Pteropine orthoreoviruses use cell surface heparan sulphate as an attachment receptor.

Pteropine orthoreoviruses (PRVs) are an emerging group of fusogenic, bat-borne viruses from the Orthoreovirus genus. Since the isolation of PRV from a patient with acute respiratory tract infections in 2006, the zoonotic potential of PRV has been further highlighted following subsequent isolation of PRV species from patients in Malaysia, Hong Kong and Indonesia. However, the entry mechanism of PRV is currently unknown. In this study, we investigated the role of previously identified mammalian orthoreovirus (MRV) receptors, sialic acid and junctional adhesion molecule-1 for PRV infection. However, none of these receptors played a significant role in PRV infection, suggesting PRV uses a distinct entry receptor from MRV. Given its broad tissue tropism, we hypothesized that PRV may use a receptor that is widely expressed in all cell types, heparan sulphate (HS). Enzymatic removal of cell surface HS by heparinase treatment and genetic ablation of HS biosynthesis genes, SLC35B2, exostosin-1, N-deacetylase/N-sulfotransferase I and beta-1,3-glucuronyltransferase 3, significantly reduced infection with multiple genetically distinct PRV species. Replication kinetic of PRV3M in HS knockout cells revealed that HS plays a crucial role in the early phase of PRV infection. Mechanistic studies demonstrated that HS is an essential host-factor for PRV attachment and internalization into cells. To our knowledge, this is the first report on the use of HS as an attachment receptor by PRVs.

A Novel Attenuated Enterovirus A71 Mutant with VP1-V238A,K244R Exhibits Reduced Efficiency of Cell Entry/Exit and Augmented Binding Affinity to Sulfated Glycans.

Enterovirus A71 (EV-A71) is one of the major etiological agents of hand, foot, and mouth disease (HFMD), and infection occasionally leads to fatal neurological complications in children. However, only inactivated whole-virus vaccines against EV-A71 are commercially available in Mainland China. Furthermore, the mechanisms underlying the infectivity and pathogenesis of EV-A71 remain to be better understood. By adaptation of an EV-A71 B5 strain in monkey Vero cells in the presence of brilliant black BN (E151), an anti-EV-A71 agent, a double mutant with VP1-V238A,K244R emerged whose infection was enhanced by E151. The growth of the reverse genetics (RG) mutant RG/B5-VP1-V238A,K244R (RG/B5-AR) was promoted by E151 in Vero cells but inhibited in other human and murine cells, while its parental wild type, RG/B5-wt, was strongly prevented by E151 from infection in all tested cells. In the absence of E151, RG/B5-AR exhibited defective cell entry/exit, resulting in reduced viral transmission and growth in vitro. It had augmented binding affinity to sulfated glycans, cells, and tissue/organs, which probably functioned as decoys to restrict viral dissemination and infection. RG/B5-AR was also attenuated, with a 355 times higher 50% lethal dose (LD50) and a shorter timing of virus clearance than those of RG/B5-wt in suckling AG129 mice. However, it remained highly immunogenic in adult AG129 mice and protected their suckling mice from lethal EV-A71 challenges through maternal neutralizing antibodies. Overall, discovery of the attenuated mutant RG/B5-AR contributes to better understanding of virulence determinants of EV-A71 and to further development of novel vaccines against EV-A71. IMPORTANCE Enterovirus A71 (EV-A71) is highly contagious in children and has been responsible for thousands of deaths in Asia-Pacific region since the 1990s. Unfortunately, the virulence determinants and pathogenesis of EV-A71 are not fully clear. We discovered that a novel EV-A71 mutant, VP1-V238A,K244R, showed growth attenuation with reduced efficiency of cell entry/exit. In the Vero cell line, which has been approved for manufacturing EV-A71 vaccines, the growth defects of the mutant were compensated by a food dye, brilliant black BN. The mutant also showed augmented binding affinity to sulfated glycans and other cellular components, which probably restricted viral infection and dissemination. Therefore, it was virulence attenuated in a mouse model but still retained its immunogenicity. Our findings suggest the mutant as a promising vaccine candidate against EV-A71 infection.

Safety and Immunogenicity of a Stable, Cold-Adapted, Temperature-Sensitive/Conditional Lethal Enterovirus A71 in Monkey Study.

Enterovirus A71 (EV-A71) and coxsackievirus A16 (CA16) are major etiological agents of hand foot and mouth disease (HFMD) in children, which may result in fatal neurological complications. The development of safe, cost effective vaccines against HFMD, especially for use in developing countries, is still a top public health priority. We have successfully generated a stable, cold-adapted, temperature sensitive/conditional lethal EV-A71 through adaptive culturing in Vero cells at incrementally lower cultivation temperatures. An additional 40 passages at an incubation temperature of 28 °C, and a temperature reversion study at an incubation temperature of 37 °C and 39.5 °C, reveals the virus's phenotypic and genetic stability at the predefined culture conditions. Six unique mutations (two in noncoding regions and four in nonstructural protein-coding genes) in combination may have contributed to its stable phenotype and inability to fully revert to its original wild phenotype. The safety and immunogenicity of this stable, cold-adapted, temperature sensitive/conditional lethal EV-A71 was performed in six monkeys. None of the inoculated monkeys developed any obvious clinical illness except one which developed a transient spike of fever. No gross postmortem lesion or abnormal histological finding was noted for all monkeys at autopsy. No virus was reisolated although EV-A71 specific RNA was detected in serum samples collected on both day 4 and day 8 postinoculation. Only EV-A71 RNA and viral antigen were detected in the spleen homogenate and peripheral blood mononuclear cells, respectively, collected on day 4. The two remaining monkeys developed good humoral immune response on day 14 and day 30 post-inoculation.

Sulfonated azo dyes enhance the genome release of enterovirus A71 VP1-98K variants by preventing the virions from being trapped by sulfated glycosaminoglycans at acidic pH.

Enterovirus A71 (EV-A71) is a causative agent of hand, foot and mouth disease and occasionally causes death in children. Its infectivity and pathogenesis, however, remain to be better understood. Three sulfonated azo dyes, including acid red 88 (Ar88), were identified to enhance the infectivity of EV-A71, especially isolates with VP1-98K, 145E (-KE), by mainly promoting viral genome release in vitro. Enzymatic removal of sulfated glycosaminoglycans (GAGs) or knockout of xylosyltransferase II (XT2) responsible for biosynthesis of sulfated GAGs weakened the Ar88 enhanced EV-A71 infection. Ar88 is proposed to prevent the -KE variants from being trapped by sulfated GAGs at acidic pH and to facilitate the viral interaction with uncoating factors for genome release in endosomes. The results suggest dual roles of sulfated GAGs as attachment factors and as decoys during host interaction of EV-A71 and caution that these artificial dyes in our environment can enhance viral infection.

In Vitro and In Vivo Inhibition of the Infectivity of Human Enterovirus 71 by a Sulfonated Food Azo Dye, Brilliant Black BN.

Hand, foot, and mouth disease (HFMD), a highly contagious disease in children, is caused by human enteroviruses, including enterovirus 71 (EV71), coxsackievirus A16 (CVA16), and coxsackievirus A6 (CVA6). Although HFMD is usually mild and self-limiting, EV71 infection occasionally leads to fatal neurological disorders. Currently, no commercial antiviral drugs for HFMD treatment are available. Here, numerous sulfonated azo dyes, widely used as food additives, were identified as having potent antiviral activities against human enteroviruses. Among them, brilliant black BN (E151) was able to inhibit all EV71, CVA16, and CVA6 strains tested. In rhabdomyosarcoma cells, the 50% inhibitory concentrations of the dye E151 for various strains of EV71 ranged from 2.39 µM to 28.12 µM, whereas its 50% cytotoxic concentration was 1,870 µM. Food azo dyes, including E151, interacted with the vertex of the 5-fold axis of EV71 and prevented viral entry. Their efficacy in viral inhibition was regulated by amino acids at VP1-98, VP1-145, and/or VP1-246. Dye E151 not only prevented EV71 attachment but also eluted attached viruses in a concentration-dependent manner. Moreover, E151 inhibited the interaction between EV71 and its cellular uncoating factor cyclophilin A. In vivo studies demonstrated that E151 at a dose of 200 mg/kg of body weight/day given on the initial 4 days of challenge protected AG129 mice challenged with 10× the 50% lethal dose of wild-type EV71 isolates. Taken together, these data highlight E151 as a promising antiviral agent against EV71 infection.IMPORTANCE Human enterovirus 71 (EV71) is one of the causative agents of hand, foot, and mouth disease in children and is responsible for thousands of deaths in the past 20 years. Food azo dyes have been widely used since the nineteenth century; however, their biological effects on humans and microbes residing in humans are poorly understood. Here, we discovered that one of these dyes, brilliant black BN (E151), was particularly effective in inhibiting the infectivity of EV71 in both cell culture and mouse model studies. Mechanistic studies demonstrated that these sulfonated dyes mainly competed with EV71 attachment factors for viral binding to block viral attachment/entry to host cells. As no commercial antiviral drugs against EV71 are currently available, our findings open an avenue to exploit the development of permitted food dye E151 as a potential anti-EV71 agent.

The Saffold Virus-Penang 2B and 3C Proteins, but not the L Protein, Induce Apoptosis in HEp-2 and Vero Cells.

Our previous work has shown that Saffold virus (SAFV) induced several rodent and primate cell lines to undergo apoptosis (Xu et al. in Emerg Microb Infect 3:1-8, 2014), but the essential viral proteins of SAFV involved in apoptotic activity lack study. In this study, we individually transfected the viral proteins of SAFV into HEp-2 and Vero cells to assess their ability to induce apoptosis, and found that the 2B and 3C proteins are proapoptotic. Further investigation indicated the transmembrane domain of the 2B protein is essential for the apoptotic activity and tetramer formation of the 2B protein. Our research provides clues for the possible mechanisms of apoptosis induced by SAFV in different cell lines. It also opens up new directions to study viral proteins (the 2B, 3C protein), and sets the stage for future exploration of any possible link between SAFV, inclusive of its related uncultivable genotypes, and multiple sclerosis.

Serological evidence of human infection by bat orthoreovirus in Singapore.

To determine whether Pteropine orthoreovirus (PRV) exposure has occurred in Singapore, we tested 856 individuals from an existing serum panel collected from 2005-2013. After an initial screen with luciferase immunoprecipitation system and secondary confirmation with virus neutralization test, we identified at least seven individuals with specific antibodies against PRV in both assays. Our findings confirm that PRV spillover into human populations is relatively common in this region of the world.

Pteropine orthoreovirus: An important emerging virus causing infectious disease in the tropics?

INTRODUCTION: Pteropine orthoreovirus (PRV) is an emerging zoonotic respiratory virus that has spilled over from bats to humans. Though initially found only in bats, further case studies have found viable virus in ill patients. METHODOLOGY: PubMed was queried with the keywords of Nelson Bay orthoreovirus OR Pteropine orthoreovirus OR Melaka orthoreovirus OR Kampar orthoreovirus, and returned 17 hits. RESULTS: Based on prevalence studies, the presence of PRV has been reported in Malaysia and Vietnam, both developing countries. Other case reports also provide further evidence of the presence of PRV in the Southeast Asian region. Despite the absence of PRV in their home countries, travellers from Hong Kong and Japan to Indonesia have returned to their countries ill with this virus, indicating that local communities in Indonesia might be affected by this virus. CONCLUSIONS: This work aims to bring to light this emerging zoonotic respiratory virus circulating among developing countries in Southeast Asia. To improve the understanding of PRV of the medical and scientific community in the Southeast Asian region, this work introduces the general features of PRV, reports of imported PRV, prevalence, and clinical features of PRV. Gaps in knowledge about PRV have also been identified in this work, and we hope that future studies can be undertaken to improve our understanding of this virus.

Enterovirus 71 infection of motor neuron-like NSC-34 cells undergoes a non-lytic exit pathway.

Enterovirus 71 (EV71) causing Hand, Foot and Mouth Disease, is regarded as the most important neurotropic virus worldwide. EV71 is believed to replicate in muscles and infect motor neurons to reach the central nervous system (CNS). To further investigate the mechanisms involved, we have employed the motor neuron cell line NSC-34. NSC-34 cells were permissive to EV71 and virus production yields were strain-dependent with differential efficacy at the entry, replication and egress steps. Furthermore, unlike all the other cell lines previously reported, EV71-infected NSC-34 cells neither displayed cytopathic effect nor underwent apoptosis. Instead, autophagy was markedly up-regulated and virus-containing autophagic vacuoles were isolated from the culture supernatant, providing the first experimental evidence that EV71 can adopt a non-lytic exit pathway. Finally, the ability of EV71 to infect productively NSC-34 cells correlated with its ability to invade the CNS in vivo, supporting the relevance of NSC-34 cells to study the intrinsic neurovirulence of EV71 strains.

Intracellular localization of Saffold virus Leader (L) protein differs in Vero and HEp-2 cells.

The Saffold virus (SAFV) genome is translated as a single long polyprotein precursor and co-translationally cleaved to yield 12 separate viral proteins. Little is known about the activities of SAFV proteins although their homologs in other picornaviruses have already been described. To further support research on functions and activities of respective viral proteins, we investigated the spatio-temporal distribution of SAFV proteins in Vero and HEp-2 cells that had been either transfected with plasmids that express individual viral proteins or infected with live SAFV. Our results revealed that, with the exception of the Leader (L) protein, all viral proteins were localized in the cytoplasm at all the time points assayed. The L protein was found in the cytoplasm at an early time point but was subsequently translocated to the nucleus of HEp-2, but not Vero, cells. This was observed in both transfected and infected cells. Further mutational analysis of L protein revealed that Threonine 58 of the Ser/Thr-rich domain of L protein is crucial for protein trafficking between the cytoplasm and nucleus in HEp-2 cells. These findings contribute to a deeper understanding and stimulate investigation of the differetial cellular responses of HEp-2 cells in comparison to other mammalian cell lines during SAFV infection.

A clinically authentic mouse model of enterovirus 71 (EV-A71)-induced neurogenic pulmonary oedema.

Enterovirus 71 (EV-A71) is a neurotropic virus that sporadically causes fatal neurologic illness among infected children. Animal models of EV-A71 infection exist, but they do not recapitulate in animals the spectrum of disease and pathology observed in fatal human cases. Specifically, neurogenic pulmonary oedema (NPE)-the main cause of EV-A71 infection-related mortality-is not observed in any of these models. This limits their utility in understanding viral pathogenesis of neurologic infections. We report the development of a mouse model of EV-A71 infection displaying NPE in severely affected animals. We inoculated one-week-old BALB/c mice with an adapted EV-A71 strain and identified clinical signs consistent with observations in human cases and other animal models. We also observed respiratory distress in some mice. At necropsy, we found their lungs to be heavier and incompletely collapsed compared to other mice. Serum levels of catecholamines and histopathology of lung and brain tissues of these mice strongly indicated onset of NPE. The localization of virally-induced brain lesions also suggested a potential pathogenic mechanism for EV-A71-induced NPE. This novel mouse model of virally-induced NPE represents a valuable resource for studying viral mechanisms of neuro-pathogenesis and pre-clinical testing of potential therapeutics and prophylactics against EV-A71-related neurologic complications.

Cooperative effect of the VP1 amino acids 98E, 145A and 169F in the productive infection of mouse cell lines by enterovirus 71 (BS strain).

Enterovirus 71 (EV71) is a neurotrophic virus that causes hand, foot and mouth disease (HFMD) and occasional neurological infection among children. It infects primate cells but not rodent cells, primarily due to the incompatibility between the virus and the expressed form of its receptor, scavenger receptor class B member 2 (SCARB2) protein, on rodent cells (mSCARB2). We previously generated adapted strains (EV71:TLLm and EV71:TLLmv) that were shown to productively infect primate and rodent cell lines and whose genomes exhibited a multitude of non-synonymous mutations compared with the EV71:BS parental virus. In this study, we aimed to identify mutations that are necessary for productive infection of murine cells by EV71:BS. Using reverse genetics and site-directed mutagenesis, we constructed EV71 infectious clones with specific mutations that generated amino acid substitutions in the capsid VP1 and VP2 proteins. We subsequently assessed the infection induced by clone-derived viruses (CDVs) in mouse embryonic fibroblast NIH/3T3 and murine neuroblastoma Neuro-2a cell lines. We found that the CDV:BS-VP1(K98E,E145A,L169F) with three substitutions in the VP1 protein-K98E, E145A and L169F-productively infected both mouse cell lines for at least three passages of the virus in murine cells. Moreover, the virus gained the ability to utilize the mSCARB2 protein to infect murine cell lines. These results demonstrate that the three VP1 residues cooperate to effectively interact with the mSCARB2 protein on murine cells and permit the virus to infect murine cells. Gain-of-function studies similar to the present work provide valuable insight into the mutational trajectory required for EV71 to infect new host cells previously non-susceptible to infection.

The Pathogenesis of Saffold Virus in AG129 Mice and the Effects of Its Truncated L Protein in the Central Nervous System.

Saffold Virus (SAFV) is a human cardiovirus that has been suggested to cause severe infection of the central nervous system (CNS). Compared to a similar virus, Theiler's murine encephalomyelitis virus (TMEV), SAFV has a truncated Leader (L) protein, a protein essential in the establishment of persistent CNS infections. In this study, we generated a chimeric SAFV by replacing the L protein of SAFV with that of TMEV. We then compared the replication in cell cultures and pathogenesis in a mouse model. We showed that both SAFV and chimeric SAFV are able to infect Vero and Neuro2a cells well, but only chimeric SAFV was able to infect RAW264.7. We then showed that mice lacking IFN-α/ß and IFN-γ receptors provide a good animal model for SAFV infection, and further identified the locality of the infection to the ventral horn of the spine and several locations in the brain. Lastly, we showed that neither SAFV nor chimeric SAFV causes persistence in this model. Overall, our results provide a strong basis on which the mechanisms underlying Saffold virus induced neuropathogenesis can be further studied and, hence, facilitating new information about its pathogenesis.

Pteropine orthoreovirus infection among out-patients with acute upper respiratory tract infection in Malaysia.

This study aims to assess the incidence rate of Pteropine orthreovirus (PRV) infection in patients with acute upper respiratory tract infection (URTI) in a suburban setting in Malaysia, where bats are known to be present in the neighborhood. Using molecular detection of PRVs directly from oropharyngeal swabs, our study demonstrates that PRV is among one of the common causative agents of acute URTI with cough and sore throat as the commonest presenting clinical features. Phylogenetic analysis on partial major outer and inner capsid proteins shows that these PRV strains are closely related to Melaka and Kampar viruses previously isolated in Malaysia. Further study is required to determine the public health significance of PRV infection in Southeast Asia, especially in cases where co-infection with other pathogens may potentially lead to different clinical outcomes.

Phenotypic and genotypic characteristics of novel mouse cell line (NIH/3T3)-adapted human enterovirus 71 strains (EV71:TLLm and EV71:TLLmv).

Since its identification in 1969, Enterovirus 71 (EV71) has been causing periodic outbreaks of infection in children worldwide and most prominently in the Asia-Pacific Region. Understanding the pathogenesis of Enterovirus 71 (EV71) is hampered by the virus's inability to infect small animals and replicate in their derived in vitro cultured cells. This manuscript describes the phenotypic and genotypic characteristics of two selected EV71 strains (EV71:TLLm and EV71:TLLmv), which have been adapted to replicate in mouse-derived NIH/3T3 cells, in contrast to the original parental virus which is only able to replicate in primate cell lines. The EV71:TLLm strain exhibited productive infection in all primate and rodent cell lines tested, while EV71:TLLmv exhibited greater preference for mouse cell lines. EV71:TLLmv displayed higher degree of adaptation and temperature adaptability in NIH/3T3 cells than in Vero cells, suggesting much higher fitness in NIH/3T3 cells. In comparison with the parental EV71:BS strain, the adapted strains accumulated multiple adaptive mutations in the genome resulting in amino acid substitutions, most notably in the capsid-encoding region (P1) and viral RNA-dependent RNA polymerase (3D). Two mutations, E167D and L169F, were mapped to the VP1 canyon that binds the SCARB2 receptor on host cells. Another two mutations, S135T and K140I, were located in the VP2 neutralization epitope spanning amino acids 136-150. This is the first report of human EV71 with the ability to productively infect rodent cell lines in vitro.

Saffold virus is able to productively infect primate and rodent cell lines and induces apoptosis in these cells.

Saffold virus (SAFV), a newly discovered human cardiovirus of the Picornaviridae family, causes widespread infection among children, as shown by previous seroprevalence studies. To determine the host cell range of SAFV and its cytopathogenicity, eight mammalian cell lines that were available in the laboratory were screened for productive SAFV infection by a laboratory-adapted SAFV of genotype 3. Five of the cell lines (Neuro2A, CHO-K1, NIH/3T3, Vero and HEp-2) were found to be permissible. The time required for SAFV to induce complete lysis as a cytopathic effect (CPE) in these permissibly infected cells and the resultant end point virus titer differed for each cell type. HEp-2 exhibited the shortest time frame to reach full CPE compared to the others. All infected cell lines produced a high virus titer at 72 h post-infection. In addition to causing lytic cell death, SAFV also induced apoptotic cell death in host cells through both extrinsic and intrinsic pathways, although the apoptotic events in HEp-2 cells appeared to have been blocked between the early and late stages. In conclusion, laboratory-adapted SAFV is able to productively infect a number of mammalian cell lines and induce apoptosis in the infected host cells. However, apoptosis in HEp-2 cells is blocked before the end stage.

Perspectives of public health laboratories in emerging infectious diseases.

The world has experienced an increased incidence and transboundary spread of emerging infectious diseases over the last four decades. We divided emerging infectious diseases into four categories, with subcategories in categories 1 and 4. The categorization was based on the nature and characteristics of pathogens or infectious agents causing the emerging infections, which are directly related to the mechanisms and patterns of infectious disease emergence. The factors or combinations of factors contributing to the emergence of these pathogens vary within each category. We also classified public health laboratories into three types based on function, namely, research, reference and analytical diagnostic laboratories, with the last category being subclassified into primary (community-based) public health and clinical (medical) analytical diagnostic laboratories. The frontline/leading and/or supportive roles to be adopted by each type of public health laboratory for optimal performance to establish the correct etiological agents causing the diseases or outbreaks vary with respect to each category of emerging infectious diseases. We emphasize the need, especially for an outbreak investigation, to establish a harmonized and coordinated national public health laboratory system that integrates different categories of public health laboratories within a country and that is closely linked to the national public health delivery system and regional and international high-end laboratories.

Genetic analysis of saffold virus-Penang in relation to other newly discovered saffold viruses.

Viruses in the family Picornaviridae are classified into nine genera. Within the family Picornaviridae, two species: Encephalomyocarditis virus and Theilovirus, are listed under the genus Cardiovirus. A novel Theilovirus, Saffold virus (SAFV), was first reported in 2007. Since then, numerous SAFV isolates have been detected around the world and genetic recombinations have been reported among them. In 2009, SAFV-Penang was isolated from a febrile child with influenza-like illness in Malaysia. SAFV-Penang is a genotype 3 SAFV. In this study we investigated the genome features of SAFV-Penang to exclude the possibility it is a recombinant variant. SAFV-Penang was found not to be a recombinant variant but to have three unique non-synonymous substitutions, alanine [A689], lysine [K708] and isoleucine [I724] in the VP1 protein.