FMD remains one of the most economically important diseases of farm animals and is widespread across the world, especially in Africa, Asia and South America. Type O FMD is prevalent in China (including Chinese Taipei, Chinese Hong Kong) and its surrounding countries [10–13]. Among the epidemic topotypes in these regions, the viruses in CHY topotype are highly adapted to pigs , which represents the biggest threat on the Chinese hog industry and the economy. For instance, in 1997, a devastating and unusual outbreak of FMD occurred in Taiwan. The outbreak, which was caused by a CHY topotype virus, rapidly developed into massive epizootic, resulting in the slaughter of more than 4 million pigs and financial losses of over 6 billion U.S. dollars [35, 36]. China is the biggest pork producer and consumer in the world and the pig industry has become the most important sector in Chinese animal husbandry . Therefore, control of FMD in swine has become high priority. Vaccination as a control tool has been gaining favor as a potentially more effective approach for controlling the virus, reducing the economic loss to animal husbandry, and contributing to improved food security in China. However, currently available FMD vaccines in China often do not provide complete protection against some variants arising in the CHY topotype.
Extensive studies has showed that the G–H loop of FMDV is the major immunodominant site [38, 39], and it can induce a strong antibody response against the virus, which is known to play a major role in protection induced by the current FMDV vaccines . Experimental peptide or recombinant vaccines [40, 41] have been based mainly on this major antigenic site of FMDV. In addition, FMDV antigenic variation also occurs within other antigenic sites, which are implicated in the full complete immunologic response [9, 42]. Based on these theories, the present study compared amino acid sequences of the capsid region of O/HN/CHA/93 vaccine strain and 18 reference isolates of CHY topotype. The results revealed that consensus amino acid substitutions occurred at VP1 (43 T → K, 48 I → V 134 C → S, 137 S → G, 139 A → T, 140 R → S, 141 V → T, 142 S → N) and VP3 (58 E → D), which are involved in antigenic site 1, 3, and 4. Among these substitutions, six were located at the G-H loop of VP1 and three were located at a position critical for antigenic sites 3 and 4. Any alteration of critical residues would confer antigenic specificity to the FMD viral variants [43–45]. Therefore, we presumed that the antigenic differences between the variants of CHY topotype and the current vaccine strain might account for incomplete protection.
Previous successes in the generation of engineered avirulent, chimeric, and thermostable FMDV vaccine candidates [33, 46, 47] have further provided insight for designing new vaccine candidate viruses with engineered modifications by reverse genetics. Therefore, the present study constructed a full-length infectious clone of O/HN/CHA/93 vaccine strain and created a genetically modified construct with amino acid substitutions (1 C 58 E → D, 1D 43 T → K, 48 I → V, 134 C → S, 137 S → G, 139 A → T, 140 R → S, 141 V → T, 142 S → N), based on vaccine strain framework using a similar strategy. As expected, a genetically modified virus was obtained from the modified full-length plasmid. Specifically, amino acid substitutions in the capsid protein did not affect the in vitro infectivity properties of the recombinant. Viability of the virus indicated that the genome of FMDV O/HN/CHA/93 can tolerate these amino acid replacements at three antigenic sites. This finding was not surprising, because previous studies have demonstrated that FMDV can accommodate replacements of G-H loop, capsid coding region as well as other gene region of inter-genotypic and intra-genotypic [16, 42, 48–50]. The recombinant viruses were genetically stable after 10 serial passages in BHK-21 cells.
As a FMD vaccine candidate, the virus should necessarily grow to high yield in tissue culture for sufficient antigenic mass to be produced . Therefore, the replicative properties of the recombinant viruses were assessed by single-step growth curves. The results indicated that the rescued viruses had similar growth properties to the wild O/HN/CHA/93 virus. In addition, an ideal vaccine candidate would induce cross protection against viruses from different antigenic groups within the subtype. Analysis of antigenic relationships between the field isolate of the three topotypes and the reference strains (O/HN/CHA/93 and rM-HN) showed that the O/HN/CHA/93 reference strains demonstrated close antigenic relationship with O/Tibet/CHA/99 and O/JX/CHA/2010 isolates. However, the O/TAW/TL/97 isolate is antigenically related to the vaccine strain. The rM-HN virus had close antigenic relationship with isolates of the three topotypes compared with the O/HN/CHA/93 strain. Thus, this recombinant virus would be used as vaccine candidate. Comparative efficacy of the inactivated vaccines prepared from the rM-HN and O/HN/CHA/93 viruses were tested in swine. The results demonstrated that pigs vaccinated with the O/HN/CHA/93 vaccine were fully protected from O/Tibet/CHA/99 and O/JX/CHA/2010 virus challenge, but only 75% of the immunized animals were against O/TAW/TL/97 infection. However, all pigs vaccinated with the rM-HN vaccine obtained complete protection against O/Tibet/CHA/99, O/TAW/TL/97, and O/JX/CHA/2010 virus challenge, which may be contribute to the more antigentic similarities of the genetically modified virus with the isolate of CHY topotype.