Human enterovirus 71 subgenotype B3 lacks coxsackievirus A16-like neurovirulence in mice infection
© Chan and AbuBakar; licensee BioMed Central Ltd. 2005
Received: 22 June 2005
Accepted: 26 August 2005
Published: 26 August 2005
At least three different EV-71 subgenotypes were identified from an outbreak in Malaysia in 1998. The subgenotypes C2 and B4 were associated with the severe and fatal infections, whereas the B3 virus was associated with mild to subclinical infections. The B3 virus genome sequences had ≥85% similarity at the 3' end to CV-A16. This offers opportunities to examine if there are characteristic similarities and differences in virulence between CV-A16, EV-71 B3 and EV-71 B4 and to determine if the presence of the CV-A16-liked genes in EV-71 B3 would also confer the virus with a CV-A16-liked neurovirulence in mice model infection.
Analysis of human enterovirus 71 (EV-71) subgenotype B3 genome sequences revealed that the 3D RNA polymerase and domain Z of the 3'-untranslating region RNA secondary structure had high similarity to CV-A16. Intracerebral inoculation of one-day old mice with the virus resulted in 16% of the mice showing swollen hind limbs and significantly lower weight gain in comparison to EV-71 B4-infected mice. None of the mice presented with hind leg paralysis typical in all the CV-A16 infected mice. CV-A16 genome sequences were amplified from the CV-A16-infected mice brain but no amplification was obtained from all the EV-71-inoculated mice suggesting that no replication had taken place in the suckling mice brain.
The findings presented here suggest that EV-71 B3 viruses had CV-A16-liked non-structural gene features at the 3'-end of the genome. Their presence could have affected virulence by affecting the mice general health but was insufficient to confer the EV-71 B3 virus a CV-A16-liked neurovirulence in mice model infection.
Enterovirus 71 (EV-71) was first described in 1969 during an outbreak with central nervous system complications in California . Since then, EV-71 infections have been associated with a number of outbreaks with wide clinical manifestations, ranging from mild hand, foot and mouth disease (HFMD) to severe neurological complications and deaths. These include outbreaks in Bulgaria , Hungary , Japan  and more recently Malaysia [5, 6], Taiwan  and Singapore . In the later three outbreaks, more than a hundred deaths in total were reported, elevating EV-71 infection as one of the most deadly virus infection to date amongst young children below the age of 3 years old in Asia. The sudden emergence of the deadly forms of EV-71 infection in Asia was puzzling, as the virus together with other human enterovirus A viruses especially coxsackievirus A5 (CV-A5), CV-A10 and CV-A16 have been noted to cause HFMD in the region for sometime . During the outbreak in Malaysia, at least three different EV-71 subgenotypes were identified. The subgenotypes C2 and B4 were associated with the severe and fatal infections, whereas, mild to subclinical infections were associated with the B3 viruses [10–12]. Unlike the earlier two subgenotypes, the B3 virus circulated for only a brief period during the outbreak and they have since not been isolated from patients from the later outbreaks [11, 12]. A recent study reported that the B3 virus genome sequences had ≥93% similarity to EV-71 at the 5' end whereas the P3 genome region and 3'UTR had ≥85% similarity to CV-A16 . CV-A16 is known to be the most common causative agent for the self-limiting HFMD. It is usually characterized by mild fever, oral ulcers and vesicular lesions on palms and soles and is not known to cause severe and fatal CNS infections. It is not presently understood why EV-71 infections tend to cause the more severe form of HFMD in comparison to CV-A16. The findings that EV-71 B3 viruses had high sequence similarity to CV-16 at the 3' end of the genome and that the viruses were not associated with the severe form of HFMD, offered opportunities to examine the potential roles of the respective genes in determining virulence. Hence, the present study was undertaken to examine if there are characteristic similarities and differences between CV-A16, EV-71 B3 and the more virulent EV-71 B4 virus and to determine if the presence of the CV-A16-liked genes in the EV-71 B3 virus genome would also confer the virus a CV-A16-liked neurovirulence in mice.
Results and Discussion
Additional File 1: Hind leg paralysis in CV-A16 infected mice. By day three post-inoculation, the mice were lethargic, tremoring and uncoordinated. (WMV 666 KB)
Results from the present study, also did not support the possibility that acquisition of CV-A16-liked genome sequences alone is sufficient to confer the EV-71 B3 virus a CVA16-liked neurovirulence in mice. The significant mice weight gain differences noted between mice infected with EV-71 B3 and EV-71 B4 viruses, with the later performing much better, however, suggested that EV-71 B3 virus infection somehow did affect mice general health. As weight gain differences are the only biological parameter that differentiate between the B3 and B4 viruses, it does appears that EV-71 B3 affected mice more than the EV-71 B4 virus. It is also worth noting that in contrast to infection in mice, CV-A16 infection in human in general does not result in severe infection as oppose to EV-71, particularly the EV-71 B4 virus infection. In parallel manner, the EV-71 B3 viruses, while they affected mice, they did not cause severe or fatal infection in humans. These implied that the EV-71 B3 virus is truly different and as its genome suggested, it has to some extent features of both EV-71 and CV-A16 infection in-vivo.
Results from the present study suggest that EV-71 B3 virus had CV-A16-liked non-structural gene 3D RNA polymerase and 3' UTR features at the 3' end of the genome. Their presence affected virulence differently from infection with EV-71 B4 and CV-A16 by affecting the mice general health. The presence of the CV-A16-liked genes, however, was insufficient to markedly influence the neurovirulence properties of EV-71 B3 virus in mice.
Materials and methods
Two EV-71 isolates identified from the 1997 HFMD outbreak in Malaysia were used. The subgenotype B3 isolate, SHA66 (EMBL: AJ238457) was isolated from a HFMD patient presented with mild infection [6, 23]. The subgenotype B4 isolate, UH1 (EMBL: AJ238455) on the other hand, was isolated from the brain of a patient who died of EV-71-associated neurogenic pulmonary edema [5, 6, 24]. The CV-A16 isolate used was previously isolated from a HFMD patient seen at the University Malaya Medical Centre. This CV-A16 isolate was identified and characterized using monoclonal antibody staining (Chemicon Cat #3323, California, USA) and amplification of partial 5' UTR gene (data not shown).
Amino acid sequence analysis
Amino acid sequences were examined after stripping the 5' UTR and 3' UTR sequences and consensus sequences of EV-71 B3 and EV-71 B4 viruses were aligned and manually edited using GeneDoc software . The previously published three-dimensional crystal structure of the 3D RNA polymerase was downloaded as template for the alignment. Using the WHAT IF program , domains that represent the conserved regions, loops, insertion or deletions were manually visualized to generate a structural alignment.
RNA secondary structure prediction
The 3' UTR RNA secondary structure was predicted using Zuker optimal and suboptimal minimal free energy folding algorithms, as implemented in RNA Structure version 3.71 software . Part of the poly A tract was incorporated into the sequences.
Determination of virulence in mice
A total of 24, 25 and 31 one-day old newborn ICR mice were inoculated intracerebrally with either CV-A16 or SHA66 (B3 virus) or UH1 (B4 virus) virus inoculum. The virus inoculum with infectivity of ~1 × 103 p.f.u. was injected in a volume of 10–20 μl into the mice brain. The mice were closely monitored for any clinical symptoms, paralysis and death and the weight of each surviving mouse was recorded daily up to day 11 post-inoculation. Another litter with at least 10 one-day old newborn mice was injected with comparable growth medium and used as controls. At selected intervals post-infection, some of the mice were sacrificed and the brain tissues were harvested for total RNA using the TRI Reagent™ (Molecular Research Centre, Inc., Cincinnati, USA) following the manufacturer's recommended protocols. The RT-PCR amplification for the detection of enterovirus sequence was performed using 1 μg of RNA. Access RT-PCR kit (Promega, USA) and primer pairs, EntabF (5'-TCC TCC GGC CCC TGA ATG CGG CTA AT-3'; nucleotide positions 449–474, based on MS87 strain, Genbank: U22522) and EVRR (5'-AAT TGT CAC CAT AAG CAG GC-3'; nucleotide positions 586–606) were used. Reverse transcription was performed at 42°C for an hour followed by amplification steps; 95°C-30 seconds, 55°C-30 seconds and 72°C-30 seconds for 30 cycles and finally with 5 minutes extension at 72°C using the PTC thermal cycler (MJ Research, Massachusetts, USA). When no amplicon was obtained, the number of cycle was increased to 40. Alternatively, a second step PCR using similar parameters was performed using ten-fold diluted RT-PCR product as template. The amplified DNA fragments were electrophoresed using 2% agarose gel in 0.5 × tris-acetate EDTA buffer (0.02 M Tris base, 0.5 mM EDTA pH 8.0, 0.057% glacial acetic acid) and sequence confirmation was made by sequencing the DNA fragment.
Student's t-test was used to evaluate if the differences in weight between the virus-inoculated mice and control mice was significant. Wilcoxon signed rank test was used to compare the survival and paralysis probability between the virus-inoculated mice and control mice. All statistical analyses were implemented using SPSS for Windows version 11.5 (SPSS Inc., Illinois, USA). All tests were two-sided and P < 0.05 was considered as statistically significant.
List of Abbreviation
- HFMD Hand:
foot and mouth disease
This study is funded in parts by grants from the Ministry of Science, Technology and Innovation, Malaysia # 06-02-09-001-BTK/TD/002.
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