Phylogenetic evidence for inter-typic recombination in the emergence of human enterovirus 71 subgenotypes

BACKGROUND: Human enterovirus 71 (EV-71) is a common causative agent of hand, foot and mouth disease (HFMD). In recent years, the virus has caused several outbreaks with high numbers of deaths and severe neurological complications. Several new EV-71 subgenotypes were identified from these outbreaks. The mechanisms that contributed to the emergence of these subgenotypes are unknown. RESULTS: Six EV-71 isolates from an outbreak in Malaysia, in 1997, were sequenced completely. These isolates were identified as EV-71 subgenotypes, B3, B4 and C2. A phylogenetic tree that correlated well with the present enterovirus classification scheme was established using these full genome sequences and all other available full genome sequences of EV-71 and human enterovirus A (HEV-A). Using the 5' UTR, P2 and P3 genomic regions, however, isolates of EV-71 subgenotypes B3 and C4 segregated away from other EV-71 subgenotypes into a cluster together with coxsackievirus A16 (CV-A16/G10) and EV-71 subgenotype C2 clustered with CV-A8. Results from the similarity plot analyses supported the clustering of these isolates with other HEV-A. In contrast, at the same genomic regions, a CV-A16 isolate, Tainan5079, clustered with EV-71. This suggests that amongst EV-71 and CV-A16, only the structural genes were conserved. The 3' end of the virus genome varied and consisted of sequences highly similar to various HEV-A viruses. Numerous recombination crossover breakpoints were identified within the non-structural genes of some of these newer EV-71 subgenotypes. CONCLUSION: Phylogenetic evidence obtained from analyses of the full genome sequence supports the possible occurrence of inter-typic recombination involving EV-71 and various HEV-A, including CV-A16, the most common causal agent of HFMD. It is suggested that these recombination events played important roles in the emergence of the various EV-71 subgenotypes

Recombinant human enterovirus 71 in hand, foot and mouth disease patients

Hand, foot and mouth disease (HFMD) is a common illness of infants and young children less than 10 years of age. It is characterized by fever, ulcers in the oral cavity, and rashes with blisters that appear on the palm and sole. The most common causal agents of HFMD are coxsackievirus A16 (CV-A16) and human enterovirus 71 (HEV71), but other enteroviruses, including CV-A5 and CV-A10, can also cause it

Enterovirus 71 uses cell surface heparan sulfate glycosaminoglycan as an attachment receptor

Enterovirus 71 (EV-71) infections are usually associated with mild hand, foot, and mouth disease in young children but have been reported to cause severe neurological complications with high mortality rates. To date, four EV-71 receptors have been identified, but inhibition of these receptors by antagonists did not completely abolish EV-71 infection, implying that there is an as yet undiscovered receptor(s). Since EV-71 has a wide range of tissue tropisms, we hypothesize that EV-71 infections may be facilitated by using receptors that are widely expressed in all cell types, such as heparan sulfate. In this study, heparin, polysulfated dextran sulfate, and suramin were found to significantly prevent EV-71 infection. Heparin inhibited infection by all the EV-71 strains tested, including those with a single-passage history. Neutralization of the cell surface anionic charge by polycationic poly-D-lysine and blockage of heparan sulfate by an anti-heparan sulfate peptide also inhibited EV-71 infection. Interference with heparan sulfate biosynthesis either by sodium chlorate treatment or through transient knockdown of N-deacetylase/N-sulfotransferase-1 and exostosin-1 expression reduced EV-71 infection in RD cells. Enzymatic removal of cell surface heparan sulfate by heparinase I/II/III inhibited EV-71 infection. Furthermore, the level of EV-71 attachment to CHO cell lines that are variably deficient in cell surface glycosaminoglycans was significantly lower than that to wild-type CHO cells. Direct binding of EV-71 particles to heparin-Sepharose columns under physiological salt conditions was demonstrated. We conclude that EV-71 infection requires initial binding to heparan sulfate as an attachment receptor

Enterovirus 71 in Malaysia: A decade later

In the last decade, Malaysia has experienced several hand, foot and mouth disease (HFMD) epidemics, complicated by fatalities due to severe neurological involvement. Enterovirus 71 (EV-71) has been implicated as the major causative agent for these epidemics. EV-71 infection is a global public health problem with pandemic potential. In many parts of Asia-Pacific, the virus has emerged as one of the most deadly virus infections amongst young children. The virus is highly transmissible through faecal-oral route and respiratory droplets. A recent rise in neurological complications and deaths suggests that the viruses currently circulating may be more virulent. The major risk factor associated with more severe EV-71 infection is young age and poor cellular immunity. Rapid laboratory diagnosis and molecular surveillance is important to closely monitor the emergence of new EV-71 subgenotypes. Since vaccine and anti-virals for EV-71 are not available, control and prevention strategies remain the only ways to combat the infection

2BC Non-Structural Protein of Enterovirus A71 Interacts with SNARE Proteins to Trigger Autolysosome Formation

Viruses have evolved unique strategies to evade or subvert autophagy machinery. Enterovirus A71 (EV-A71) induces autophagy during infection in vitro and in vivo. In this study, we report that EV-A71 triggers autolysosome formation during infection in human rhabdomyosarcoma (RD) cells to facilitate its replication. Blocking autophagosome-lysosome fusion with chloroquine inhibited virus RNA replication, resulting in lower viral titres, viral RNA copies and viral proteins. Overexpression of the non-structural protein 2BC of EV-A71 induced autolysosome formation. Yeast 2-hybrid and co-affinity purification assays showed that 2BC physically and specifically interacted with a N-ethylmaleimide-sensitive factor attachment receptor (SNARE) protein, syntaxin-17 (STX17). Co-immunoprecipitation assay further showed that 2BC binds to SNARE proteins, STX17 and synaptosome associated protein 29 (SNAP29). Transient knockdown of STX17, SNAP29, and microtubule-associated protein 1 light chain 3B (LC3B), crucial proteins in the fusion between autophagosomes and lysosomes) as well as the lysosomal-associated membrane protein 1 (LAMP1) impaired production of infectious EV-A71 in RD cells. Collectively, these results demonstrate that the generation of autolysosomes triggered by the 2BC non-structural protein is important for EV-A71 replication, revealing a potential molecular pathway targeted by the virus to exploit autophagy. This study opens the possibility for the development of novel antivirals that specifically target 2BC to inhibit formation of autolysosomes during EV-A71 infection.Peer reviewe

Viral load and sequence analysis reveal the symptom severity, diversity, and transmission clusters of Rhinovirus infections

Background:Rhinovirus (RV) is one of the main viral etiologic agents of acute respiratory illnesses. Despite the heightened disease burden caused by RV, the viral factors that increase the severity of RV infection, the transmission pattern, and seasonality of RV infections remain unclear. Methods: An observational study was conducted among 3935 patients presenting with acute upper respiratory illnesses in the ambulatory settings between 2012 and 2014. Results: The VP4/VP2 gene was genotyped from all 976 RV-positive specimens, where the predominance of RV-A (49%) was observed, followed by RV-C (38%) and RV-B (13%). A significant regression in median nasopharyngeal viral load (VL) (P < .001) was observed, from 883 viral copies/µL at 1-2 days after symptom onset to 312 viral copies/µL at 3-4 days and 158 viral copies/µL at 5-7 days, before declining to 35 viral copies/µL at ≥8 days. In comparison with RV-A (median VL, 217 copies/µL) and RV-B (median VL, 275 copies/µL), RV-C-infected subjects produced higher VL (505 copies/µL; P < .001). Importantly, higher RV VL (median, 348 copies/µL) was associated with more severe respiratory symptoms (Total Symptom Severity Score ≥17, P = .017). A total of 83 phylogenetic-based transmission clusters were identified in the population. It was observed that the relative humidity was the strongest environmental predictor of RV seasonality in the tropical climate. Conclusions: Our findings underline the role of VL in increasing disease severity attributed to RV-C infection, and unravel the factors that fuel the population transmission dynamics of R

Risk Factors for Enterovirus A71 Seropositivity in Rural Indigenous Populations in West Malaysia

Enterovirus A71 (EV-A71), which is transmitted by the fecal-oral route, causes hand, foot and mouth disease and, rarely, severe neurological complications. In Malaysia, the indigenous rural community (Orang Asli) has a high prevalence of parasitic diseases due to poor sanitation, water supply and hygiene practices. This cross-sectional study compared the seroepidemiology of EV-A71 among rural Orang Asli and urban Kuala Lumpur populations in West Malaysia, and determined the risk factors associated with EV-A71 seropositivity in rural Orang Asli. Seropositive rates were determined by neutralization assay. EV-A71 seropositivity was strongly associated with increasing age in both populations. Rural Orang Asli children �12 years had significantly higher EV-A71 seropositivity rates than urban Kuala Lumpur children (95.5% vs 57.6%, P < 0.001), and also higher rates in the age groups of 1–3, 4–6 and 7–12 years. Multivariate analysis confirmed that age �12 years (adjusted OR 8.1, 95% CI 3.2–20.7, P < 0.001) and using untreated water (adjusted OR 6.2, 95% CI 2.3– 16.6, P < 0.001) were independently associated with EV-A71 seropositivity in the Orang Asli population. Supply of clean drinking water may reduce the risk of EV-A71 infection. With significantly higher EV-A71 seropositive rates, younger rural children should be a priority target for future vaccination programs in Malaysia

Establishment of Asia-Pacific Network for Enterovirus Surveillance

Enteroviruses (EV), the major pathogens of hand, foot, and mouth disease (HFMD) and herpangina, affect millions of children each year. Most human enteroviruses cause self-limited infections except polioviruses, enterovirus A71 (EV-A71), enterovirus D68 (EV-D68), and several echoviruses (Echo) and coxsackieviruses (CV). Especially, EV-A71 has repeatedly caused large-scale outbreaks in the Asia-Pacific region since 1997. Some Asian countries have experienced cyclical outbreaks of severe EV-A71 infections and initiated development of EV-A71 vaccines. Five EV-A71 vaccine candidates have been clinically evaluated and three of them were approved for marketing in China. However, none of the China-approved products seek marketing approval in other countries. This situation supports a role for collaboration among Asian countries to facilitate clinical trials and licensure of EV-A71 vaccines. Additionally, enterovirus D68 outbreaks have been reported in the US and Taiwan currently and caused severe complications and deaths. Hence, an Asia-Pacific Network for Enterovirus Surveillance (APNES) has been established to estimate disease burden, understand virus evolution, and facilitate vaccine development through harmonizing laboratory diagnosis and data collection. Founded in 2017, the APNES is comprised of internationally recognized experts in the field of enterovirus in Asian countries working to raise awareness of this potentially fatal and debilitating disease. This article demonstrated the summaries of the first expert meeting, 2017 International Workshop on Enterovirus Surveillance and Vaccine Development, held by APNES in Taipei, Taiwan, March 2017

Characterization of Full-Length Enterovirus 71 Strains from Severe and Mild Disease Patients in Northeastern China

Human enterovirus 71 (EV71)-associated hand, foot, and mouth disease (HFMD) has been a leading cause of childhood infection in China since 2008. Epidemic and molecular characteristics of HFMD have been examined in many areas of China, including the central and southern regions. However, clinical and genetic characterization of EV71 in the northeastern region of China is scarce. In this study, a series of analyses were performed on seven full-length EV71 sequences from HFMD patients who had either severe or mild disease. We have determined that these seven circulating EV71 viruses from Changchun, China are actually complex recombinant viruses involving multiple type A human enterovirus (HEV). Classified as EV71 subtype C4 (EV71 C4), these Changchun EV71 viruses contain genetic recombination events between the CA4, CA5, EV71B4 and EV71C1 strains. Most of the structural protein region (P1) of these viruses resembled that of the prototype EV71 C1 strains. The non-structural protein domains (P2 and P3) showed a high degree of similarity with CA4, CA5 and EV71 B4 in different regions. The 5′UTR had unclassified recombination,while partial 3D region of these viruses showed a high degree of similarity to CA16. Phylogenetic analysis of full-length or partial sequences of isolates from severe or mild disease patients in Changchun always formed a single cluster in various phylogenetic analyses of different genomic regions, suggesting that all seven strains originated from one single common ancestor. There was no correlation between viral genomic sequence and virulence. Thus, we found that circulating recombinant forms of EV71 are prevalent among HFMD patients in Northeastern China. The existence of a unique cluster of EV71 related viruses in Northeast China has important implications for vaccine development that would address the increasing prevalence of HFMD