the rCVS-11 strain, including passaging stability in vitro. qPCR results showed that the two G genes were over-expressed in BHK-21 cells infected with the rCVS-11-G strain. However, the rCVS-11-G strain presented an 80 % lower LD50 than the wtCVS-11 strain when intracranially (i.c.) inoculated in adult mice. Adult mice that were either intracranially (i.c.) or intramuscularly (i.m.) inoculated with rCVS-11-G strain developed more acute neurological symptoms and greater mortality than those inoculated with the wtCVS-11 strain. Furthermore, the rCVS-11-G strain was more easily and rapidly taken up by neuroblastoma cells. These data indicated that the rCVS-11-G strain might have increased neurotropism because of the over-expression of the pathogenic G protein. The inactivated rCVS-11-G strain induced significantly higher levels of virus neutralization antibodies and provided better protection from street rabies virus challenge in mice. Therefore, the rCVS-11-G strain may be a promising inactivated vaccine strain due to its better immunogenicity.

Selection of an aptamer against rabies virus: a new class of molecules with antiviral activity.

Rabies is a fatal central nervous system (CNS) disease caused by the neurotropic rabies virus (RABV). The therapeutic management of RABV infections is still problematic, and novel antiviral strategies are urgently required. We established the RVG-BHK-21 cell line, which expresses RABV glycoprotein on the cell surface, to select aptamers. Through 28 iterative rounds of selection, single-stranded DNA (ssDNA) aptamers were generated by exponential enrichment (SELEX). A virus titer assay and a real-time quantitative reverse transcription PCR (qRT-PCR) assay revealed that four aptamers could inhibit the replication of RABV in cultured baby hamster kidney (BHK)-21 cells. However, the aptamers did not inhibit the replication of other virus, e.g., canine distemper virus (CDV) and canine parvovirus (CPV). In addition, the GE54 aptamer was found to effectively protect mice against lethal RABV challenge. After inoculation with aptamers for 24h or 48h, followed by inoculation with CVS-11, approximately 25-33% of the mice survived. In summary, we selected aptamers that could significantly protect from a lethal dose of RABV in vitro and in vivo.

Canine parvovirus VP2 protein expressed in silkworm pupae self-assembles into virus-like particles with high immunogenicity.

The VP2 structural protein of parvovirus can produce virus-like particles (VLPs) by a self-assembly process in vitro, making VLPs attractive vaccine candidates. In this study, the VP2 protein of canine parvovirus (CPV) was expressed using a baculovirus expression system and assembled into parvovirus-like particles in insect cells and pupae. Electron micrographs of VLPs showed that they were very similar in size and morphology when compared to the wild-type parvovirus. The immunogenicity of the VLPs was investigated in mice and dogs. Mice immunized intramuscularly with purified VLPs, in the absence of an adjuvant, elicited CD4(+) and CD8(+) T cell responses and were able to elicit a neutralizing antibody response against CPV, while the oral administration of raw homogenates containing VLPs to the dogs resulted in a systemic immune response and long-lasting immunity. These results demonstrate that the CPV-VLPs stimulate both cellular and humoral immune responses, and so CPV-VLPs may be a promising candidate vaccine for the prevention of CPV-associated disease.

Aptamers targeting rabies virus-infected cells inhibit viral replication both in vitro and in vivo.

Rabies is an acute fatal encephalitis disease that affects many warm-blooded mammals. The causative agent of the disease is Rabies virus (RABV). Currently, no approved therapy is available once the clinical signs have appeared. Aptamers, oligonucleotide ligands capable of binding a variety of molecular targets with high affinity and specificity, have recently emerged as promising therapeutic agents. In this study, sixteen high-affinity single-stranded DNA (ssDNA) aptamers were generated by cell-SELEX. Viral titer assays revealed aptamers could specifically inhibit the replication of RABV in cells but did not inhibit the replication of canine distemper virus or canine parvovirus. In addition, the FO21 and FO24 aptamers, with and without PEGylation, were found to effectively protect mice against lethal RABV challenge. When mice were inoculated with aptamers for 24h prior to inoculation with CVS-11, approximately 87.5% of the mice survived. Here, we report aptamers that could significantly protect the mice from a lethal dose of RABV in vitro and in vivo, as demonstrated by the results for survival rate, weight loss and viral titers. These results indicate that FO21 and FO24 aptamers are a promising agent for specific antiviral against RABV infections.