A rabies mRNA vaccine with H270P mutation in its glycoprotein induces strong cellular and humoral immunity.

Rabies is a lethal zoonotic disease that kills approximately 60,000 people each year. As the sole virion-surface protein, the rabies virus glycoprotein (RABV-G) mediates its host-cell entry. RABV-G's pre-fusion conformation displays major known neutralizing antibody epitopes, which can be used as immunogen for prophylaxis. H270P targeted mutation can stabilize RABV-G in the pre-fusion conformation. Herein, we report the development of a highly promising rabies mRNA vaccine composed of H270P targeted mutation packaged in lipid nanoparticle (LNP), named LNP-mRNA-G-H270P. Humoral and cellular immunity of this vaccine were assessed in mice comparing to the unmodified LNP-mRNA-G and a commercially available inactivated vaccine using one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test. The results show the titer of RABV-G-specific IgG and virus-neutralization antibody titers (VNTs) in LNP-mRNA-G-H270P group were significant higher than those in LNP-mRNA-G and inactivated vaccine groups. Likewise, IFN-γ-secreting splenocytes, level of IL-2 in the supernatant of spleen cells, as well as IFN-γ-producing CD4+ T cells in LNP-mRNA-G-H270P group were significant higher than those in the other two vaccine groups. Hence, these results demonstrated that targeting the H270P mutation in RABV-G through an mRNA-LNP vaccine platform represents a promising strategy for developing a more efficacious rabies vaccine.

Epidemiological and etiological characteristics of viral meningitis for hospitalized pediatric patients in Yunnan, China.

BACKGROUND: Viral infection is the most common cause of aseptic meningitis. The purpose of this study was to identify the viruses responsible for aseptic meningitis to better understand the clinical presentations of this disease. METHOD: Between March 2009 and February 2010, we collected 297 cerebrospinal fluid specimens from children with aseptic meningitis admitted to a pediatric hospital in Yunnan (China). Viruses were detected by using "in house" real-time quantitative polymerase chain reaction or reverse-transcription real-time quantitative polymerase chain reaction from these samples. Phylogenetic analyses were conducted using the Molecular Evolutionary Genetic Analysis version 7.0 software, with the neighbor-joining method. RESULTS: Viral infection was diagnosed in 35 of the 297 children (11.8%). The causative viruses were identified to be enteroviruses in 25 cases (71.4%), varicella-zoster virus in 5 cases (14.3%), herpes simplex virus 1 in 2 cases (5.7%), and herpes simplex virus 2, Epstein-Barr virus, and human herpesvirus 6 in 1 case each (2.9% each). Of the enteroviruses, coxsackievirus B5 was the most frequently detected serotype (10/25 cases; 40.0%) and all coxsackievirus B5 strains belonged to C group. CONCLUSIONS: In the study, a causative virus was only found in the minority of cases, of them, enteroviruses were the most frequently detected viruses in patients with viral meningitis, followed by varicella-zoster virus and herpes simplex virus. Our findings underscore the need for enhanced surveillance and etiological study of aseptic meningitis.

Identification of a new recombinant strain of echovirus 33 from children with hand, foot, and mouth disease complicated by meningitis in Yunnan, China.

BACKGROUND: Hand, foot, and mouth disease (HFMD) is a common childhood disease, which is usually caused by enterovirus A (EV-A) serotypes. Enterovirus A71 (EV-A71) and coxsackievirus A16 (CV-A16) are the main etiologic agents. Multiple serotypes of enterovirus B serotypes (EV-B) have been detected in outbreaks or sporadic cases of HFMD. RESULTS: During HFMD surveillance in Yunnan, China in 2013, two echovirus 33 (E-33) isolates were recovered in cell culture and typed by molecular methods from the cerebrospinal fluid (CSF) and feces of two sporadic cases of HFMD complicated by meningitis. Sequence analysis indicated that the study isolates, YNK35 and YNA12, formed an independent branch, and belonged to E-33 genotype H. Recombination analysis indicated multiple recombination events in the genomic sequence of isolate YNK35. The recombination mainly occurred in the non-structural coding region of P2 and P3, and involved intra-species recombination of species B. CONCLUSION: In this study, the complete sequences of two E-33 isolates were determined. This is the first report of severe HFMD associated with E-33 in Yunnan China, and it enriches the number of full-length genome sequences of E-33 in the GenBank database.

Ongoing change of severe hand, foot, and mouth disease pathogens in Yunnan, China, 2012 to 2016.

Hand, foot, and mouth disease (HFMD) is a common infectious disease caused by enteroviruses (EVs). In this study, a total of 341 children with serious HFMD were admitted to a pediatric hospital in Yunnan, China in 2012 to 2016. EVs were detected in 283 specimens (83.0%) and were assigned to 17 EV types. Enterovirus A71 (EV-A71) was predominant, accounting for 41.6%, and was followed by coxsackievirus A16 (CV-A16; 18.8%), CV-A6 (9.1%), CV-A10 and E-9 (2.9%), CV-B5 (1.8%), CV-A9 (1.2%), E-30 (0.9%), E-18, CV-A4, C-B3, and CV-A2 (0.6%) and other EV types such as CV-A8, CV-A14, E-14, E-11, and CV-B4 (0.3%). All of the EV-A71 isolates belonged to C4a; the CV-A16 belonged to B1b or B1a, although the B1b strains were predominant; and CV-A6 belonged to D3. In 2012 to 2014, E-9 was the third most frequent serotype (8.2%, 5.0%, and 6.5%, respectively). E-9 was not detected in 2015 and 2016. CV-A6 was not detected in 2012 but was the second most frequent serotype (25.3%) in 2015. Active etiological surveillance of HFMD makes it necessary to be aware of these emerging pathogens.

Molecular characterization of Coxsackievirus A16 strains isolated from children with severe hand, foot, and mouth disease in Yunnan, Southwest China, during 2009-2015.

Coxsackievirus A16 (CV-A16) commonly causes mild symptoms, but severe diseases, such as aseptic meningitis, encephalitis, and even fatal cases, have been reported. Thirteen CV-A16 strains were isolated from patients with severe hand, foot, and mouth disease in Yunnan, Southwest China, from 2009 to 2015. Subgenotype B1a and B1b of CV-A16 were predominantly circulating the region with B1b the predominant strain in recent years. The mean rate of nucleotide substitution based on the VP1 gene sequence was 4.545 × 10 -3 substitution per site per year from 2009 to 2015. These results may help in understanding the genetic diversity of CV-A16 and develop a CV-A16 vaccine.

Molecular characterization of echovirus 12 strains isolated from healthy children in China.

Human echovirus 12 (E-12) belongs to the enterovirus B species. To date, only one full-length genome sequence of E-12 (prototype strain Travis) is available in the GenBank database. This study determined the complete sequence of three E-12 strains, which were isolated from the stools of three healthy children in Yunnan, China, in 2013. We revealed that the three Yunnan E-12 strains had only 80.8-80.9% nucleotide identity and 96.4-96.8% amino acid identity with the Travis strain based on pairwise comparisons of the complete genome nucleotide and amino acid sequences. The three Yunnan strains shared 99.7% nucleotide identity and 99.1-99.5% amino acid similarity. Phylogenetic and similarity plot analyses showed that intertypic recombination occurred in the non-structural regions of the three Yunnan E-12 strains. This is the first report of the complete genome sequence of E-12 in China and it enriches the complete genome sequences of E-12 in the GenBank database.

Molecular characteristics of hand, foot, and mouth disease for hospitalized pediatric patients in Yunnan, China.

Hand, foot, and mouth disease (HFMD) is a common infectious disease caused by multiple enteroviruses (EVs) in China. To better define the etiologic agents and clinical characteristics of HFMD, we conducted this study in Yunnan, China.In this study, 1280 stool specimens were collected from pediatric patients hospitalized for treatment of HFMD in 2010. EV was detected with nested reverse transcription polymerase chain reaction and directly genotyped by gene sequencing of the viral protein 1 (VP1) region. Phylogenetic analysis was performed based on the VP1 partial gene and the clinical characteristics were analyzed using SPSS Software.Of 1280 specimens, 1115 (87.1%) tested positive for EV. Seventeen different EV serotypes were detected. Coxsackievirus A16 (CA16) was the most frequently detected serotype (615/1115 cases, 55.1%), followed by enterovirus 71 (EV71; 392/1115, 35.2%), CA10 (45/1115, 4.0%), and CA4 (23/1115, 2.1%). Among the 709 severe cases, CA16, EV71, CA10, and CA4 accounted for 48.0%, 42.0%, 3.5%, and 2.3%, respectively. Of the 26 critical cases, 13 were caused by EV71, 9 by CA16, 2 by CA4, and 1 each were the result of CA10 and E9, respectively. All EV71, CA16, CA10, and CA4 isolates were highly homologous to the strains isolated from mainland China, and belonged to the C4a, B1a, G, and C genotypes, respectively.Our study showed that EV71 and CA16 were the main causative agents for severe and critical HFMD, but other serotypes can also cause severe and critical cases.

Molecular characterization of two novel echovirus 18 recombinants associated with hand-foot-mouth disease.

Human echovirus 18 (E-18) is a member of the enterovirus B species. To date, sixteen full-length genome sequences of E-18 are available in the GenBank database. In this study, we describe the complete genomic characterization of two E-18 strains isolated in Yunnan, China. Pairwise comparisons of the nucleotide sequences and the deduced amino acid sequences revealed that the two Yunnan E-18 strains had 87.5% nucleotide identity and 96.3-96.5% amino acid identity with the Chinese strain. Phylogenetic and bootscanning analyses revealed the two E-18 strains had the highest identity with other several EV-B serotypes than the other E-18 strains in the P3 coding region, especially, 3B region of the Swine Vesicular disease virus (SVDV) strain HK70, indicated that frequent intertypic recombination might have occurred in the two Yunnan strains. This study contributes the complete genome sequences of E-18 to the GenBank database and provides valuable information on the molecular epidemiology of E-18 in China.

Complete Genome Sequence of Human Echovirus 20 Strain 812/YN/CHN/2010, Associated with Severe Hand-Foot-and-Mouth Disease.

Human echovirus 20 (E-20) belongs to the Human enterovirus B (HEV-B) species and is often detected in nonpolio enterovirus cases of acute flaccid paralysis. We determined the complete genome of strain 812/YN/CHN/2010, isolated from a child with severe hand-foot-and-mouth disease in Yunnan, China, in 2010.

Complete Genome Sequence of an Enterovirus 71 Strain Isolated from the Cerebrospinal Fluid of a Child with Severe Hand-Foot-and-Mouth Disease in Yunnan, China, 2013.

The complete genome sequence of the enterovirus 71 strain CSF15/YN/CHN/2013, first isolated from cerebrospinal fluid of a child in Yunnan, China, in 2013, was determined. According to the phylogenetic and homogeneity analyses, the isolate was assigned to subgenotype C4a.

Molecular characterization of a new human coxsackievirus B2 associated with severe hand-foot-mouth disease in Yunnan Province of China in 2012.

Human coxsackievirus B2 (CVB2) belongs to the species Human enterovirus B and can cause aseptic meningitis, myocarditis and hand-foot-mouth disease (HFMD). We first determined the complete genome of the RW41-2/YN/CHN/2012 strain, isolated from a patient with HFMD and aseptic meningitis in the Yunnan Province, China in 2012. The strain shared 83.5 % and 82.2 % nucleotide similarity with CVB2 prototype strain Ohio-1, in the complete VP1 gene and the complete genome, respectively. Using phylogenetic and homogeneity analyses for the complete VP1 gene, CVB2 strains could be divided into four genogroups (A-D); the RW41-2/YN/CHN/2012 strain belonging to genogroup D. The amino acid sequence of VP1 is highly conserved. Recombination analyses showed the newly isolated RW41-2/YN/CHN/2012 strain was probably a recombinant, which was closely related to strain CVB2 (KM386639) in the genomic P1 and P2 regions and strains of other human enterovirus B (HEV-B) viruses (KT353721, JX644073, and KP262053) in the P3 region.

Characterization of four vaccine-related polioviruses including two intertypic type 3/type 2 recombinants associated with aseptic encephalitis.

BACKGROUND: Four vaccine-related polioviruses (VRPV) were isolated from aseptic encephalitis cases in Yunnan, China in 2010. The genomic sequences of these VRPVs were investigated to gain a better understanding of their molecular characteristics. METHODS: Molecular typing was performed by amplification and sequencing of the VP1 region. The genomic sequences of the four VRPV3 strains were compared to vaccine strain and wild strain sequences to study genetic drift and recombination. RESULTS: All four isolates could be entirely neutralized by polyclonal poliovirus 3 (PV3) antisera but not by PV1 and PV2 antisera and displayed a temperature-sensitive phenotype. The genomic sequences of all four isolates contained two Sabin 3-specific attenuating mutations at nucleotides 472(C → T) and 2034(C → T), but a third Sabin 3-specific attenuating mutation at position 2493 (T → C) had reverted back to a T. Recombination analyses showed RF108/YN/CHN/2010 and RF134/YN/CHN/2010 strain recombined with Sabin 2 at the 3'-end of the 2C to 3'-untranslated region (3'-UTR) and at the 5'-end of the 3D to 3'-UTR, respectively. CONCLUSION: Four VRPV3 strains including two type 3/type 2 intertypic recombinants were identified. The recombination of Sabin vaccine strains with other Sabin serotypes or human enterovirus C species could be a critical factor in the potential of emerging viruses and related disease outbreaks. Therefore, it is essential to be persistent in the surveillance of EVs (including PV).

Complete genome analysis of coxsackievirus A24 isolated in Yunnan, China, in 2013.

Human coxsackievirus A24 (CVA24) belongs to the species Enterovirus C, and variants of this virus frequently cause acute hemorrhagic conjunctivitis (AHC). The complete genome of the K282/YN/CHN/2013 strain, isolated from a healthy child in Yunnan, China, in 2013, is reported here for the first time. The strain showed 80.0 % and 79.9 % nucleotide sequence identity to CVA24 prototype strain Joseph and CVA24 variant prototype EH24, respectively. The K282/YN/CHN/2013 strain belongs to the CVA24 serotype. Twelve amino acid differences, most of which are in structural regions, were found between the CVA24 and CVA24v strains. In the whole-length genome sequence, only the structural region of K282/YN/CHN/2013 was similar to that of the CVA24 strains; the other genome regions were more similar to those of other members of the species Enterovirus C. Recombination analysis showed evidence of recombination with other viruses of the same species.

Classification of the hydrogen-bonding species in a series of novel hydrazide based azobenzene derivatives investigated by two-dimensional correlation infrared spectroscopy and molecular modeling.

Classification of hydrogen-bonding species in a series of novel hydrazide modified p-methoxyazobenzene derivatives, 4-{n-[4-(4-methoxy-phenylazo)-phenoxy]-alkoxy}-benzoic acid hydrazide (Dn, n = 3, 6, 10) are performed in the present study. Temperature-dependent infrared (IR) spectra of Dn have been measured to investigate the thermal stability of the weak intermolecular interactions, such as hydrogen bonding among hydrazide moieties, pi-pi stacking among aromatic groups, and hydrophobic interaction between alkyl chains. In order to reveal the hydrogen bonding formed between NH, NH2, and CONH groups efficiently, two-dimensional (2D) correlation spectra have been constructed in the thermal sensitive spectral regions of (a) 3500-3100 cm(-1) and (b) 1700-1450 cm(-1), separately, and it have also been constructed between these two spectral regions. Based on the experimental data, the ab initio computational models have been developed to the proposed patterns of hydrogen bonding related to intermolecular interactions in Dn. The intermolecular hydrogen bondings and molecular alignments patterns result from both the experimental data and the computational models are performed for D3, D6, and D10, respectively, in the present study.