Human enterovirus 71 subgenotype B3 lacks coxsackievirus A16-like neurovirulence in mice infection

Abstract

Background: 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.

Results: 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.

Conclusion: 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.

Figure 1

Structural alignment of EV-71 and CV-A16 3D RNA polymerase amino acid sequences. EV-71 subgenotype B3, B4 and CV-A16/G10 amino acid sequences were aligned against the poliovirus 1 Mahoney 3D RNA polymerase template sequences (PDB: 1RDR). Conserved residues are indicated as (●) and each domain are boxed and labeled. Residues shared by EV-71 B3 virus and CV-A16 were highlighted in grey and residues unique for EV-71 B3 virus were highlighted in pink.

Figure 2

Predicted RNA secondary structures of EV-71 B3, EV-71 B4 and CV-A16/G10 3' UTR. RNA structures were predicted based on the lowest free energy, using the Zuker algorithm as implemented in RNA Structure (version 3.71). The predicted 3' UTR structures consisted of nucleotides from position 7326–7407 and additional 12 nucleotides of the poly-A tail.

Figure 3

EV-71 and CV-A16 infections of newborn mice. One-day old newborn mice were intracerebrally inoculated with 1 × 10³ PFU virus per mouse and monitored daily. CV-A16-infected mice had floppy tails on day two post-inoculation (a) and hind leg paralysis beginning on day three post-inoculation (arrow, b). Mice with swollen limbs were noted in EV-71 B3 virus infection (arrow, c) and the EV-71 B3-infected mice had significantly reduced body weight gain in comparison to the mock-infected mice (d, V = B3-infected mouse, C = mock-infected mouse). Mice with floppy tails, swollen limbs and paralysis (e) and death (f) were recorded. The weight gain of the surviving mice was also determined (g).

Figure 4

Detection of enterovirus genome sequences in infected newborn mice brain. At selected intervals post-inoculation (indicated by the number above each lane), mice were sacrificed (each mouse indicated by the alphabet above each lane) and RT-PCR was performed using an enterovirus specific primers. The presence of a 150 bp amplified DNA fragment indicates the presence of enterovirus genome, which was later confirmed by DNA sequencing.