Yaqing He

Emergence, circulation and spatiotemporal phylogenetic analysis of Coxsackievirus A6 and Coxsackievirus A10 associated hand, foot and mouth disease infections from 2008 to 2012 in Shenzhen, China

ABSTRACT Sporadic hand, foot, and mouth disease (HFMD) outbreaks and other infectious diseases in recent years have frequently been associated with certain human enterovirus (HEV) serotypes. This study explored the prevalences and genetic characteristics of non-HEV71 and non-coxsackievirus A16 (CV-A16) human enterovirus-associated HFMD infections in Shenzhen, China. A total of 2,411 clinical stool specimens were collected from hospital-based surveillance for HFMD from 2008 to 2012. The detection of HEV was performed by real-time reverse transcription-PCR (RT-PCR) and RT-seminested PCR, and spatiotemporal phylogenetic analysis was performed based on the VP1 genes. A total of 1,803 (74.8%) strains comprising 28 different serotypes were detected. In the past 5 years, the predominant serotypes were HEV71 (60.0%), followed by CV-A16 (21.2%) and two uncommon serotypes, CV-A6 (13.0%) and CV-A10 (3.3%). However, CV-A6 replaced CV-A16 as the second most common serotype between 2010 and 2012. As an emerging pathogen, CV-A6 became as common a causative agent of HFMD as HEV71 in Shenzhen in 2012. Phylogenetic analysis revealed that little variation occurred in the Chinese HEV71 and CV-A16 strains. The genetic characteristics of the Chinese CV-A6 and CV-A10 strains displayed geographic differences. The CV-A6 and CV-A10 strains circulating in Shenzhen likely originated in Europe. It was found that human enteroviruses have a high mutation rate due to evolutionary pressure and frequent recombination (3.2 × 10−3 to 6.4 ×10−3 substitutions per site per year for HEV71, CV-A6, CV-A16, and CV-A10). Since certain serotypes are potential threats to the public health, this study provides further insights into the significance of the epidemiological surveillance of HFMD.

Potent inhibition of human enterovirus 71 replication by type I interferon subtypes.

BACKGROUND Enterovirus 71 (EV71) infection can induce a series of syndromes including herpangina, viraemia, hand-foot-and-mouth disease and even death. Outbreaks of EV71 infection have been reported periodically over the world and have caused a great number of casualties and a high medical expenditure. Some interferons (IFNs) have been used for the treatment of viral infections for decades; however, conventional IFNs only display mild anti-EV71 activities. No effective drug is currently available for the treatment of EV71 infection. Here, we aimed to investigate whether some IFN subtypes display potent anti-EV71 activities. METHODS The antiviral activities of 17 type I IFNs were assayed in Vero cells using the cytopathic effect method. Cells were incubated with different concentrations of type I IFNs before or after virus infection. Viral replication was determined by quantitative real-time PCR (qRT-PCR). The expression levels of IFN downstream antiviral genes were also measured by qRT-PCR. RESULTS Out of 17 type I IFNs, 4 IFNs (IFN-α4, IFN-α6, IFN-α14 and IFN-α16) displayed potent antiviral activity. Compared with conventional IFN-α2a, IFN-α14 displayed approximately 20× higher antiviral activity. The superior antiviral effect of IFN-α14 was caused by a strong induction of the downstream antiviral effectors. CONCLUSIONS IFN-α14 and three other IFNs could be considered for the treatment of EV71 infection.

Viral kinetics of Enterovirus 71 in human abdomyosarcoma cells

AIM To characterise the viral kinetics of enterovirus 71 (EV71). METHODS In this study, human rhabdomyosarcoma (RD) cells were infected with EV71 at different multiplicity of infection (MOI). After infection, the cytopathic effect (CPE) was monitored and recorded using a phase contrast microscope associated with a CCD camera at different time points post viral infection (0, 6, 12, 24 h post infection). Cell growth and viability were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in both EV71 infected and mock infected cells at each time point. EV71 replication kinetics in RD cells was determined by measuring the total intracellular viral RNA with real-time reverse-transcription polymerase chain reaction (qRT-PCR). Also, the intracellular and extracellular virion RNA was isolated and quantified at different time points to analyze the viral package and secretion. The expression of viral protein was determined by analyze the levels of viral structure protein VP1 with Western blotting. RESULTS EV71 infection induced a significant CPE as early as 6 h post infection (p.i.) in both RD cells infected with high ratio of virus (MOI 10) and low ratio of virus (MOI 1). In EV71 infected cells, the cell growth was inhibited and the number of viable cells was rapidly decreased in the later phase of infection. EV71 virions were uncoated immediately after entry. The intracellular viral RNA began to increase at as early as 3 h p.i. and the exponential increase was found between 3 h to 6 h p.i. in both infected groups. For viral structure protein synthesis, results from western-blot showed that intracellular viral protein VP1 could not be detected until 6 h p.i. in the cells infected at either MOI 1 or MOI 10; and reached the peak at 9 h p.i. in the cells infected with EV71 at both MOI 1 and MOI 10. Simultaneously, the viral package and secretion were also actively processed as the virus underwent rapid replication. The viral package kinetics was comparable for both MOI 1 and MOI 10 infected groups. It was observed that at 3 h p.i, the intracellular virions obviously decreased, thereafter, the intracellular virions began to increase and enter into the exponential phase until 12 h p.i. The total amounts of intracellular virons were decreased from 12 to 24 h p.i. Consistent with this result, the increase of virus secretion occurred during 6 to 12 h p.i. CONCLUSION The viral kinetics of EV71 were established by analyzing viral replication, package and secretion in RD cells.

Norovirus GII.P16/GII.2–Associated Gastroenteritis, China, 2016

During October–December 2016, the number of norovirus outbreaks in China increased sharply from the same period during the previous 4 years. We identified a recombinant norovirus strain, GII.P16-GII.2, as the cause of 44 (79%) of the 56 outbreaks, signaling that this strain could replace the predominant GII.4 viruses.

Characterization of severe hand, foot, and mouth disease in Shenzhen, China, 2009–2013

Hand, foot, and mouth disease (HFMD) is caused by human enteroviruses, especially by enterovirus 71 (EV71) and coxsackievirus A16 (CA16). Patients infected with different enteroviruses show varied clinical symptoms. The aim of this study was to determine whether the etiological spectrum of mild and severe HFMD changed, and the association between pathogens and clinical features. From 2009 to 2013, a total of 2,299 stool or rectal specimens were collected with corresponding patient data. A dynamic view of the etiological spectrum of mild and severe HFMD in Shenzhen city of China was provided. EV71 accounted for the majority proportion of severe HFMD cases and fatalities during 2009–2013. CA16 and EV71 were gradually replaced by coxsackievirus A6 (CA6) as the most common serotype for mild HFMD since 2010. Myoclonic jerk and vomiting were the most frequent severe symptoms. Nervous system complications, including aseptic encephalitis and aseptic meningitis were observed mainly in patients infected by EV71. Among EV71, CA16, CA6, and CA10 infection, fever and pharyngalgia were more likely to develop, vesicles on the hand, foot, elbow, knee and buttock were less likely to develop in patients infected with CA10. Vesicles on the mouth more frequently occurred in the patients with CA6, but less in the patient with EV71. Associations between diverse enterovirus serotypes and various clinical features were discovered in the present study, which may offer further insight into early detection, diagnosis and treatment of HFMD. J. Med. Virol. 87:1471–1479, 2015.

Two gastroenteritis outbreaks caused by GII Noroviruses: host susceptibility and HBGA phenotypes.

Noroviruses (NoVs) cause epidemic acute gastroenteritis, in which histo-blood group antigens (HBGAs) may play an important role in the host susceptibility. To further explore this issue, two outbreaks of acute gastroenteritis caused by a GII.4 and a GII.3 NoV, respectively, in China in 2009 were studied. Stool and saliva samples from symptomatic patients and water samples from the outbreak facilities were collected. RT-PCR showed that 23 out of 33 (GII.4 outbreak) and 12 out of 13 (GII.3outbreak) stool samples were NoV positive. For the GII.4 outbreak the NoV sequences of stool and water samples were from an identical GII.4 strain, while the same GII.3 NoV sequences were found in five stool samples from the GII.3 outbreak. The HBGA phenotypes (A, B, Lea, Leb, Lex, and Ley) of all saliva samples were determined, which revealed both secretors and nonsecretors in the symptomatic groups of the two outbreaks. In the GII.3 outbreak, type O individuals appeared less susceptible, while the type A may be more at risk of infection. However, No preference of HBGAs was observed in the GII.4 outbreak. The observation that nonsecretors were infected in both outbreaks differed from the previous results that nonsecretors are resistant to these two GII NoVs.

Molecular Phylogeny of Coxsackievirus A16 in Shenzhen, China, from 2005 to 2009

ABSTRACT Phylogenetic analysis of a Coxsackievirus A16 (CA16) sequence from Shenzhen, China, and other Chinese and international CA16 sequences revealed a pattern of endemic cocirculation of strains of clusters B2a and B2b within subtype B2 viruses. Amino acid evolution and nucleotide variation in the VP1 region were slight for 5 years.

Seroprevalence of rubella in female migrant factory workers in Shenzhen, China.

BACKGROUND Rubella remains a common disease in Mainland China and is a major cause of severe birth defects from Congenital Rubella Syndrome (CRS). Rubella-containing vaccines were not included in Chinas National Expanded Program of Immunization (NEPI) until December 2007. In Shenzhen, women of childbearing age make up a large percentage of its migrant factory worker population. Understanding their immunity to rubella is critical in furthering efforts towards rubella vaccination programs. OBJECTIVES To investigate the seronegativity of rubella antibodies and evaluate potential associates of rubella immunity among female migrant factory workers in Shenzhen, China. SUBJECTS AND METHODS Serum samples were collected from 518 female migrant workers, aged 18-55, working in 44 randomly selected factories in Shenzhen, China during May through June of 2009. Samples were tested for Rubella Immunoglobulin G (IgG) using a commercial Enzyme-linked immunosorbant assay kit. Self-reported vaccination histories and socio-demographic information were also collected. RESULTS Of 518 female workers, 402 (77.6%) were immune to rubella. Significant differences in seronegativity were dependent on region of origin, being without a job contract, age group, marital status and seronegativity of measles. CONCLUSIONS Seroprevalence of antibodies to rubella in Shenzhen, China amongst female migrant workers is too low to provide immunity in the population. Given the high numbers of women of childbearing age amongst Shenzhen migrant factory workers coming from many provinces across China, local health authorities in Shenzhen should consider combining new rubella immunization programs with existing measles immunization efforts in this population.

Characterization of the new GII.17 norovirus variant that emerged recently as the predominant strain in China.

Human noroviruses are the most important viral pathogens causing epidemic acute gastroenteritis, in which the GII.4 viruses have been predominant worldwide for the past decades. During 2014-2015 winter season, a new GII.17 variant emerged as the predominant virus in China surpassing the GII.4 virus in causing significantly increased acute gastroenteritis outbreaks. Genome sequences of the new GII.17 variant was determined and compared with other GII.17 noroviruses, revealing residue substitutions at specific locations, including the histo-blood group antigen-binding site and the putative antigenic epitopes. Further study of GII.17 outbreaks focusing on host susceptibility showed that the new GII.17 variant infected secretor individuals of A, B, O and Lewis types. Accordingly, the P particles of the new GII.17 variant bound secretor saliva samples of A, B, O and Lewis types with significantly higher binding signals than those of the P particles of the previous GII.17 variants. In addition, human sera collected from the outbreaks exhibited stronger blockade against the binding of the new GII.17 P particles to saliva samples than those against the binding between the P particles of previous GII.17 variants and saliva samples. Taken together, our data strongly suggested that the new GII.17 variant gained new histo-blood group antigen-binding ability and antigenic features, which may contribute to its predominance in causing human norovirus epidemics.

Molecular Identification and Analysis of Human Enteroviruses Isolated from Healthy Children in Shenzhen, China from 2010 to 2011

Objective To determine the prevalence and distribution of human enteroviruses (HEVs) among healthy children in Shenzhen, China. Method Clinical specimens were obtained from 320 healthy children under 5 years old in Shenzhen, China from 2010 to 2011. The specimens were evaluated using real-time PCR and cell cultures. The positive specimens were further tested using reverse transcription-seminested PCR (RT-snPCR). Molecular typing and phylogenetic analysis were based on the sequence determined. Results Among the 320 samples, 34 were tested positive for HEVs (10.6%) and 22 different serotypes were identified using RT-snPCR. PV1 and PV2 were also detected. The predominant serotype observed was EV71 (17.6%), followed by CV-B4 (14.7%). HEV-B was detected most frequently, with an overall prevalence of 47.1%. HEV-A and HEV-C were found in 32.3% and 20.6% of the samples, respectively. No HEV-D was identified. Molecular phylogeny indicated that all EV71 strains were of C4 genotype. Conclusion Although a variety of HEVs was detected in healthy children, HEV-B was relatively more prevalent than other HEV species. Considering HEV-A is more prevalent than HEV-B among patients with hand-foot-mouth disease, additional long-term surveillance of HEV is warranted in both asymptomatic and symptomatic populations.