Severity of enterovirus A71 infection in a human SCARB2 knock-in mouse model is dependent on infectious strain and route.

Enterovirus A71 (EV-A71) is a major etiological agent of human hand, foot and mouth disease, and it can cause severe neurological complications. Although several genotypes of EV-A71 strains are prevalent in different regions of the world, the genotype C4 has circulated in mainland China for more than 20 years. The pathogenicity of different EV-A71 clinical isolates varies and needs to be explored. In this study, hSCARB2 knock-in mice (N = 181) with a wide range of ages were tested for their susceptibility to two EV-A71 strains with the subgenotypes C4 and C2, and two infection routes (intracranial and venous) were compared. The clinical manifestations and pathology and their relationship to the measured viral loads in different tissues were monitored. We observed that 3 weeks is a crucial age, as mice younger than 3-week-old that were infected became extremely ill. However, mice older than 3 weeks displayed diverse clinical symptoms. Significant differences were observed in the pathogenicity of the two strains with respect to clinical signs, disease incidence, survival rate, and body weight change. We concluded that hSCARB2 knock-in mice are a sensitive model for investigating the clinical outcomes resulting from infection by different EV-A71 strains. The intracranial infection model appears to be suitable for evaluating EV-A71 neurovirulence, whereas the venous infection model is appropriate for studying the pathogenicity of EV-A71.

[Prevalence and Analyses of the Changing Etiology of Hand, Foot and Mouth Disease in China].

Hand, foot, and mouth disease (HFMD) is a viral infectious disease regarded to be a public-health problem worldwide. Since the 1990s, HFMD began to spread in the Asia-Pacific region (especially in South-East Asia). HFMD outbreaks have occurred in mainland China frequently since 2008, and the morbidity and mortality of HFMD has continued to increase in recent years. In mainland China, enterovirus A serotype enterovirus A71 (EV-A71) and coxsackievirus A16 (CVA16) have been the major pathogens of HFMD during these years. However, the etiological spectrum of HFMD changes with time. This review focuses mainly on the etiological spectrum of HFMD and changes in epidemic patterns in mainland China.

Phylogenetic analysis of Enterovirus 71 circulating in Beijing, China from 2007 to 2009.

The major pathogens of hand, foot and mouth disease (HFMD) in Beijing, China from 2007 to 2009 were identified in this study. A total of 186 HFMD cases were included, and 136 cases (73%) were positive for enterovirus (EV). In 2007, 75% (27/36) were Coxsackievirus A16 (CA16) positive and 19% (7/36) were Enterovirus 71 (EV71) positive cases. However, EV71 was the predominant virus in 2008, when 56% (31/55) of the cases were positive for EV71 and 22% (12/55) were positive for CA16. In 2009, EV71 and CA16, with positive rates of 36% (16/45) and 29% (13/45), respectively, were still the major pathogens of HFMD. Phylogenetic analysis revealed that the dominant genotype of EV71 was C4, with co-circulation of genotype A in 2009. The prevalent cluster of the EV71 subgenotype C4 changed over time. A proposed new sublineage of EV71, C4a-2, was the predominant virus associated with the Beijing and nationwide HFMD outbreaks since 2008 and amino acid substitution, which possibly link to the central nervous system tropism of EV71, was found in genotype A viruses. Persistent surveillance of HFMD-associated pathogens is required for predicting potential emerging viruses and related disease outbreaks.

NS2B/3 proteolysis at the C-prM junction of the tick-borne encephalitis virus polyprotein is highly membrane dependent.

The replication of tick-borne encephalitis virus (TBEV), like that of all flaviviruses, is absolutely dependent on proteolytic processing. Production of the mature proteins C and prM from their common precursor requires the activity of the viral NS2B/3 protease (NS2B/3(pro)) at the C-terminus of protein C and the host signal peptidase I (SPaseI) at the N-terminus of protein prM. Recently, we have shown in cell culture that the cleavage of protein C and the subsequent production of TBEV particles can be made dependent on the activity of the foot-and-mouth disease virus 3C protease, but not on the activity of the HIV-1 protease (HIV1(pro)) (Schrauf et al., 2012). To investigate this failure, we developed an in vitro cleavage assay to assess the two cleavage reactions performed on the C-prM precursor. Accordingly, a recombinant modular NS2B/3(pro), consisting of the protease domain of NS3 linked to the core-domain of cofactor NS2B, was expressed in E. coli and purified to homogeneity. This enzyme could cleave a C-prM protein synthesised in rabbit reticulocyte lysates. However, cleavage was only specific when protein synthesis was performed in the presence of canine pancreatic microsomal membranes and required the prevention of signal peptidase I (SPaseI) activity by lengthening the h-region of the signal peptide. The presence of membranes allowed the concentration of NS2B/3(pro) used to be reduced by 10-20 fold. Substitution of the NS2B/3(pro) cleavage motif in C-prM by a HIV-1(pro) motif inhibited NS2B/3(pro) processing in the presence of microsomal membranes but allowed cleavage by HIV-1(pro) at the C-prM junction. This system shows that processing at the C-terminus of protein C by the TBEV NS2B/3(pro) is highly membrane dependent and will allow the examination of how the membrane topology of protein C affects both SPaseI and NS2B/3(pro) processing.

[Primary detection of pathogen from children with hand, foot, and mouth disease in Beijing, 2007].

We investigated the causative agents of hand, foot and mouth disease (HFMD) in children in Beijing epidemic from April to June of 2007. Totally, 82 specimens (including throat swabs, rectal swabs and vesicular swabs) were collected from 51 patients with HFMD. All patients had typical skin lesions, but no neurological manifestations. These clinical specimens were directly tested by RT-PCR assay with three primer sets: universal enterovirus, CA16-specific and EV71-specific primers. Enterovirus, CA16 and EV71 were identified depending on the size of PCR products. The percentage of the specimens identified as positive for enterovirus was 70.6%. Of the 51 cases, 25 were positive for CA16, 4 were positive for EV71, and 7 were enterovirus positive of non-CA16 or EV71. The ratio among them was about 6:1:2. EV71 strains belonged to genotype C4 after sequencing and phylogenetic analysis.

Genetic characteristics of human enterovirus 71 and coxsackievirus A16 circulating from 1999 to 2004 in Shenzhen, People's Republic of China.

The genetic and phylogenetic characteristics of human enterovirus 71 (EV71) and coxsackievirus A16 (CA16) sampled from children with hand, foot, and mouth disease in Shenzhen, People's Republic of China, over a 6-year period (1999 to 2004) were examined with reverse transcription-PCR and DNA sequencing. Out of 147 stool specimens, 60 showed positive signals when screened with EV71- and CA16-specific primers. EV71 was identified in 19 specimens, and CA16 was identified in 41 specimens; coinfection by EV71 and CA16 was not observed. Phylogenetic analysis of all EV71 strains isolated from the mainland Chinese samples established C4 as the predominant genotype. Only one other known strain (3254-TAI-98; AF286531), isolated in Taiwan in 1998, was identified as belonging to genotype C4. Phylogenetic analysis of CA16 strains allowed us to identify three new genetic lineages (A, B, and C), with lineage C recently predominating in Asian countries, such as the People's Republic of China, Malaysia, and Japan. These new observations indicate that CA16 circulating in the People's Republic of China is genetically diverse, and additional surveillance is warranted.