Pressure-inactivated yellow fever 17DD virus: implications for vaccine development

The successful Yellow Fever (YF) vaccine consists of the live attenuated 17D-204 or 17DD viruses. Despite its excellent record of efficacy and safety, serious adverse events have been recorded and influenced extensive vaccination in endemic areas. Therefore, alternative strategies should be considered, which may include inactivated whole virus. High hydrostatic pressure has been described as a method for viral inactivation and vaccine development. The present study evaluated whether high hydrostatic pressure would inactivate the YF 17DD virus. YF 17DD virus was grown in Vero cells in roller bottle cultures and subjected to 310 MPa for 3 h at 4 °C. This treatment abolished YF infectivity and eliminated the ability of the virus to cause disease in mice. Pressure-inactivated virus elicited low level of neutralizing antibody titers although exhibited complete protection against an otherwise lethal challenge with 17DD virus in the murine model. The data warrant further development of pressure-inactivated vaccine against YF.     

Neurovirulence of yellow fever 17DD vaccine virus to rhesus monkeys

The yellow fever 17D virus is attenuated and used for human vaccination. Two of its substrains, 17D-204 and 17DD, are used for vaccine production. One of the remarkable properties of this vaccine is limited viral replication in the host but with significant dissemination of the viral mass, yielding a robust and long-lived neutralizing antibody response. The vaccine has excellent records of efficacy and safety and is cheap, used as a single dose, and there are well-established production methodology and quality control procedures which include the monkey neurovirulence test (MNTV). The present study aims at a better understanding of YF 17DD virus attenuation and immunogenicity in the MNVT with special emphasis on viremia, seroconversion, clinical and histological lesions scores, and their intrinsic variability across the tests. Several MNVTs were performed using the secondary seed lot virus 17DD 102/84 totaling 49 rhesus monkeys. Viremia was never higher than the accepted limits established in international requirements, and high levels of neutralizing antibodies were observed in all animals. None of the animals showed visceral lesions. We found that the clinical scores for the same virus varied widely across the tests. There was a direct correlation between the clinical scores in animals with clinical signs of encephalitis and a higher degree of central nervous system (CNS) histological lesions, with an increase of lesions in areas of the CNS such as the substantia nigra, nucleus caudatus, intumescentia cervicalis, and intumescentia ventralis. The histological scores were shown to be less prone to individual variations and had a more homogeneous value distribution among the tests. Since 17DD 102/84 seed virus has been used for human vaccine production and immunization for 16 years with the vaccine being safe and efficacious, it demonstrates that the observed variations across the MNVTs do not influence its effect on humans.     

北京市4例输入性黄热病病例病毒全基因组特征分析

目的 通过对2016年北京市4例输入性黄热病病毒全基因组深度测序分析,以期对病毒进行溯源、分析疫苗有效性以及防控策略提供分子依据.方法 将荧光PCR检测为阳性的血液、尿液样本利用Ion Torrent PGM平台进行全基因组深度测序,通过MEGA 6.0软件中邻接法进行遗传进化分析,并采用Lasergene Protean软件进行E蛋白氨基酸变异分析及抗原性分析.结果 从首例病人的血液样本和其他3个病例的尿液样本中分别获得了黄热病病毒的全基因组序列.遗传进化树分析结果表明,4例黄热病病毒基因组与安哥拉71株(AY968064)高度同源,相似度分别达99.21％、98.11％、98.02％、98.39％.4例黄热病病毒E蛋白的氨基酸序列与安哥拉71株的序列分析相似度分别达99.6％、99.39％、99.01％、99.59％.4例黄热病病毒E蛋白抗原性与17D疫苗株(X03700)的E蛋白抗原性比较分析无明差异.结论 4例黄热病病毒都是安哥拉流行株类似株,目前使用的17D疫苗具有有效性.     

Detection of yellow fever virus: a comparison of quantitative real-time PCR and plaque assay

Yellow fever virus quantitation is performed routinely by cultivation of virus containing samples using susceptible cells. Counting of the resulting plaques provides a marker for the number of infectious particles present in the sample. This assay usually takes up to 5 days before results are obtained and must be carried out under L2 or L3 laboratory conditions, depending on the yellow fever virus strain used. For clinical diagnosis of yellow fever virus infections the cell culture-based approach takes too long and is of limited practical relevance. Recently, due to its considerable sensitivity, PCR has become a promising method for virus detection. However, whilst PCR can detect virus-specific nucleic acids, it does not allow conclusions to be drawn regarding the infectious potential of the virus detected. Nonetheless, for diagnostic purposes, a rapid, specific and sensitive virus PCR is preferable. Therefore, two independent yellow fever virus-specific real-time PCR assays were established and compared the viral RNA loads to the results of a traditional plaque assay. The estimated ratio of yellow fever virus genomes to infectious particles was between 1000:1 and 5000:1; both approaches displayed a comparable precision of <45%. A significant correlation between genome number as determined by real-time PCR and the corresponding number of plaques in paired samples was found with a Pearson coefficient of correlation of r=0.88 (P<0.0001).     

Antibody responses against wild-type yellow fever virus and the 17D vaccine strain: characterization with human monoclonal antibody fragments and neutralization escape variants

Human monoclonal antibody fragments neutralizing wild-type and vaccine strains of yellow fever (YF) virus (genotypes West Africa I + II, East/Central Africa, 17D-204-WHO) were generated by repertoire cloning from YF patients. Analysis of virus escape variants identified amino acid (aa) 71 in domain II of the envelope glycoprotein (E) as the most critical residue for neutralization, with aa 153-155 in domain I contributing to the epitope. These data confirm the previous mapping of YFV neutralizing epitopes using mouse monoclonal antibodies but suggest that a conformational epitope could be formed by amino acids from domains I and II opposing each other in the dimeric form of the E protein. While the sera of the YF patients showed up to 10-fold reduced neutralizing activity against the 17D escape variants, sera from 17D vaccinees retained their neutralizing titers. Mutations in this major neutralizing epitope of YFV thus do not seem to carry the risk of immune escape in persons immunized with the YFV-17D vaccine.     

Steroidal saponins from the roots of Solanum sisymbriifolium Lam. (Solanaceae) have inhibitory activity against dengue virus and yellow fever virus

Dengue is the most important arthropod-borne viral disease worldwide. Infection with any of the four dengue virus (DENV) serotypes can be asymptomatic or lead to disease with clinical symptoms ranging from undifferentiated and self-limiting fever to severe dengue disease, which can be fatal in some cases. Currently, no specific antiviral compound is available for treating DENV. The aim of this study was to identify compounds in plants from Paraguayan folk medicine with inhibitory effects against DENV. We found high virucidal activity (50% maximal effective concentration (EC₅₀) value of 24.97 µg/mL) against DENV-2 in the ethanolic extract of the roots of Solanum sisymbriifolium Lam. (Solanaceae) without an evident cytotoxic effect on Vero E6 cells. Three saponins isolated from the root extract showed virucidal effects (EC₅₀ values ranging from 24.9 to 35.1 µg/mL) against DENV-2. Additionally, the saponins showed inhibitory activity against yellow fever virus (EC₅₀ values ranging from 126 to 302.6 µg/mL), the prototype virus of the Flavivirus genus, suggesting that they may also be effective against other members of this genus. Consequently, these saponins may be lead compounds for the development of antiviral agents.     

Differential cytokine responses from primary human Kupffer cells following infection with wild-type or vaccine strain yellow fever virus

Wild-type yellow fever virus (YFV) infections result in a hepatotropic disease which is often fatal, while vaccination with the live-attenuated 17-D strain results in productive infection yet is well-tolerated with few adverse events. Kupffer cells (KCs) are resident liver macrophages that have a significant role in pathogen detection, clearance and immune signaling. Although KCs appear to be an important component of YF disease, their role has been under-studied. This study examined cytokine responses in KCs following infection with either wild-type or vaccine strains of YFV. Results indicate that KCs support replication of both wild-type and vaccine strains, yet wild-type YFV induced a prominent and prolonged pro-inflammatory cytokine response (IL-8, TNF-α and RANTES/CCL5) with little control by a major anti-inflammatory cytokine (IL-10). This response was significantly reduced in vaccine strain infections. These data suggest that a differentially regulated infection in KCs may play a critical role in development of disease.     

Molecular detection and characterization of yellow fever virus in blood and liver specimens of a non-vaccinated fatal human case

A yellow fever virus of a South American genotype was identified in the liver and blood samples of a non-vaccinated European patient after his return from Brazil. ELISA tests were negative for IgG and positive for IgM against yellow fever. Yellow fever proteins in the formalin-fixed and paraffin-embedded liver biopsy were detected by immunohistochemical procedures. Viral RNA extracted from the liver tissue was also detected using an RT-semi-nested PCR procedure and molecular hybridization. Alignment of the sequence obtained from a gene fragment amplified by RT-semi-nested PCR directly from a blood sample with those of African and South American yellow fever virus strains identified a Brazilian topotype as being responsible for the disease. RT-semi-nested PCR may be used advantageously for clinical specimens for rapid and specific diagnosis, and with archival biopsy material for retrospective studies. J. Med. Virol. 53:212–217, 1997. © 1997 Wiley-Liss, Inc.     

Immunogenicity and duration of protection after yellow fever vaccine in people living with human immunodeficiency virus: a systematic review

BackgroundWe lack the rationale on which to base the development of a yellow fever (YF) vaccination schedule for people living with human immunodeficiency virus (PLWHIV).ObjectivesTo report on the current evidence regarding the seroconversion rate and the duration of humoral protection after YF vaccine, as well as the impact of revaccination in PLWHIV.Data sourcesMEDLINE, Google Scholar, LILACS and Cochrane CENTRAL were searched.MethodsWe selected studies on PLWHIV of all ages (including perinatally HIV-infected patients) and all settings (YF endemic and non-endemic zones). Intervention investigated was vaccination against YF, at least once after the HIV diagnosis. The research questions were the seroconversion rate, duration of humoral immunity after YF vaccine and impact of revaccination in PLWHIV. Selected studies were assessed for quality using the Newcastle–Ottawa scale.ResultsTen, six and six studies were selected for the systematic review of each question, respectively. Only one study addressed the first question in perinatally HIV-infected children. The quality of the studies was assessed as Poor (n = 16), Fair (n = 2) or Good (n = 4). A meta-analysis demonstrated that 97.6% (95% CI 91.6%–100%) of the included population seroconverted. Between 1 and 10 years after YF vaccine, reported persistence of neutralizing antibodies was 72% (95% CI 53.6%–91%), and it was 62% (95% CI 45.4%–78.6%) more than 10 years after YF vaccine. No conclusions could be drawn on impact of revaccination because of the small number of patients.ConclusionsThe current evidence regarding seroconversion rate, duration of humoral protection after YF vaccine and impact of revaccination in PLWHIV is limited by the low number and quality of studies. Based on the presently available data, it is difficult to rationally develop yellow fever vaccination guidelines for PLWHIV.     

Simian hemorrhagic fever virus infection of rhesus macaques as a model of viral hemorrhagic fever: clinical characterization and risk factors for severe disease

Simian Hemorrhagic Fever Virus (SHFV) has caused sporadic outbreaks of hemorrhagic fevers in macaques at primate research facilities. SHFV is a BSL-2 pathogen that has not been linked to human disease; as such, investigation of SHFV pathogenesis in non-human primates (NHPs) could serve as a model for hemorrhagic fever viruses such as Ebola, Marburg, and Lassa viruses. Here we describe the pathogenesis of SHFV in rhesus macaques inoculated with doses ranging from 50 PFU to 500,000 PFU. Disease severity was independent of dose with an overall mortality rate of 64% with signs of hemorrhagic fever and multiple organ system involvement. Analyses comparing survivors and non-survivors were performed to identify factors associated with survival revealing differences in the kinetics of viremia, immunosuppression, and regulation of hemostasis. Notable similarities between the pathogenesis of SHFV in NHPs and hemorrhagic fever viruses in humans suggest that SHFV may serve as a suitable model of BSL-4 pathogens.     

Comprehensive analysis of T cell epitope discovery strategies using 17DD yellow fever virus structural proteins and BALB/c (H2d) mice model

Immunomics research uses in silico epitope prediction, as well as in vivo and in vitro approaches. We inoculated BALB/c (H2d) mice with 17DD yellow fever vaccine to investigate the correlations between approaches used for epitope discovery: ELISPOT assays, binding assays, and prediction software. Our results showed a good agreement between ELISPOT and binding assays, which seemed to correlate with the protein immunogenicity. PREDBALB/c prediction software partially agreed with the ELISPOT and binding assay results, but presented low specificity. The use of prediction software to exclude peptides containing no epitopes, followed by high throughput screening of the remaining peptides by ELISPOT, and the use of MHC-biding assays to characterize the MHC restrictions demonstrated to be an efficient strategy. The results allowed the characterization of 2 MHC class I and 17 class II epitopes in the envelope protein of the YF virus in BALB/c (H2d) mice.     

中国首次输入性黄热病疫情处置及相关问题探讨

目的 描述中国首次输入性黄热病疫情处置过程及防控效果,并深入探讨防控的经验和教训,以期对今后黄热病疫情处置有所启示.方法 依据国家卫计委《黄热病预防控制技术指南》(2008年),同时参考登革热和寨卡病毒病相关规定对病例实施隔离、开展蚊媒应急监测、划定共同暴露者和密切接触者并对其实施为期6日的健康监测.结果 此次疫情是我国首次输入性黄热病人间疫情.对输入性黄热病病例实施防蚊隔离和血液-体液隔离,对13例共同暴露者和11例密切接触者健康监测6日,均未出现黄热病相关症状.北京市蚊媒应急监测未发现蚊虫,对病例可能污染的环境使用含氯制剂进行消毒,公布疫情后及时对公众开展媒体信息发布,并对公众开展防控措施宣传.结论 此次疫情是我国首次输入黄热病疫情,疫情处置措施得当,未出现疫情扩散和公众恐慌.采取的措施将对今后我国输入性黄热病疫情的规范处置有所帮助.     

Biological and molecular characterization of Soybean yellow common mosaic virus, a new species in the genus Sobemovirus

A novel soybean-infecting sobemovirus termed Soybean yellow common mosaic virus (SYCMV) was characterized. The virus has a single, positive-strand RNA genome of 4152 nucleotides. The virus contains four putative open reading frames encoding P1 (78-566 nt), polyprotein ORF2a (524-2248 nt), polymerase domain ORF2b (1852-3417 nt), and CP (3227-4030 nt). The entire nucleotide sequence of SYCMV showed 31.2-71.3% nucleotide identity with the previously known eleven species of sobemovirus. In host range analysis of SYCMV, in which twenty one species and three different Nicotiana tabacum cultivars belonging to seven families were inoculated with the virus, SYCMV had a narrow host range, infecting only Glycine max and G. soja. Based on the obtained sequence, full-length clones of SYCMV were constructed. Symptoms produced by inoculation with clones were indistinguishable from those produced by inoculation with sap from symptomatic plants. Viral RNA accumulation of SYCMV was detected in the upper leaves by Northern blotting. This indicated that full-length clones of SYCMV were sufficient to produce disease symptoms. Genomic organization, the predicted amino acid sequence, and phylogenetic analyses with known sobemoviruses confirmed the assignment of SYCMV as a new member of the genus Sobemovirus.     

Genetic characterization of the M RNA segment of a Balkan Crimean-Congo hemorrhagic fever virus strain

Crimean-Congo hemorrhagic fever (CCHF) virus causes one of the most severe diseases in humans, with a mortality rate of up to 30%. It is transmitted to humans by the bite of hard ticks or by contact with blood or tissues from human patients or infected livestock. Balkan Peninsula is an endemic region of the disease, and sporadic cases or even outbreaks are observed every year. The M RNA segment encodes for the glycoprotein precursor of two surface glycoproteins Gn and Gc. Up to now complete M RNA CCHF virus sequences have been published from strains isolated in Nigeria, China, Pakistan, Tajikistan, and Russia. In the present study, the genetic characterization of the complete nucleotide sequence of the M RNA segment of a Balkan CCHF virus strain, Kosovo/9553/2001, isolated in summer of 2001 from a human fatal case in Kosovo is reported. This is the first published complete M nucleotide sequence of a CCHF virus strain isolated in Balkans. It was found that the Balkan strain is similar to the Russian strain, both strains differing from all other completely sequenced CCHF virus strains by approximately 22% at the nucleotide level forming an independent clade in the phylogenetic tree.     

Yellow fever virus infection in Syrian golden hamsters: relationship between cytokine expression and pathologic changes

Infection of primates by yellow fever virus (YFV) often results in severe multi-organ failure with marked histologic abnormalities. However, the role of host's immune response, particularly innate immunity, in disease process is unclear. In this study, we used a well established hamster model of yellow fever to examine the dynamic changes of cytokine expression and histopathology in the liver, spleen, kidney, and heart during the course of YFV infection. We observed that the levels of inflammatory cytokines (IFN-gamma, IL-2, TNF-alpha) in the liver were significantly reduced in the mid-stage of infection (8 days), but were elevated later (12 days). In contrast, IL-12p40 was elevated throughout the infection. The levels of IFN-gamma, IL-2, and TNF-alpha were increased in the spleen, kidney, and heart throughout the study period. For regulatory cytokines, IL-10 was significantly increased, and TGF-beta was reduced in the liver, spleen and heart in both early and mid-stages of infection, but was elevated in the kidney during the entire course of infection. In view of the pathologic changes, the observed cytokine profiles suggest that YFV has immunosuppressive effects, which contribute to liver damage in the mid-stage of infection, followed by an immunopathogenic mechanism that leads to disease progression during the late-stages of infection. Our findings support the hypothesis that organ injury by YFV is probably due to a combination of multiple factors, including direct viral injury and host innate immune responses.