Genomic Characterization of a Relative of Mumps Virus in Lesser Dawn Bats of Southeast Asia.

The importance of genomic surveillance on emerging diseases continues to be highlighted with the ongoing SARS-CoV-2 pandemic. Here, we present an analysis of a new bat-borne mumps virus (MuV) in a captive colony of lesser dawn bats (Eonycteris spelaea). This report describes an investigation of MuV-specific data originally collected as part of a longitudinal virome study of apparently healthy, captive lesser dawn bats in Southeast Asia (BioProject ID PRJNA561193) which was the first report of a MuV-like virus, named dawn bat paramyxovirus (DbPV), in bats outside of Africa. More in-depth analysis of these original RNA sequences in the current report reveals that the new DbPV genome shares only 86% amino acid identity with the RNA-dependent RNA polymerase of its closest relative, the African bat-borne mumps virus (AbMuV). While there is no obvious immediate cause for concern, it is important to continue investigating and monitoring bat-borne MuVs to determine the risk of human infection.

Single-cell transcriptome analysis of the in vivo response to viral infection in the cave nectar bat Eonycteris spelaea.

Bats are reservoir hosts of many zoonotic viruses with pandemic potential. We utilized single-cell transcriptome sequencing (scRNA-seq) to analyze the immune response in bat lungs upon in vivo infection with a double-stranded RNA virus, Pteropine orthoreovirus PRV3M. Bat neutrophils were distinguished by high basal IDO1 expression. NK cells and T cells were the most abundant immune cells in lung tissue. Three distinct CD8+ effector T cell populations could be delineated by differential expression of KLRB1, GFRA2, and DPP4. Select NK and T clusters increased expression of genes involved in T cell activation and effector function early after viral infection. Alveolar macrophages and classical monocytes drove antiviral interferon signaling. Infection expanded a CSF1R+ population expressing collagen-like genes, which became the predominant myeloid cell type post-infection. This work uncovers features relevant to viral disease tolerance in bats, lays a foundation for future experimental work, and serves as a resource for comparative immunology studies.

Presence of Recombinant Bat Coronavirus GCCDC1 in Cambodian Bats.

Bats have been recognized as an exceptional viral reservoir, especially for coronaviruses. At least three bat zoonotic coronaviruses (SARS-CoV, MERS-CoV and SARS-CoV-2) have been shown to cause severe diseases in humans and it is expected more will emerge. One of the major features of CoVs is that they are all highly prone to recombination. An extreme example is the insertion of the P10 gene from reoviruses in the bat CoV GCCDC1, first discovered in Rousettus leschenaultii bats in China. Here, we report the detection of GCCDC1 in four different bat species (Eonycteris spelaea, Cynopterus sphinx, Rhinolophus shameli and Rousettus sp.) in Cambodia. This finding demonstrates a much broader geographic and bat species range for this virus and indicates common cross-species transmission. Interestingly, one of the bat samples showed a co-infection with an Alpha CoV most closely related to RsYN14, a virus recently discovered in the same genus (Rhinolophus) of bat in Yunnan, China, 2020. Taken together, our latest findings highlight the need to conduct active surveillance in bats to assess the risk of emerging CoVs, especially in Southeast Asia.

Orthogonal genome-wide screens of bat cells identify MTHFD1 as a target of broad antiviral therapy.

Bats are responsible for the zoonotic transmission of several major viral diseases, including those leading to the 2003 SARS outbreak and likely the ongoing COVID-19 pandemic. While comparative genomics studies have revealed characteristic adaptations of the bat innate immune system, functional genomic studies are urgently needed to provide a foundation for the molecular dissection of the viral tolerance in bats. Here we report the establishment of genome-wide RNA interference (RNAi) and CRISPR libraries for the screening of the model megabat, Pteropus alecto. We used the complementary RNAi and CRISPR libraries to interrogate P. alecto cells for infection with two different viruses: mumps virus and influenza A virus, respectively. Independent screening results converged on the endocytosis pathway and the protein secretory pathway as required for both viral infections. Additionally, we revealed a general dependence of the C1-tetrahydrofolate synthase gene, MTHFD1, for viral replication in bat cells and human cells. The MTHFD1 inhibitor, carolacton, potently blocked replication of several RNA viruses, including SARS-CoV-2. We also discovered that bats have lower expression levels of MTHFD1 than humans. Our studies provide a resource for systematic inquiry into the genetic underpinnings of bat biology and a potential target for developing broad-spectrum antiviral therapy.

Molecular Analysis of the Bloodmeals of Culex spp. Mosquitoes at Natural Habitats in Singapore to Investigate the Potential Risk of Japanese Encephalitis Virus and West Nile Virus Transmission.

Japanese encephalitis virus (JEV) and West Nile virus (WNV) are arboviruses primarily transmitted by Culex spp. mosquitoes. Birds are the primary hosts for JEV and WNV. Recent WNV outbreaks in Europe and United States and their association with migratory birds highlight the importance of understanding the feeding host preference of potential vectors for outbreak preparedness, especially in nonendemic settings. Singapore is nonendemic to JEV and WNV, but is a stopover site for migratory birds of the East Asian-Australasian Flyway. Therefore, we elucidated the feeding host range of Culex spp. mosquitoes captured in four natural (bird) habitats in Singapore from January 2011 to December 2012. We characterized feeding host DNA in field-caught mosquitoes using a PCR sequencing-based assay targeting the mitochondrial gene regions. Of 22,648 mosquitoes captured, 21,287 belonged to the Culex vishnui subgroup. The host DNA analysis showed that mosquitoes from the Cx. vishnui subgroup are opportunistic biters, feeding on a range of birds and mammals. Cx. vishnui subgroup, Culex sitiens and Culex bitaeniorhynchus, was primarily ornithophagic, although they fed opportunistically on mammals, including humans. Culex gelidus and Culex quinquefasciatus, in contrast, fed mainly on mammals. The presence of ornitho- and anthropophilic mosquito vectors and susceptible avian and mammalian hosts poses a risk spill-over transmission of JEV and WNV among humans, should these viruses be introduced through migratory birds and establish persistent transmission in resident birds and animal hosts in Singapore.

Detection of Japanese Encephalitis Virus in Culex Mosquitoes in Singapore.

Mosquito-borne flaviviruses are emerging pathogens of an increasing global public health concern because of their rapid increase in geographical range and the impact of climate change. Japanese encephalitis virus (JEV) and West Nile virus (WNV) are of concern because of the risk of reemergence and introduction by migratory birds. In Singapore, human WNV infection has never been reported and human JEV infection is rare. Four sentinel vector surveillance sites were established in Singapore to understand the potential risk posed by these viruses. Surveillance was carried out from August 2011 to December 2012 at Pulau Ubin, from March 2011 to March 2013 at an Avian Sanctuary (AS), from December 2010 from October 2012 at Murai Farmway, and from December 2010 to December 2013 at a nature reserve. The present study revealed active JEV transmission in Singapore through the detection of JEV genotype II in Culex tritaeniorhynchus collected from an Avian Sanctuary. Culex flavivirus (CxFV), similar to the Quang Binh virus isolated from Cx. tritaeniorhynchus in Vietnam and CxFV-LSFlaviV-A20-09 virus isolated in China, was also detected in Culex spp. (vishnui subgroup). No WNV was detected. This study demonstrates the important role that surveillance plays in public health and strongly suggests the circulation of JEV among wildlife in Singapore, despite the absence of reported human cases. A One Health approach involving surveillance, the collaboration between public health and wildlife managers, and control of mosquito populations remains the key measures in risk mitigation of JEV transmission in the enzootic cycle between birds and mosquitoes.

Detection of Recombinant Rousettus Bat Coronavirus GCCDC1 in Lesser Dawn Bats (Eonycteris spelaea) in Singapore.

Rousettus bat coronavirus GCCDC1 (RoBat-CoV GCCDC1) is a cross-family recombinant coronavirus that has previously only been reported in wild-caught bats in Yúnnan, China. We report the persistence of a related strain in a captive colony of lesser dawn bats captured in Singapore. Genomic evidence of the virus was detected using targeted enrichment sequencing, and further investigated using deeper, unbiased high throughput sequencing. RoBat-CoV GCCDC1 Singapore shared 96.52% similarity with RoBat-CoV GCCDC1 356 (NC_030886) at the nucleotide level, and had a high prevalence in the captive bat colony. It was detected at five out of six sampling time points across the course of 18 months. A partial segment 1 from an ancestral Pteropine orthoreovirus, p10, makes up the recombinant portion of the virus, which shares high similarity with previously reported RoBat-CoV GCCDC1 strains that were detected in Yúnnan, China. RoBat-CoV GCCDC1 is an intriguing, cross-family recombinant virus, with a geographical range that expands farther than was previously known. The discovery of RoBat-CoV GCCDC1 in Singapore indicates that this recombinant coronavirus exists in a broad geographical range, and can persist in bat colonies long-term.

Detection of air and surface contamination by SARS-CoV-2 in hospital rooms of infected patients.

Understanding the particle size distribution in the air and patterns of environmental contamination of SARS-CoV-2 is essential for infection prevention policies. Here we screen surface and air samples from hospital rooms of COVID-19 patients for SARS-CoV-2 RNA. Environmental sampling is conducted in three airborne infection isolation rooms (AIIRs) in the ICU and 27 AIIRs in the general ward. 245 surface samples are collected. 56.7% of rooms have at least one environmental surface contaminated. High touch surface contamination is shown in ten (66.7%) out of 15 patients in the first week of illness, and three (20%) beyond the first week of illness (p = 0.01, χ2 test). Air sampling is performed in three of the 27 AIIRs in the general ward, and detects SARS-CoV-2 PCR-positive particles of sizes >4 µm and 1-4 µm in two rooms, despite these rooms having 12 air changes per hour. This warrants further study of the airborne transmission potential of SARS-CoV-2.

Salmonella in Retail Food and Wild Birds in Singapore-Prevalence, Antimicrobial Resistance, and Sequence Types.

Non-typhoidal salmonellosis is a leading cause of foodborne zoonosis. To better understand the epidemiology of human salmonellosis, this study aimed to determine the prevalence, antimicrobial resistance and sequence types of Salmonella in retail food and wild birds (proximity to humans) in Singapore. We analyzed 21,428 cooked and ready-to-eat food and 1,510 residual faecal samples of wild birds collected during 2010-2015. Thirty-two Salmonella isolates from food and wild birds were subjected to disc diffusion and multi-locus sequence typing (MLST). Salmonella was isolated from 0.08% (17/21,428) of food and 0.99% (15/1510) of wild birds. None of the isolates from wild birds (n = 15) exhibited phenotypic resistance, while the isolates from food (47.1%, 8/17) showed a high prevalence of phenotypic resistance to, at least, one antimicrobial. These findings suggested that the avian Salmonella isolates had been subjected to less antimicrobial selection pressure than those from food samples. MLST revealed specific sequence types found in both food and wild birds. The study can guide future studies with whole-genome analysis on a larger number of isolates from various sectors for public health measures.

Detection and characterization of a novel bat-borne coronavirus in Singapore using multiple molecular approaches.

Bats are important reservoirs and vectors in the transmission of emerging infectious diseases. Many highly pathogenic viruses such as SARS-CoV and rabies-related lyssaviruses have crossed species barriers to infect humans and other animals. In this study we monitored the major roost sites of bats in Singapore, and performed surveillance for zoonotic pathogens in these bats. Screening of guano samples collected during the survey uncovered a bat coronavirus (Betacoronavirus) in Cynopterus brachyotis, commonly known as the lesser dog-faced fruit bat. Using a capture-enrichment sequencing platform, the full-length genome of the bat CoV was sequenced and found to be closely related to the bat coronavirus HKU9 species found in Leschenault's rousette discovered in the Guangdong and Yunnan provinces.

Serological evidence of continued Japanese encephalitis virus transmission in Singapore nearly three decades after end of pig farming.

BACKGROUND: Singapore used to report an annual average of 14 cases of Japanese encephalitis, but ever since the abolishment of pig farms in the early 1990s, the local incidence rate for Japanese encephalitis virus (JEV) infections has reduced drastically. Studies done in the early 2000s demonstrated the presence of JEV-specific antibodies in animals such as wild boars, dogs, chickens and goats on the offshore island and peripheral parts of the Singapore, indicative of prior JEV exposure. A JEV wildlife and sentinel chicken surveillance system was initiated in 2010 through to 2017 to study the animal host seroprofiles. RESULTS: A total of 12/371 (3.23%) of resident bird samples, 24/254 (9.45%) of migratory bird samples and 10/66 (15.16%) of wild boar samples were positive for the presence of JEV antibodies. Seroconversions in sentinel chickens were observed at two time points. Through this study, two sites with active transmission of JEV amongst avian or porcine hosts were identified. CONCLUSIONS: JEV transmission in animal hosts has continued despite the phasing out of pig farming nearly thirty years ago; however, the public health risk of transmission remains low. Environmental management for mosquito population remains key to keeping this risk low.

Serological evidence and experimental infection of cynomolgus macaques with pteropine orthoreovirus reveal monkeys as potential hosts for transmission to humans.

Pteropine orthoreoviruses (PRV) are emerging bat-borne viruses with proven zoonotic transmission. We recently demonstrated human exposure to PRV in Singapore, which together with previous reports from Malaysia and Vietnam suggest that human infection of PRV may occur periodically in the region. This raises the question whether bats are the only sources of human infection. In this study, we screened 517 cynomolgus macaques caught in Singapore for evidence of exposure to PRV3M (also known as Melaka virus), which was first isolated from human patients in Melaka, Malaysia. We found that 67 serum samples were PRV3M positive by ELISA and 34 were also positive by virus neutralization assay. To investigate whether monkeys could act as hosts for PRV transmission, we experimentally infected cynomolgus macaques with PRV3M and housed these animals with uninfected monkeys. Although no clinical signs of infection were observed in infected animals, viral RNA was detected in nasal and rectal swabs and all infected macaques seroconverted. Additionally, one of the uninfected animals seroconverted, implying active shedding and transmission of PRV3M. We provide evidence that PRV exposure in the macaque population in Singapore occurs at a relatively high prevalence and this study suggests that cynomolgus macaques may be an intermediate or reservoir host for PRVs.

Characterization of Fowlpox virus in chickens and bird-biting mosquitoes: a molecular approach to investigating Avipoxvirus transmission.

Avian pox is a highly contagious avian disease, yet relatively little is known about the epidemiology and transmission of Avipoxviruses. Using a molecular approach, we report evidence for a potential link between birds and field-caught mosquitoes in the transmission of Fowlpox virus (FWPV) in Singapore. Comparison of fpv167 (P4b), fpv126 (VLTF-1), fpv175-176 (A11R-A12L) and fpv140 (H3L) gene sequences revealed close relatedness between FWPV strains obtained from cutaneous lesions of a chicken and four pools of Culex pseudovishnui, Culex spp. (vishnui group) and Coquellitidea crassipes caught in the vicinity of the study site. Chicken-derived viruses characterized during two separate infections two years later were also identical to those detected in the first event, suggesting repeated transmission of closely related FWPV strains in the locality. Since the study location is home to resident and migratory birds, we postulated that wild birds could be the source of FWPV and that bird-biting mosquitoes could act as bridging mechanical vectors. Therefore, we determined whether the FWPV-positive mosquito pools (n=4) were positive for avian DNA using a polymerase chain reaction-sequencing assay. Our findings confirmed the presence of avian host DNA in all mosquito pools, suggesting a role for Cx. pseudovishnui, Culex spp. (vishnui group) and Cq. crassipes mosquitoes in FWPV transmission. Our study exemplifies the utilization of molecular tools to understand transmission networks of pathogens affecting avian populations, which has important implications for the design of effective control measures to minimize disease burden and economic loss.

Author Correction: Characterization of a filovirus (Menglà virus) from Rousettus bats in China.

In the version of this Letter originally published, in the 'Phylogenetic analysis' section of the Methods, the authors mistakenly stated that the GenBank accession number for the Ravn virus genome sequence was FJ750958. The correct accession number is DQ447649 for Ravn virus, Kenya, 1987. Accordingly, the label 'RAVN2007' in Fig. 1b should have been 'RAVV1987'. This mistake does not change any conclusions in this study. This statement and figure have now been amended in all versions of the Letter, and the Supplementary Information file has been updated accordingly.

Characterization of a filovirus (Menglà virus) from Rousettus bats in China.

Filoviruses, especially Ebola virus (EBOV) and Marburg virus (MARV), are notoriously pathogenic and capable of causing severe haemorrhagic fever diseases in humans with high lethality1,2. The risk of future outbreaks is exacerbated by the discovery of other bat-borne filoviruses of wide genetic diversity globally3-5. Here we report the characterization of a phylogenetically distinct bat filovirus, named Menglà virus (MLAV). The coding-complete genome of MLAV shares 32-54% nucleotide sequence identity with known filoviruses. Phylogenetic analysis places this new virus between EBOV and MARV, suggesting the need for a new genus taxon. Importantly, despite the low amino acid sequence identity (22-39%) of the glycoprotein with other filoviruses, MLAV is capable of using the Niemann-Pick C1 (NPC1) as entry receptor. MLAV is also replication-competent with chimeric MLAV mini-genomes containing EBOV or MARV leader and trailer sequences, indicating that these viruses are evolutionally and functionally closely related. Finally, MLAV glycoprotein-typed pseudo-types transduced cell lines derived from humans, monkeys, dogs, hamsters and bats, implying a broad species cell tropism with a high risk of interspecies spillover transmission.

Development of a sensitive and specific epitope-blocking ELISA for universal detection of antibodies to human enterovirus 71 strains.

BACKGROUND: Human Enterovirus 71 (EV71) is a common cause of hand, foot and mouth disease (HFMD) in young children. It is often associated with severe neurological diseases and mortalities in recent outbreaks across the Asia Pacific region. Currently, there is no efficient universal antibody test available to detect EV71 infections. METHODOLOGY/PRINCIPAL FINDING: In the present study, an epitope-blocking ELISA was developed to detect specific antibodies to human EV71 viruses in human or animal sera. The assay relies on a novel monoclonal antibody (Mab 1C6) that specifically binds to capsid proteins in whole EV71 viruses without any cross reaction to any EV71 capsid protein expressed alone. The sensitivity and specificity of the epitope-blocking ELISA for EV71 was evaluated and compared to microneutralization using immunized animal sera to multiple virus genotypes of EV71 and coxsackieviruses. Further, 200 serum sample from human individuals who were potentially infected with EV71 viruses were tested in both the blocking ELISA and microneutralization. Results indicated that antibodies to EV71 were readily detected in immunized animals or human sera by the epitope blocking ELISA whereas specimens with antibodies to other enteroviruses yielded negative results. This assay is not only simpler to perform but also shows higher sensitivity and specificity as compared to microneutralization. CONCLUSION: The epitope-blocking ELISA based on a unique Mab 1C6 provided highly sensitive and 100% specific detection of antibodies to human EV71 viruses in human sera.

Characterization of a novel monoclonal antibody reactive against the N-terminal region of Enterovirus 71 VP1 capsid protein.

Hand, foot and mouth disease (HFMD) is a viral infectious disease caused by human Enterovirus A, particularly Enterovirus 71 (EV71) and Coxsackievirus 16 (CA16) serotypes, with EV71 infection associated with severe neurological complications and mortality. Lots of attention has been placed on elucidating viral epitopes, which is useful for EV71 viral research. In this study, a murine monoclonal antibody (mAb 4) specific for EV71 was generated and mapped to target the N-terminal region of VP1 capsid protein, spanning amino acid residues 12-19 (IGDSVSRA). mAb 4 can cross-react with all the 11 representative EV71 subgenotypes (A, B1-5, C1-5), but not with the representative strain of CA16 as demonstrated by immunofluorescence assay (IFA). BLAST analyses of this epitope against all Enterovirus entries in Genbank also demonstrated that this epitope is unique in EV71, but not other Enterovirus such as CA16 It may be useful for structural study of VP1 morphogenesis during infection and also applications for identification of EV71 infection.

Characterization and specificity of the linear epitope of the enterovirus 71 VP2 protein.

BACKGROUND: Enterovirus 71 (EV71) has emerged as a major causative agent of hand, foot and mouth disease in the Asia-Pacific region over the last decade. Hand, foot and mouth disease can be caused by different etiological agents from the enterovirus family, mainly EV71 and coxsackieviruses, which are genetically closely related. Nevertheless, infection with EV71 may occasionally lead to high fever, neurologic complications and the emergence of a rapidly fatal syndrome of pulmonary edema associated with brainstem encephalitis. The rapid progression and high mortality of severe EV71 infection has highlighted the need for EV71-specific diagnostic and therapeutic tools. Monoclonal antibodies are urgently needed to specifically detect EV71 antigens from patient specimens early in the infection process. Furthermore, the elucidation of viral epitopes will contribute to the development of targeted therapeutics and vaccines. RESULTS: We have identified the monoclonal antibody 7C7 from a screen of hybridoma cells derived from mice immunized with the EV71-B5 strain. The linear epitope of 7C7 was mapped to amino acids 142-146 (EDSHP) of the VP2 capsid protein and was characterized in detail. Mutational analysis of the epitope showed that the aspartic acid to asparagine mutation of the EV71 subgenogroup A (BrCr strain) did not interfere with antibody recognition. In contrast, the serine to threonine mutation at position 144 of VP2, present in recently emerged EV71-C4 China strains, abolished antigenicity. Mice injected with this virus strain did not produce any antibodies against the VP2 protein. Immunofluorescence and Western blotting confirmed that 7C7 specifically recognized EV71 subgenogroups and did not cross-react to Coxsackieviruses 4, 6, 10, and 16. 7C7 was successfully used as a detection antibody in an antigen-capture ELISA assay. CONCLUSIONS: Detailed mapping showed that the VP2 protein of Enterovirus 71 contains a single, linear, non-neutralizing epitope, spanning amino acids 142-146 which are located in the VP2 protein's E-F loop. The S/T(144) mutation in this epitope confers a loss of VP2 antigenicity to some newly emerged EV71-C4 strains from China. The corresponding monoclonal antibody 7C7 was used successfully in an AC-ELISA and did not cross-react to coxsackieviruses 4, 6, 10, and 16 in immunofluorescence assay and Western blots. 7C7 is the first monoclonal antibody described, that can differentiate Coxsackievirus 16 from Enterovirus 71.

Characterization of a monoclonal antibody against the 3D polymerase of enterovirus 71 and its use for the detection of human enterovirus A infection.

Over the last decade, frequent epidemic outbreaks of hand, foot and mouth disease have been observed in the Asia-Pacific region. Hand, foot and mouth disease is caused by different viruses from the enterovirus family, mainly coxsackievirus A16 and enterovirus 71 (EV71) from the human enterovirus A family. Severe disease and neurological complications are associated more often with EV71 infection, and can lead occasionally to fatal brain stem encephalitis in young children. The rapid progression and high mortality of severe hand, foot and mouth disease makes the direct detection of antigens early in infection essential. The best method for virus detection is the use of specific monoclonal antibodies. The generation and characterization of a monoclonal antibody specific for the 3D polymerase of human enterovirus A and the development of a virus detection dot blot assay are described. A recombinant 3CD protein from EV71 C4 strain was used as an immunogen to generate monoclonal antibodies (MAbs). Screening of hybridoma cells led to the isolation of monoclonal antibody 4B12 of the immunoglobulin IgG1 isotype. MAb 4B12 recognizes the linear epitope DFEQALFS close to the active site of the 3D polymerase, corresponding to amino acid positions 53-60 of 3D and 1784-1791 of enterovirus 71 polyprotein. The presence of 3D polymerase and its precursor 3CD proteinase in purified virus particles was confirmed. MAb 4B12 was used successfully to detect all enterovirus 71 subgenotypes in a denaturing dot blot assay with a sensitivity of 10 pg of 3D protein and 10(4) tissue culture infective dose of virus particles.

Characterization of an isotype-dependent monoclonal antibody against linear neutralizing epitope effective for prophylaxis of enterovirus 71 infection.

BACKGROUND: Enterovirus 71 (EV71) is the main causative agent of Hand, Foot and Mouth disease (HFMD) and is associated with severe neurologic complications and mortalities. At present, there is no vaccine or therapeutic available for treatment. METHODOLOGY/PRINCIPAL FINDING: In this study, we generated two mAbs, denoted as mAb 51 and 53, both targeting the same linear epitope on VP1 capsid protein, spanning amino acids 215-219. In comparison, mAb 51 belonging to isotype IgM possesses neutralizing activity in vitro, whereas, mAb 53 belonging to isotype IgG1 does not have any neutralizing ability, even towards its homologous strain. When mAb 51 at 10 µg/g of body weight was administered to the 2-week-old AG129 mice one day prior to lethal challenge, 100% in vivo passive protection was observed. In contrast, the isotype control group mice, injected with an irrelevant IgM antibody before the challenge, developed limb paralysis as early as day 6 post-infection. Histological examination demonstrated that mAb 51 was able to protect against pathologic changes such as neuropil vacuolation and neuronal loss in the spinal cord, which were typical in unprotected EV-71 infected mice. BLAST analyses of that epitope revealed that it was highly conserved among all EV71 strains, but not coxsachievirus 16 (CA16). CONCLUSION: We have defined a linear epitope within the VP1 protein and demonstrated its neutralizing ability to be isotype dependent. The neutralizing property and highly conserved sequence potentiated the application of mAb 51 and 53 for protection against EV71 infection and diagnosis respectively.