Juying Yan

Application of a loop-mediated isothermal amplification method for the detection of pathogenic Leptospira

Leptospirosis is an emerging infectious disease, which is considered to be the most widespread zoonotic disease in the world. There are more than 230 known serovars in the genus Leptospira. A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of pathogenic Leptospira spp. was developed and evaluated through amplification of the lipL41 gene coding for the outer membrane protein LipL41. The LAMP assay did not rely on the isolation and culture of leptospires, and no cross-reactivity was observed with other bacterial species. A SYBR Green I-based LAMP assay was also carried out for the real-time detection of DNA amplification. The lower detection limit of the LAMP assay was approximately 100 copies, which was the same as the polymerase chain reaction (PCR) and real-time PCR assays. The accuracy of the LAMP reaction was confirmed by restriction endonuclease analysis of the amplified product. The LAMP assay is easy to perform and inexpensive, and so may be applied in the rapid and specific diagnosis of Leptospira.

Rapid detection of H5 avian influenza virus by TaqMan‐MGB real‐time RT‐PCR

Aims:  Real‐time reverse transcription‐polymerase chain reaction (RT‐PCR) assay based on a TaqMan‐minor groove binder (MGB) probe was developed for the rapid detection of avian influenza virus subtype H5.

Origin and Characteristics of Internal Genes Affect Infectivity of the Novel Avian-Origin Influenza A (H7N9) Virus

Background Human infection with a novel avian-origin influenza A (H7N9) virus occurred continuously in China during the first half of 2013, with high infectivity and pathogenicity to humans. In this study, we investigated the origin of internal genes of the novel H7N9 virus and analyzed the relationship between internal genes and infectivity of the virus. Methodology and Principal findings We tested the environmental specimens using real-time RT-PCR assays and isolated five H9N2 viruses from specimens that were positive for both H7 and H9. Results of recombination and phylogeny analysis, performed based on the entire sequences of 221 influenza viruses, showed that one of the Zhejiang avian H9N2 isolates, A/environment/Zhejiang/16/2013, shared the highest identities on the internal genes with the novel H7N9 virus A/Anhui/1/2013, ranging from 98.98% to 100%. Zhejiang avian H9N2 isolates were all reassortant viruses, by acquiring NS gene from A/chicken/Dawang/1/2011-like viruses and other five internal genes from A/brambling/Beijing/16/2012-like viruses. Compared to A/Anhui/1/2013 (H7N9), the homology on the NS gene was 99.16% with A/chicken/Dawang/1/2011, whereas only 94.27-97.61% with A/bramnling/Beijing/16/2012-like viruses. Analysis on the relationship between internal genes and the infectivity of novel H7N9 viruses were performed by comparing amino acid sequences with the HPAI H5N1 viruses, the H9N2 and the earlier H7N9 avian influenza viruses. There were nine amino acids on the internal genes found to be possibly associated with the infectivity of the novel H7N9 viruses. Conclusions These findings indicate that the internal genes, sharing the highest similarities with A/environment/Zhejiang/16/2013-like (H9N2) viruses, may affect the infectivity of the novel H7N9 viruses.

Characterization of the complete genome of chikungunya in Zhejiang, China, using a modified virus discovery method based on cDNA-AFLP.

Background Chikungunya (CHIK) virus is a mosquito-borne emerging pathogen presenting great health challenges worldwide, particularly in tropical zones. Here we report a newly detected strain of CHIK, Zhejiang/chik-sy/2012, in China, a nonindigenous region for CHIK, using a modified approach based on the classic cDNA-AFLP. We then performed etiological and phylogenetic analyses to better understand its molecular characterization and phylogenetic pattern, and also to aid in further evaluating its persistence in Southeast Asia. Methods By using this modified procedure, we determined for the first time the complete genome sequence of the chikungunya virus strain, Zhejiang/chik-sy/2012, isolated in 2012 from a patient in Zhejiang, China. Sequence analyses revealed that this positive single strand of RNA is 12,017 bp long. We found no single amino acid mutation in A226V, D284E and A316V. Phylogenetic analysis showed that our strain shared the greatest homology with a strain isolated in Taiwan, which was derived from a strain from Indonesia. Chik-sy/2012 is in a different clade from other CHIK viruses found in China previously. Conclusions A modified cDNA-AFLP in virus discovery was used to isolate the first CHIK and the first complete genome sequence of virus strain chik-sy/2012 in 2012 from a patient with CHIK fever in Zhejiang, China. The infection displayed great phylogenetic distance from viruses detected in Guangdong, China, in 2008 and 2010, since they were derived from another evolutionary lineage. Additional molecular epidemiology data are needed to further understand, monitor and evaluate CHIK in China.

Inapparent Infection During an Outbreak of Dengue Fever in Southeastern China

Dengue fever (DF) is often asymptomatic in endemic areas. Asymptomatic infection during a DF outbreak in China, where DF is not endemic, has not been reported until now. In this study a total of 365 subjects from 6 villages were recruited from October 4-7, 2009. Overall, 102 subjects (27.95%) were positive for dengue virus (DENV) IgM, and 14 subjects (3.84%) were positive for DENV IgG and IgM. In different age groups, seropositive rates varied from 12.50% to 50.00% for DENV IgM, and from 0% to 11.76% for DENV IgG. Seroprevalence of DENV IgM was significantly higher than that of DENV IgG. Seroprevalence rates of DENV IgM differed among different villages. However, the seroprevalence of DENV IgM was not statistically significantly different among gender and age groups. Asymptomatic DF infection is prevalent in non-endemic areas.

Isolation and characterization of H7N9 avian influenza A virus from humans with respiratory diseases in Zhejiang, China.

In 2013, the novel reassortant avian-origin influenza A (H7N9) virus was reported in China. Through enhanced surveillance, infection by the H7N9 virus in humans was first identified in Zhejiang Province. Real-time reverse-transcriptase-polymerase-chain-reaction (RT-PCR) was used to confirm the infection. Embryonated chicken eggs were used for virus isolation from pharyngeal swabs taken from infected human patients. The H7N9 isolates were first identified by the hemagglutination test and electron microscopy, then used for whole genome sequencing. Bioinformatics software was used to construct the phylogenetic tree and for computing the mean rate of evolution of the HA gene in H7Nx and NA in HxN9. Two novel H7N9 avian influenza A viruses (A/Zhejiang/1/2013 and A/Zhejiang/2/2013) were isolated from the positive infection cases. Substitutions were found in both Zhejiang isolates and were identified as human-type viruses. All phylogenetic results indicated that the novel reassortant in H7N9 originated in viruses that infected birds. The sequencing and phylogenetic analysis of the whole genome revealed the mean rate of evolution of the HA gene in H7NX to be 5.74E-3 (95% Highest posterior density: 3.8218E-3 to 7.7873E-3) while the NA gene showed 2.243E-3 (4.378E-4 to 3.79E-3) substitutions per nucleotide site per year. The novel reassortant H7N9 virus was confirmed by molecular methods to have originated in poultry, with the mutations occurring during the spread of the H7N9 virus infection. Live poultry markets played an important role in whole H7N9 circulation.

Sero-Molecular Epidemiology of Japanese Encephalitis in Zhejiang, an Eastern Province of China.

Background Sporadic Japanese encephalitis (JE) cases still have been reported in Zhejiang Province in recent years, and concerns about vaccine cross-protection and population-level immunity have been raised off and on within the public health sphere. Genotype I (GI) has replaced GIII as the dominant genotype in Asian countries during the past few decades, which caused considerable concerns about the potential change of epidemiology characteristics and the vaccine effectiveness. The aim of this study was to investigate the prevalence of JE neutralizing antibody and its waning antibody trend after live attenuated JE vaccine immunization. Additionally, this study analyzed the molecular characteristics of the E gene of Zhejiang Japanese encephalitis virus (JEV) strains, and established genetic relationships with other JEV strains. Methodology/Principal Findings A total of 570 serum specimens were sampled from community population aged from 0 to 92 years old in Xianju county of Zhejiang Province in 2013–2014. Microseroneutralization test results were analyzed to estimate the population immunity and to observe antibody dynamics in vaccinated children. E genes of 28 JEV strains isolated in Zhejiang Province were sequenced for phylogenetic tree construction and molecular characteristics analysis with other selected strains. Positive JE neutralizing antibody rates were higher in residents ≥35 years old (81%~98%) and lower in residents <35 years old (0~57%). 7 or 8 years after the 2nd live attenuated vaccine dose, the antibodies against for 4 different strains with microseroneutralization test were decreased by 55%~73% on seropositive rates and by 25%~38% on GMTs respectively. JEV strains isolated in recent years were all grouped into GI, while those isolated in the 1980s belonged to GIII. On important amino acid sites related to antigenicity, there was no divergence between the Zhejiang JE virus strains and the vaccine strain (SA14-14-2). Conclusion/Significances JE neutralizing antibody positive rates increase in age ≥10 years old population, likely reflecting natural infection or natural boosting of immunity through exposure to wild virus. JE seropositivity rates were quite low in <35 years old age groups in Zhejiang Province. Waning of neutralizing antibody after live attenuated vaccine immunization was observed, but the clinical significance should be further investigated. Both the peripheral antibody response and genetic characterization indicate that current live attenuated JE vaccine conferred equal neutralizing potency against GI or GIII of wild strains. GI has replaced GIII as the dominant genotype in Zhejiang in the past few decades. Although the chance of exposure to wild JE virus has reduced, the virus still circulates in nature; therefore, it is necessary to implement immunization program for children continually and to conduct surveillance activity periodically.

Epidemiological Characterization of the 2017 Dengue Outbreak in Zhejiang, China and Molecular Characterization of the Viruses

Dengue, a mosquito-borne disease caused by the dengue virus (DV), has been recognized as a global public health threat. In 2017, an unexpected dengue outbreak occurred in Zhejiang, China. To clarify and characterize the causative agent of this outbreak, data on dengue fever cases were collected from the China Information System for Disease Control and Prevention in Zhejiang province for subsequent epidemiological analysis. A total of 1,229 cases were reported, including 1,149 indigenous and 80 imported cases. Most indigenous cases (1,128 cases) were in Hangzhou. The epidemic peak occurred in late August and early September, and the incidence rate of elderly people (4.34 per 100,000) was relatively high. Imported cases were reported all year round, and most were from South-East Asia and Western Pacific regions. Young people and men accounted for a large fraction of the cases. Acute phase serums of patients were collected for virus isolation. And 35 isolates (including 25 DV-2, 8 DV-1, 1 DV-3, and 1 DV-4) were obtained after inoculation and culture in mosquito C6/36 cells. The E genes of the 35 new DV isolates and the complete genome of a DV-2 isolate (Zhejiang/HZ33/2017), and the E gene of a DV-2 isolate from Ae. albopictus (Zhejiang/Aedes-1/2017) were determined. Phylogenetic analyses were performed using the neighbor-joining method with the Tajima-Nei model. Phylogenetically, DVs of all four serotypes with multiple genotypes (mainly including 21 Cosmopolitan genotype DV-2, 4 Asian I genotype DV-2, 6 genotype I DV-1, and 2 genotype V DV-1) were present in the indigenous and imported cases in Zhejiang during the same period. Most of the isolates probably originated from South-East Asia and Western Pacific countries. The imported cases, high density of mosquito vector, and missed diagnosis might contribute to the 2017 outbreak in Zhejiang.

Molecular Epidemiology and Prevalence of Echovirus 30 in Zhejiang Province, China, from 2002 to 2015

Echovirus serotype 30 (ECHO30) has been responsible for several recent worldwide outbreaks of viral meningitis. In Zhejiang Province, China, ECHO30 has been one of the main causes of viral meningitis for years. This study, using phylogenetic analysis of the VP1 gene, was performed to investigate the general molecular epidemiology and genetic patterns of ECHO30 circulating in Zhejiang Province between the years 2002 and 2015. The nucleotide sequences of ECHO30 VP1 showed that they were 64.8% identical with the prototype strain, Bastianni, while the amino acids were 84.9% identical. Phylogenetic analyses showed that ECHO30 in the Zhejiang area has diverged into two genotypes. Genotype I consists of strains isolated since 2002, whereas genotype II includes strains that were mainly isolated during the 2002 to 2004 outbreak. ECHO30 has been endemically circulating in both humans and the environment for a long period of time. Additionally, we evaluated the significance of recombination presented during the years 2005 to 2007 to demonstrate that recombination plays an important role in the prevalence of ECHO30 in the Zhejiang area.