Bacterial outer membrane vesicles (OMV) hold promise as vaccine platforms due to their natural adjuvant properties and nanoscale dimensions. However, their inherent inflammatory properties can induce excessive immune reactions, potentially harming the host, while their variability in size and low yield pose challenges for large-scale applications. To address these challenges, we developed synthetic bacterial vesicles (SyBV) through bacterial cell lysis followed by detergent treatment and sonication to eliminate nucleic acids, producing high-purity vesicles with minimal cytoplasmic residue. SyBV triggered comparable antigen-specific adaptive immunity but avoided strong inflammatory responses. We engineered SyBV to display the prefusion F protein (preF) of respiratory syncytial virus (RSV) with cytotoxic lysin A (ClyA). This approach effectively induced preF-specific antibodies and robust immune responses. Our findings suggest that SyBV could provide a safer, more efficient vaccine platform for preventing viral infections, overcoming the limitations associated with traditional OMV while preserving immunogenicity.

Background: Human metapneumovirus (HMPV) causes respiratory tract infections among infant, elderly, and immunocompromised patients, with significant mortality. Currently no licensed vaccines or therapeutic agents of HMPV exist.Methods: HMPV virus-like particle (VLP) was constructed by co-expressing fusion protein of HMPV and matrix 1 protein of influenza virus using the baculovirus expression. Mice were immunized with VLP with or without aluminum hydroxide (alum) adjuvant by intramuscular route respectively. Sera were determined for titers of IgG and neutralizing antibody. Splenic lymphocytes were determined by IFN-gamma and IL-4 ELISPOT. Mice were chal-lenged with HMPV, and protective efficacy was evaluated.Results: We generated HMPV VLP in baculovirus expression system. After three times immunization, IgG anti-body titers induced by VLP formulated with or without alum adjuvant group were 273,066 +/- 100,331 and 136,533 +/- 47,269 respectively, there was no difference (p > 0.05); the neutralizing antibody titers vaccinated with VLP plus with alum adjuvant (266 +/- 92) were higher than those of the VLP alone group (106 +/- 37). For IFN-gamma, mice vaccinated with VLP with or without alum adjuvant are 151 +/- 36.4 and 77.0 +/- 17.1SFC/106 respectively, there was difference (p = 0.03); For IL-4, they are 261.3 +/- 38.7 versus 125.67 +/- 29.78SFC/106 respectively, the difference was significant (p = 0.009). After challenge, in pathological analysis, the overall lesion scores in the VLP plus with and without alum adjuvant were 3.25 and 5.6 respectively, those of control group is 8. For immunohistochemical analyses, the average optical density of the lungs in the VLP immunized group containing adjuvant (9.07 +/- 1.74) was lower than that in the VLP group without adjuvant (12.83 +/- 2.31, p = 0.14).Conclusions: This is the first study to demonstrate that HMPV VLP was successfully prepared in the baculovirus expression system. HMPV VLP could induce specific humoral and cellular immune responses as well as protective efficacy, and aluminum hydroxide may be an effective adjuvant in mice.

Abstract Background The type I human interferon (IFN) family consists of a group of cytokines with a multiplicity of biological activities, including antiviral, antitumor, and immunomodulatory effects. However, because the half-life of IFN is short, its clinical application is limited. Increasing the yield and biological activity of IFN while extending its half-life is currently the focus of IFN research. Results Two novel long-acting recombinant human IFN-α2b (rhIFN-α2b) proteins were designed in which the carboxyl-terminal peptide (CTP) of the human chorionic gonadotropin β su bunit and N-linked glycosylation sequences were linked to rhIFN-α2b. They were designated IFN-1CTPON (fused at the C-terminus of rhIFN-α2b) and IFN-2CTPON (fused at both the C-terminus and N-terminus of rhIFN-α2b). Monoclonal CHO cell strains stably and efficiently expressing the IFNs were successfully selected with methotrexate (MTX), and the highest expression levels were 1468 mg/l and 1196 mg/l for IFN-1CTPON and IFN-2CTPON, respectively. The proteins were purified with affinity chromatography and molecular sieve chromatography. IFN-1CTPON and IFN-2CTPON showed antiviral and antiproliferative activities in vitro. Notably, the half-life of IFN-1CTPON and IFN-2CTPON in vivo were three-fold and two-fold longer than that of commercially available rhIFN-α2b. Conclusions CHO cell strains stably expressing long-acting rhIFN-α2b were screened. The purified IFN-CTPON protein has biological activity and an extended half-life, and therefore potential applications.

自2007年GaynorA M等人通过高通量测序在肺炎患儿呼吸道样本中发现了WU多瘤病毒(Washington University polyomavirus,WUPyV)以来,WUPyV在世界各地被广泛检出.为了解北京地区急性呼吸道感染住院儿童中WUPyV感染情况及其临床特征,收集北京地区2017年4月至2018年3月共1 276份呼吸道感染住院患儿的鼻咽抽吸物样本,使用real time PCR方法对样本进行WUPyV检测,同时对WUPyV阳性样本进行17种常见呼吸道病毒筛查.共检出WUPyV阳性样本76份(5.96％,76/1276),4岁以下儿童居多(92.11％,70/76);WUPyV感染全年可见,无显著季节性;多伴随其他呼吸道病毒混合感染(60.53％,46/76),最常见混合感染为鼻病毒和A型流感病毒;WUPyV单一感染者与混合感染者病毒载量无显著性差异,临床诊断和表现基本一致;WUPyV感染患儿的常见诊断为支气管炎(68.42％,52/76)和肺炎(30.26％,23/76);临床症状主要表现为高热、咳嗽、咳痰.研究结果提示WUPyV是北京地区急性呼吸道感染住院患儿呼吸道样本中常见病毒之一,多见于4岁以下儿童.

ON1 is a novel genotype of human respiratory syncytial virus (HRSV) subtype A, in children with acute respiratory tract infections (ARTIs). However, there is not much data on the prevalence and clinical and molecular characterization in China. Our study is based on the children who had respiratory infections positive for RSV-A admitted by Gansu Provincial Maternity and Child-care Hospital in Lanzhou (northwestern China) during the last 7 epidemic seasons from 2010 to 2017. In our study, different strains of the novel RSV-A genotype ON1, first identified in Canada in December 2010, were first detected in Gansu Provincial Maternity and Child-care Hospital in August 2012 and then followed by an abrupt expansion in the number of ON1 variants in the beginning of 2014 and eventually replaced all other RSV-A strains from 2015 to 2017. ON1 is characterized by a 72-nt duplication in the C-terminal region of the highly variable attachment glycoprotein (G), predicted to lengthen the polypeptide with 24 amino acids, including a 23-aa duplication, which likely changes antigenicity. New N-glycosylation sites occurred within the 23-aa duplication and 24-aa insertion of the ON1 viruses in our study. Notably, RSV infections occurred later, but peaked sooner from the 2014/2015 to 2016/2017 epidemic seasons, compared with the previous 4 seasons. Our study concluded that genotype ON1 has caused larger outbreaks and became the predominate genotype for HRSV subgroup A in Lanzhou from 2013 to 2017, and became the sole genotype of RSV-A in 2015/2016 and 2016/2017. Our data indicate that northwest of China and the world will eventually be dominated by the ON1 RSV-A genotype, including the possibility for vaccine development. Based on trends seen in RSV-B BA genotype, which predominated for decades, there is a possibility to develop a vaccine for children in the next 10 years.

Background Lower respiratory tract illness is a major cause of morbidity and mortality in children worldwide, however, information about the epidemiological and clinical characteristics of LRTI s caused by HMPV and HB oV in China is limited. Objectives Human bocavirus ( HB oV) and human metapneumovirus ( HMPV ) are two important viruses for children with lower respiratory tract infections ( LRTI ). We aimed to assay the correlation between viral load and clinical characteristics of HB oV and HMPV with LRTI in Changsha, China. Methods Nasopharyngeal aspirates ( NPA s) from children with LRTI were collected. Real‐time PCR was used to screen HB oV and HMPV . Analyses were performed using SPSS 16.0 software. Results Pneumonia was the most frequent diagnosis. There was no significant difference between HB oV‐ and HMPV ‐positive patients in age ( P = .506) or hospitalization duration ( P = .280); 24.1% and 18.2% were positive for HB oV and HMPV . HB oV infections peaked in summer (32.2%), and HMPV infections peaked in winter (28.9%). The HB oV‐positive patients had a shorter hospitalization duration than the HB oV‐negative patients ( P = .021), and the HMPV ‐positive patients had a higher prevalence of fever than the HMPV ‐negative patients ( P = .002). The HB oV viral load was significantly higher among patients aged <1 year ( P = .006). The mean HB oV and HMPV viral loads were not significantly different between patients with single infections and coinfections. Patients infected with HB oV only were older than those coinfected with HB oV and other respiratory viruses ( P = .005). No significant difference was found in the clinical characteristics of patients infected with HMPV only and those coinfected with HMPV and other respiratory viruses. Conclusion Pneumonia was the most frequent diagnosis caused by HB oV and HMPV . Neither HB oV nor HMPV viral load was correlated with disease severity.

Pneumonia is a serious public health issue and is concerned around the world. This study is to investigate the association between viral load in children with human adenovirus (HAdV) pneumonia and disease severity.

AbstractLanzhou lamb rotavirus vaccine (LLR) is an oral live attenuated vaccine first licensed in China in 2000. To date, > 60 million doses of LLR have been distributed to children. However, very little is known about faecal shedding of LLR in children. Therefore, faecal samples (n = 1,184) were collected from 114 children for 15 days post-vaccination in September–November 2011/2012. Faecal shedding and viral loads were determined by an enzyme immunoassay kit (EIA) and real-time RT-PCR. The complete genome was sequenced and the vaccine strain was isolated by culture in MA104 cells. Approximately 14.0% (16/114) of children had rotavirus-positive samples by EIA for at least 1 day post-vaccination. Viral loads in EIA-positive samples ranged from < 1.0 × 103 to 1.9 × 108 copies/g. Faecal shedding occurred as early as post-vaccination day 2 and as late as post-vaccination day 13 and peaked on post-vaccination day 5–10. One LLR strain was isolated by culture in MA104 cells. Sequence analysis showed 99% identity with LLR prototype strain. Faecal shedding of LLR in stool is common within 15 days of LLR vaccination, indicating vaccine strains can replicate in human enteric tissues.

Objective To investigate the clinical and epidemiological characteristics of Coronavirus HKU1 ( Human CoV-HKU1) and NL63 ( Human CoV-NL63 ) in children with acute respiratory tract infection in Nanjing. Methods From August 2009 to July 2011, 1286 respiratory samples were collected from the outpatient and hospitalized children in the Children′ s Hospital of Nanjing Medical University. Reverse transcription polymerase chain reaction ( RT-PCR) was used to detect HCoV-HKU1 and NL63 genes, besides,positive samples were used for common respiratory virus screening. The positive amplification products were cloned, sequenced, homologous and phylogenetic analysis was conducted by molecular biological method . Results The detection rate of HCoV-HKU1 was 1. 1％ (14 / 1286), the positive sequences shared a 98. 2％ -100％ nucleotide identity with the HCoV-HKU1 strains and mixed infection rate was 92. 9％ . The main clinical diagnoses were bronchitis, bronchopneumonia and bronchiolitis. The clinical manifestations were cough, fever, wheezing. The detection rate of HCoV-NL63 was 1. 5％ (19 / 1286), the positive sequences shared a 95. 6％ -100％ nucleotide identity with the HCoV-NL63 strains and mixed infection rate were 63. 2％ . The main clinical diagnosis were acute upper respiratory tract infection, bronchitis, bronchopneumonia. The clinical manifestations were fever, cough, expectoration. No deaths were found in both HCoV-HKU1 and NL63 infections. Conclusions From August 2009 to July 2011, HCoV-HKU1 and NL63 were detected in children with respiratory tract infection in Nanjing area. HCoV-HKU1 infected cases were lower respiratory tract infection, epidemic in winter and spring, infected cases were mainly under 1 years of age, HCoV-NL63 infected cases including upper respiratory and lower respiratory tract infection, epidemic in the season of summer and autumn. The infected cases were mainly at the age rank from 1 year to 3 years. The clinical manifestations of children infected with coronavirus HKU1 and NL63 are not specificity.

To investigate the current genotypes of circulating human adenovirus (HAdV) strains, we molecularly genotyped HAdV in the nasopharyngeal aspirates (NPAs) of patients with acute lower respiratory tract infections (ALRTIs) and attempted to determine their associations with clinical symptoms. A total of 4751 NPA samples were collected from 4751 patients admitted to Hunan Provincial People's Hospital from September 2007 to March 2014, of which 447 (9.4%) samples were HAdV positive. Fourteen different HAdV types were identified; HAdV types 1 to 7 (HAdV 1-7) were identified in 95.7% of the 447 NPA samples with HAdV-7 and HAdV-3 being the most prevalent. In addition, 93.3% (417 of 447) of patients were younger than 5 years. The incidence of HAdV infection peaked in summer. Different HAdV types showed a predilection for different age groups and different seasonal distribution patterns. Coinfection of HAdVs and other respiratory viruses was detected in 63.3% (283 of 447) of the HAdV-positive samples. The most common clinical diagnosis was pneumonia and the most common symptoms were fever and cough. In comparison with children infected with HAdV-3 alone, those infected with HAdV-7 alone had an increased frequency of severe pneumonia involvement (11.6% vs 32.4%; P = 0.031), higher intensive care unit admission rates (7.0% vs 26.5%; P = 0.019), and a longer length of hospital stay (P = 0.03). Mixed infections in younger children were associated with a longer hospital stay (P = 0.023). Our results demonstrate the recent changes in the trends of circulating HAdV genotypes associated with ALRTIs in Hunan China.

Objective To investigate the human bocavirus (HBoV) infection in the children diagnosed with brochiolitis,incomparison with the clinical characteristics of bronchiolitis of HBoV vs.respiratory syncytial virus (RSV) infection alone.Methods A total of 396 throat swabs were obtained from children diagnosed with bronchiolitis from January 2015 to December 2016 in Pediatric Asthma Center of Gansu Maternal and Child Health Care Hospital,Lanzhou.Specimens were tested for the nucleic acids of HBoV,RSV and other 6 common respiratory viruses by nested-polymerase chain reaction (nested PCR ()and reverse transcription-polymerase chain reaction (RT-PCR),and the epidemic status,clinical characteristics of HBoV and RSV were analyzed and compared with each other.Results The positive rate of virus infection was 53.54％,and HBoV and RSV infections were 9.84％ and 24.49％ in 396 cases.The numbers of cases of HBoV single infection,RSV single infection,HBoV and RSV mixed infection were 29,86 and 5.The number of boys infected with HBoV was 27.There was statistical significance in the difference between the genders.The statistical significance also existed in difference of age.The babies at ages ranging from 6 to 12 months had the hifgest positive rate.The first incidence peak of the infection of HBoV was in October 2015.The children with HBoV infection,compared with RSV patients,presented more often with vomiting,diarrhea (P ＜ 0.05),while symptoms such as the occurrence of dyspnea,hypoxemia and the hospitalization time were all lower.Conclusions HBoV is one of the possible causes of brochiolitis in infants,its infection distributed over the whole year.The clinical symptoms,laboratory and radio-graphic changes are similar between the HBoV and RSV infections,the only difference was that the HBoV infection was comparatively mild.

Objective To express envelope protein of ZIKA virus in baculovirus expression system.Methods Full-length E gene of ZIKA virus was obtained by DNA synthesis and inserted into vector pFastBac1.The constructed recombinant baculovirus transfer vector pFB1-E was transformed to competent DH10Bac cells.The obtained skeleton plasmid rBacmid-E was transfected to sf9 cells,and the constructed recombinant baculovirus rBac-E was determined for titer,for insertion of E gene by PCR,and for expression of E protein by IFA and Western blotting.Results PCR proved that skeleton plasmid rBacmid-E was constructed correctly.The titer of rBac-E of passage 3 was 2.58 × 105 pfu/ ml.The genome of infected cells virus was extracted,the gene band at length of 3 830 bp was observed after PCR amplification.Indirect immunofluorescence of the infected cells showed the specific green fluorescence,55 × 103 specific band was determined by Western blotting identification in the cell pellet of the infected recombinant baculovirus rBacE.Conclusions The recombinant baculovirus with E gene of ZIKA virus was successfully constructed,which laid a foundation of further study on the function of E protein and the vaccine of ZIKA virus.

Objective To explore the viral pathogens of acute respiratory tract infection in Shanghai,and to provide reference data for diagnosis and treatment.Methods Between January 2012 and December 2013,643 nasopharyngeal aspiration (NPA) samples were collected from children who had been hospitalized for acute respiratory tract infection at Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine,the Ninth People's Hospital,Affiliated to Medical College of Shanghai Jiaotong University School and Putuo District Center Hospital Shanghai,China.Human rhinoviruses,Respiratory syncytial virus,Adenovirus,Influenza viruses A and B,Parainfluenza viruses 1-4,Human metapneumovirus,Human bocavirus,Human coronavirus HKU1 and Human coronavirus NL-63 were detected by reverse transcirption-polymerase chain reaction (RT-PCR) and standard polymerase chain reaction (PCR) methods.Epidemiology and clinical manifestations of each virus were analyzed.Results Six hundred and forty-three NPAs samples were collected from children (402 boys,241 girls) with ARTI,The mean age was 12 months(range from 11 days to 12 years).Viral pathogens were identified in 369 (57.4％) samples.RSV was the highest detection rate in 2012 and ADV was the highest detection rate in 2013,HCoV-NL63 was not detected.6 months to 1 year old age group virus detection rate was highest,along with the age growth,virus detection rate was falling,virus detection throughout the year,diagnosis includes upper and lower respiratory tract infection.Conclusions Viral pathogens are the main etiology of ARTI in children in Shanghai area.RSV and ADV are the most frequent viral pathogens.

Human parainfluenza viruses (HPIVs) are an important cause of acute lower respiratory tract infections (ALRTIs). HPIV‐4, a newly identified virus, has been associated with severe ALRTIs recently. A total of 771 nasopharyngeal aspirate samples were collected from hospitalized children between March 2010 and February 2011. HPIVs were detected by Nest‐PCR, and other known respiratory viruses were detected by RT‐PCR and PCR. All amplification products were sequenced. HPIVs were detected in 151 (19.58%) patients, of whom 28 (3.63%) were positive for HPIV‐4, 12(1.55%) for HPIV‐1, 4 (0.51%) for HPIV‐2, and 107 (13.87%) for HPIV‐3. Only three were found to be co‐infected with different types of HPIVs. All HPIV‐positive children were under 5 years of age, with the majority being less than 1 year. Only the detection rate of HPIV‐3 had a significant statistical difference (χ 2 = 29.648, P = 0.000) between ages. HPIV‐3 and HPIV‐4 were detected during the summer. Sixty (39.74%) were co‐infected with other respiratory viruses, and human rhinovirus (HRV) was the most common co‐infecting virus. The most frequent clinical diagnosis was bronchopneumonia, and all patients had cough; some patients who were infected with HPIV‐3 and HPIV‐4 had polypnea and cyanosis. No significant difference was found in clinical manifestations between those who were infected with HPIV‐4 and HPIV‐3. Two genotypes for HPIV‐4 were prevalent, although HPIV‐4a dominated. HPIV‐4 is an important virus for children hospitalized with ALRTIs in China. HRV was the most common co‐infecting virus. Two genotypes for HPIV‐4 are prevalent, HPIV‐4a dominated. J. Med. Virol. 88:2085–2091, 2016 . © 2016 Wiley Periodicals, Inc.

Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are two common viral pathogens in acute lower respiratory tract infections (ALRTI). However, the association of viral load with clinical characteristics is not well‐defined in ALRTI. To explore the correlation between viral load and clinical characteristics of RSV and HMPV in children hospitalized for ALRTI in Lanzhou, China. Three hundred and eighty‐seven children hospitalized for ALRTI were enrolled. Nasopharyngeal aspirates (NPAs) were sampled from each children. Real‐time PCR was used to screen RSV, HMPV, and twelve additional respiratory viruses. Bronchiolitis was the leading diagnoses both in RSV and HMPV positive patients. A significantly greater frequency of wheezing (52% vs. 33.52%, P = 0.000) was noted in RSV positive and negative patients. The RSV viral load was significant higher in children aged <1 year (P = 0.003), children without fever and wheezing (P = 0.015 and P = 0.000), days of illness <14 days (P = 0.002), children with bronchiolitis (P = 0.012) and children with RSV single infections (P = 0.000). No difference was found in the clinical features of HMPV positive and negative patients. The HMPV viral load had no correlation with any clinical characteristics. The incidences of severe disease were similar between single infection and coinfection for the two viruses (RSV, P = 0.221; HMPV, P = 0.764) and there has no statistical significance between severity and viral load (P = 0.166 and P = 0.721). Bronchiolitis is the most common disease caused by RSV and HMPV. High viral load or co‐infection may be associated with some symptoms but neither has a significant impact on disease severity for the two viruses. J. Med. Virol. 89:589–597, 2017. © 2016 Wiley Periodicals, Inc.

Objective The study was to investigation the contribution of 9 respiratory viruses and clinical features of refractory pneumonia in children in Lanzhou area.Methods In total,100 bronchoalveolar lavage fluid were collected from children younger than 14 years with refractory pneumonia between January 2013 and December 2013.The polymerase chain reaction (PCR) was used to screen human respiratory syncytial virus (HRSV),human metapneumovirus (HMPV),influenza viruses A and B (IFVA,IFVB),parainfluenza virus types 1 to 3 (PIV1-3),human rhinoviruses (HRVs),and human coronaviruses (HCoV-NL63 and HKU1),enterovirus (EV) using a standard reverse-transcription PCR technique,adenovirus(AdVs) and human Bocavirus (Bcov) using traditional PCR methods.All PCR-positive products were sequenced.Results Viral agents were identified in 53％ (53/100) of specimens,including AdV in 40％ (40/100),followed by RSV in 18％ (18/100),IFVB in 5％ (5/100),HRV in 4％ (4 /100),EV-68 was found in one case.Among AdV positive cases,AdV7 (33/40) was detected most frequently,followed by AdV2 in 15％ (6/40),AdV1 in 2.5％ (1/40),The coinfection rate of positive sample was 24.53％ (13/53).Conclusions Viruses play an important role in refractory pneumonia in children in Lanzhou area.AdV is an important viral agent in children with refractory pneumonia in Lanzhou City,especially AdV7.The frequency of fever,respiratory failure,cyanosis,and pulmonary consolidation were significant between AdV positive sample and negative sample.

Twelve cases of acute measles encephalitis without rash were identified from October 2011 to July 2013 in Changsha city, China; 5 were found to be genotype H1 and 2 were B3. Our data suggest that screening for measles virus is necessary in children with viral encephalitis, to eliminate the disease.

Hepatitis A virus (HAV) is a hepatotropic picornavirus that causes acute liver disease worldwide. Here, we report on the identification of a novel hepatovirus tentatively named Marmota Himalayana hepatovirus (MHHAV) in wild woodchucks (Marmota Himalayana) in China. The genomic and molecular characterization of MHHAV indicated that it is most closely related genetically to HAV. MHHAV has wide tissue distribution but shows tropism for the liver. The virus is morphologically and structurally similar to HAV. The pattern of its codon usage bias is also consistent with that of HAV. Phylogenetic analysis indicated that MHHAV groups with known HAVs but forms an independent branch, and represents a new species in the genus Hepatovirus within the family Picornaviridae. Antigenic site analysis suggested MHHAV has a new antigenic property to other HAVs. Further evolutionary analysis of MHHAV and primate HAVs led to a most recent common ancestor estimate of 1,000 years ago, while the common ancestor of all HAV-related viruses including phopivirus can be traced back to 1800 years ago. The discovery of MHHAV may provide new insights into the origin and evolution of HAV and a model system with which to explore the pathogenesis of HAV infection.