我国分离的两株病毒为重组甲病毒

目的 明确我国分离的XJ－90260和XJ－91006病毒的分类地位、种系发生和遗传型。方法 特异引物逆转录－聚合酶链反应（RT－PCR）扩增两株病毒的NSP4、E1基因区和3′端非编码区,测序,进行核苷酸序列同源性比较和3′端非编码区核苷酸序列分析,并结合同属其他病毒这些基因区的核苷酸序列进行种系进化分析。结果 两株病毒的核苷酸序列同源性为100％,与西方马脑炎病毒的核种酸序列同源性最高,具有西方马脑炎病毒3′端非编码区结构特征,其NSP4基因区与东方马脑炎病毒同源,E1基因区与辛德毕斯病毒同源,两株病毒均位于西方马脑炎病毒B组,与西方马脑炎病毒俄罗斯分离株进化关系最近。结论 我国分离的XJ－90260和91006病毒属于西方马脑炎病毒同一遗传型,均为重组甲病毒。     

Bivalent Junin & Machupo experimental vaccine based on alphavirus RNA replicon vector

Junin (JUNV) and Machupo (MACV), two mammalian arenaviruses placed on the 2018 WHO watch list, are prevalent in South America causing Argentine and Bolivian hemorrhagic fevers (AHF and BHF), respectively. The live attenuated JUNV vaccine, Candid #1, significantly reduced the incidence of AHF. Vaccination induces neutralizing antibody (nAb) responses which effectively target GP1 (the viral attachment glycoprotein) pocket which accepts the tyrosine residue of the cellular receptor, human transferrin receptor 1 (TfR1). In spite of close genetic relationships between JUNV and MACV, variability in the GP1 receptor binding site (e.g., MACV GP1 loop 10) results in poor MACV neutralization by Candid #1-induced nAbs. Candid #1 is not recommended for vaccination of children younger than 15 years old (a growing “at risk” group), pregnant women, or other immunocompromised individuals. Candid #1’s primary reliance on limited missense mutations for attenuation, genetic heterogeneity, and potential stability concerns complicate approval of this vaccine in the US. To address these issues, we applied alphavirus RNA replicon vector technology based on the human Venezuelan equine encephalitis vaccine (VEEV) TC-83 to generate replication restricted virus-like-particles vectors (VLPVs) simultaneously expressing cellular glycoprotein precursors (GPC) of both viruses, JUNV and MACV. Resulting JV&MV VLPVs were found safe and immunogenic in guinea pigs. Immunization with VLPVs induced humoral responses which correlated with complete protection against lethal disease after challenge with pathogenic strains of JUNV (Romero) and MACV (Carvallo).     

Alphavirus vector-based replicon particles expressing multivalent cross-protective Lassa virus glycoproteins

Lassa virus (LASV) is the most prevalent rodent-borne arenavirus circulated in West Africa. With population at risk from Senegal to Nigeria, LASV causes Lassa fever and is responsible for thousands of deaths annually. High genetic diversity of LASV is one of the challenges for vaccine R&D. We developed multivalent virus-like particle vectors (VLPVs) derived from the human Venezuelan equine encephalitis TC-83 IND vaccine (VEEV) as the next generation of alphavirus-based bicistronic RNA replicon particles. The genes encoding VEEV structural proteins were replaced with LASV glycoproteins (GPC) from distantly related clades I and IV with individual 26S promoters. Bicistronic RNA replicons encoding wild-type LASV GPC (GPCwt) and C-terminally deleted, non-cleavable modified glycoprotein (ΔGPfib), were encapsidated into VLPV particles using VEEV capsid and glycoproteins provided in trans. In transduced cells, VLPVs induced simultaneous expression of LASV GPCwt and ΔGPfib from 26S alphavirus promoters. LASV ΔGPfib was predominantly expressed as trimers, accumulated in the endoplasmic reticulum, induced ER stress and apoptosis promoting antigen cross-priming. VLPV vaccines were immunogenic and protective in mice and upregulated CD11c⁺/CD8⁺ dendritic cells playing the major role in cross-presentation. Notably, VLPV vaccination resulted in induction of cross-reactive multifunctional T cell responses after stimulation of immune splenocytes with peptide cocktails derived from LASV from clades I-IV. Multivalent RNA replicon-based LASV vaccines can be applicable for first responders, international travelers visiting endemic areas, military and lab personnel.     

Non-pathogenic Sindbis virus causes hemorrhagic fever in the absence of alpha/beta and gamma interferons

The role of interferon-gamma (IFNgamma) in antiviral innate immune responses during acute alphavirus infection is not well defined. We examined the contribution of IFNgamma to the protection of adult mice from Sindbis virus (SB)-induced disease by comparing subcutaneous infection of mice lacking receptors for either IFNalpha/beta (A129), IFNgamma (G129) or both (AG129) to normal mice (WT129). While neither G129 nor WT129 mice exhibited clinical signs of disease, infection of A129 or AG129 mice was fatal with AG129 mice succumbing more rapidly. Furthermore, AG129 mice developed signs of viral hemorrhagic fever (VHF), including extensive hepatocellular damage, inflammatory infiltrates in multiple organs and vascular leakage, which were significantly delayed and/or partially ameliorated during fatal A129 infections. We conclude that: (i) IFNalpha/beta is the primary mediator of innate immunity to SB infection, however; (ii) IFNgamma is directly antiviral in vivo, acting before the adaptive immune response appears and; (iii) development of VHF may involve viral suppression of both IFNalpha/beta and IFNgamma responses.     

Genomic analysis of a Chinese isolate of Getah-like virus and its phylogenetic relationship with other Alphaviruses

An alphavirus, M-1 strain, was isolated from a pool of culicine mosquitoes collected in Hainan island of China during an arbovirus survey in 1964. In the present study, we determined the complete nucleotide sequence of the M-1 strain using RT-PCR and RACE techniques. The M-1 genome is 11,690 nucleotides (nt) in length and contains two open reading frames (ORFs) encoding four nonstructural proteins and five structural proteins, respectively. Searches using Blast and comparison analyses suggested that M-1 is closely linked to Sagiyama virus (SAGV, AB032553) with 98% identity and Getah viruse (GETV, AY702913) with 97.8% identity in the full-length nucleotide sequence. However, compared with SAGV, there is 1 deletion (3 nucleotides in length) in the Capsid region, a deletion in the 3′ untranslated region (10 nucleotides in length) and 2 insertions in the 3′ untranslated region involving a total of 5 nucleotides. Interestingly, from the 5′ UTR to the end of coding region, M-1 share the highest identity with GETV, even though the identity of 3′ UTR drops dramatically to 76.2%. Furthermore, phylogenetic analysis based on the complete genomic sequences and sequences for structural or non-structural proteins of M-1 and 15 alphaviruses showed that M-1 grouped with GETV first and then grouped together with SAGV. Based on the comparison analysis and phylogenetic analysis, we conclude that M-1 strain can be considered as a strain that is a Chinese isolate of Getah-like virus. Jin-Sheng Wen and Wen-Zhong Zhao equally contributed to this work. The nucleotide sequence data reported in this article have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number: EF011023.     

Alphavirus replicon-based enhancement of mucosal and systemic immunity is linked to the innate response generated by primary immunization

Venezuelan equine encephalitis virus replicon particles (VRP) function as an effective systemic, cellular and mucosal adjuvant when codelivered with antigen, and show promise for use as a component in new and existing human vaccine formulations. We show here that VRP are effective at low dose and by intramuscular delivery, two useful features for implementation of VRP as a vaccine adjuvant. In mice receiving a prime and boost with antigen, we found that VRP are required in prime only to produce a full adjuvant effect. This outcome indicates that the events triggered during prime with VRP are sufficient to establish the nature and magnitude of the immune response to a second exposure to antigen. Events induced by VRP in the draining lymph node after prime include robust secretion of many inflammatory cytokines, upregulation of CD69 on leukocytes, and increased cellularity, with a disproportionate increase of a cell population expressing CD11c, CD11b, and F4/80. We show that antigen delivered 24 h after administration of VRP does not benefit from an adjuvant effect, indicating that the events which are critical to VRP-mediated adjuvant activity occur within the first 24 h. Further studies of the events induced by VRP will help elucidate the mechanism of VRP adjuvant activity and will advance the safe implementation of this adjuvant in human vaccines.     

Characterization of purified Sindbis virus nsP4 RNA-dependent RNA polymerase activity in vitro

The Sindbis virus RNA-dependent RNA polymerase (nsP4) is responsible for the replication of the viral RNA genome. In infected cells, nsP4 is localized in a replication complex along with the other viral non-structural proteins. nsP4 has been difficult to homogenously purify from infected cells due to its interactions with the other replication proteins and the fact that its N-terminal residue, a tyrosine, causes the protein to be rapidly turned over in cells. We report the successful expression and purification of Sindbis nsP4 in a bacterial system, in which nsP4 is expressed as an N-terminal SUMO fusion protein. After purification the SUMO tag is removed, resulting in the isolation of full-length nsP4 possessing the authentic N-terminal tyrosine. This purified enzyme is able to produce minus-strand RNA de novo from plus-strand templates, as well as terminally add adenosine residues to the 3′ end of an RNA substrate. In the presence of the partially processed viral replicase polyprotein, P123, purified nsP4 is able to synthesize discrete template length minus-strand RNA products. Mutations in the 3′ CSE or poly(A) tail of viral template RNA prevent RNA synthesis by the replicase complex containing purified nsP4, consistent with previously reported template requirements for minus-strand RNA synthesis. Optimal reaction conditions were determined by investigating the effects of time, pH, and the concentrations of nsP4, P123 and magnesium on the synthesis of RNA.     

Augmentation of alphavirus vector-induced human papilloma virus-specific immune and anti-tumour responses by co-expression of interleukin-12

To enhance the efficacy of a therapeutic immunisation strategy against human papillomavirus-induced cervical cancer we evaluated the adjuvant effect of interleukin-12 (IL12) expressed by a Semliki Forest virus vector (SFV) in mice. Depending on the dose and schedule, SFV-IL12 stimulated antigen-specific CTL responses elicited upon immunisation with recombinant SFV expressing HPV16-E6E7 (SFVeE6,7). SFVeE6,7–CTL and anti-tumour activity were enhanced by a low dose of SFV-IL12 to the prime immunisation. Using higher dosages these activities were reduced. Addition of SFV-IL12 to the booster immunisation further reduced the efficacy of the SFVeE6,7 immunisation. In transgenic mice, tolerant for HPV16-E6E7, SFV-IL12 also stimulated SFVeE6,7-induced CTL responses. In conclusion, SFV-IL12 can enhance antigen-specific immune responses. Yet, prudence is called for when considering co-administration of SFV-IL12 to a vaccine, as the enhancement of cell-mediated immune responses greatly depends on dosage and schedule.