The genus bocavirus includes bovine parvovirus (BPV), minute virus of canines (MVC), and a group of human bocaviruses (HBoV1-4). Using sequence-independent single primer amplification (SISPA), a novel bocavirus group was discovered with high prevalence (12.59%) in piglet stool samples. Two nearly full-length genome sequences were obtained, which were approximately 5,100 nucleotides in length. Multiple alignments revealed that they share 28.7–56.8% DNA sequence identity with other members of Parvovirinae. Phylogenetic analyses indicated their closest neighbors were members of the genus bocavirus. The new viruses had a putative non-structural NP1 protein, which was unique to bocaviruses. They were provisionally named porcine bocavirus 1 and 2 (PBoV1, PBoV2). PBoV1 and PBoV2 shared 94.2% nucleotide identity in NS1 gene sequence, suggesting that they represented two different bocavirus species. Two additional samples (6V, 7V) were amplified for 2,407 bp and 2,434 bp products, respectively, including a partial NP1 gene and the complete VP1 gene; Phylogenetic analysis indicated that 6Vand 7V grouped with PBoV1 and PBoV2 in the genus of bocavirus, but were in the separate clusters. Like other parvoviruses, PBoV1, PBoV2, 6Vand 7V also contained a putative secretory phospholipase A₂ (sPLA₂) motif in the VP1 unique region, with a conserved HDXXY motif in the catalytic center. The conserved motif YXGXF of the Ca²⁺-binding loop of sPLA2 identified in human bocavirus was also found in porcine bocavirus, which differs from the YXGXG motif carried by most other parvoviruses. The observation of PBoV and potentially other new bocavirus genus members may aid in molecular and functional characterization of the genus bocavirus.

Enterovirus 71 (EV71) has emerged as one of the most important enteroviruses since the eradication of poliovirus, and it causes severe neurological symptoms for which no effective antiviral drugs are available. Type I interferons (IFN) α/β have been used clinically as antiviral therapy as the first line of defense against virus infections successfully for decades. However, treatment with type I interferons has not been effective in patients with EV71 infection. In this study, we found that in cells pretreated with IFN-β, EV71 infection could still lead to a cytopathic effect, and the viral replication was not affected. The mechanism by which EV71 antagonizes interferon signaling, however, has been controversial. Our study indicated that EV71 infection did not inhibit phosphorylation of STAT1/2 induced by IFN-β stimulation, but p-STAT1/2 transport into the nucleus was significantly blocked. We showed that EV71 infection reduced the formation of STAT/karyopherin-α1 (KPNA1) complex upon interferon stimulation and that the virus down-regulated the expression of KPNA1, a nuclear localization signal receptor for p-STAT1. Using specific caspase inhibitors and siRNA for caspase-3, we demonstrated that EV71 infection induced degradation of cellular KPNA1 in a caspase-3-dependent manner, which led to decreased induction of interferon-inducible genes and IFN response. Viral 2A and 3C proteases did not degrade KPNA1, inhibit the activity of ISRE or suppress the transcription of interferon-inducible genes induced by IFN-β. Our study demonstrates a novel mechanism by which antiviral signaling is suppressed through degradation of KPNA1 by activated caspase-3 induced in an enteroviral infection.

Severe complications associated with EV71 infections caused many infants death. However, the pathogenesis of EV71 infection in the severe cases remained poorly understood. In this study we collected plasma and cerebrospinal fluid (CSF) specimens drawn in the acute and/or recovery phases from EV71-infected individuals, and plasma specimens from healthy children served as normal controls. We compared the levels of cytokines and chemokines determined by a Luminex-based cytokine bead array. The plasma levels of IL-1β and IL-6 were significantly higher in severe and critical cases than in mild patients and normal controls. Higher plasma levels of IL-6, IL-10, and IL-8 were evident in critical than severe cases. The CSF levels of IL-6, IL-8, and IP-10 were higher, and that of RANTES lower (compared to plasma), in severe and critical patients. Significantly lower CSF levels of cytokines and chemokines were recorded in the recovery than the acute phase in severe and critical cases treated with intravenous immunoglobulin (IVIG) and glucocorticoids. Only the CSF levels of IL-6, IP-10, and IL-8 were significantly correlated with white blood cell counts, and absolute neutrophil and monocyte counts, in severe cases. Furthermore, the CSF levels of IL-6 were correlated with temperature in both cases. These data indicate that a major cytokine response and inflammation, in both plasma and the CNS, are features of disease caused by EV71 infection. Systemic inflammation caused by EV71 infection exacerbated the deterioration of the disease, and resulted in the disease progression to the critical illness stage.

Severe hand, foot, and mouth disease (HFMD) is a common childhood illness caused by various enteroviruses. The disease has imposed increased burden on children younger than 5 years old. We aimed to determine the epidemiology, CNS complication, and etiology among severe HFMD patients, in Jiangsu, China. Epidemiological, clinical, and laboratory data of severe HFMD cases were extracted from 2009 to 2015. The CNS complication, annually severe illness rates, mortality rates, severity-PICU admission rates, severity-hospitalization rates, and so on were analyzed to assess the disease burden of severe HFMD. All analyses were stratified by time, region, population, CNS involvement and serotypes. The VP1 gene from EV-A71, CV-A16, CV-A6, CV-A10 and other enteroviruses isolates was amplified. Phylogenetic analysis was performed using MEGA5.0. Seven thousand nine hundred ninety-four severe HFMD cases were reported, of them, 7224 cases were inpatients, 611 were PICU inpatients, and 68 were fatal. The average severe illness rate, mortality rate, severity−fatality rate, severity-PICU admission rate, and severity-hospitalization rate were 14.54, 0.12,8506, 76,430, and 903,700 per 1 million, respectively. The severe illness rate was the highest in the 12–23 months age group, and the greatest mortality rate was in the 6–11 months age group. Geographical difference in severe illness rate and mortality were found. Patients infected with EV-A71 were at a higher proportion in different CNS involvement even death. EV-A71, CV-A16 and other enteroviruses accounted for 79.14, 6.49, and 14.47%, respectively. A total of 14 non-EV-A71/ CV-A16 genotypes including CV-A2, CV-A4, CV-A 6, CV-A9, CV-A10, CV-B1, CV-B2, CV-B3, CV-B4, CV-B5, E-6, E-7, E-18, and EV-C96 were identified. Phylogentic analyses demonstrated that EV-A71 strains belonged to subgenotype C4a, while CV-A16 strains belonged to sub-genotype B1a and sub-genotype B1b of genotype B1. CV-A6 strains were assigned to genogroup F, and CV-A10 strains belonged to genogroup D. Future mitigation policies should take into account the age, region heterogeneities, CNS conditions and serotype of disease. Additional a more rigorous study between the mild and severe HFMD should be warranted to elucidate the difference epidemiology, pathogen spectrum and immunity patterns and to optimize interventions in the following study.

Background: The levels of proinflammatory cytokine or chemokine in blood and cerebrospinal fluid are thought to be one of predictors for clinical severity of enterovirus 71 (EV71) infection, yet the cellular sources or signalling mechanism remain undefined. Here, we focused on the response of human primary monocyte-derived macrophages (MDMs) to EV71 virus and its possible mechanisms. Methods: Human primary MDMs were infected by EV71 virus in vitro. Infectivity and viral replication were assayed, and cytokine responses were determined by Cytometric Bead Array(CBA) analysis. The relative changes of Toll-like receptors, retinoic acid-inducible gene I (RIG-I) and melamoma differentiation associated gene 5 (MDA5) mRNA expression were detected by real-time RT-PCR. Results: Effective infection and viral replication were detected in EV71-infected MDMs. The titters of progeny virus released from EV71-infected MDMs gradually increased from 6-h to 48-h point of infection (POI.). Proinflammatory cytokines: IL-1, IL-6, TNF-α but not IFN-α and γ were induced in MDMs by EV71. EV71 infection significantly increased the release of IL-8, IP-10 and RANTES at 12-h or 24-h POI. Upregulation of TLR2, TLR7 and TLR8 mRNA expression rather than TLR3, TLR4, TLR6, TLR9, TLR10, RIG-I, MDA5 were found at different time points in EV71-infected MDMs. Conclusions: Our findings suggested that macrophages are not only the important target cells but also the effectors during EV71 infection, and they may play an important role in the pathogenesis of EV71 infection. And the proinflammatory cytokine and chemokine responses in EV71-infected MDMs may be mediated by the activation of differential pattern of TLRs.