Enterovirus 71 (EV71) was first isolated in 1969 in California, USA. Several epidemic outbreaks with high mortality rates have occurred in European and Asian Countries (Bulgaria in 1975, Hungary in 1978, Malaysia in 1997, Taiwan in 1998 and China in 2008). EV71 CNS involvement may be associated with neurological sequelae, delayed neurodevelopment and reduced cognitive functioning. Since poliovirus was nearly eradicated by vaccination, EV71 is now considered as one of the top candidates for new vaccine development against human enteroviruses. Recently, several EV71 vaccine candidates, including live-attenuated virus, inactivated whole virus, recombinant viral protein, virus-like particle and DNA vaccines, have been evaluated in animals but no clinical trial has yet been conducted. Based on historical experiences with poliovirus vaccines and animal studies, the inactivated whole-virus vaccines are feasible and could be licensed readily, so these are targeted for preparing clinical trials in several organizations in Asia.

Recently, enterovirus 71 (EV71) has caused life-threatening outbreaks involving neurological and cardiopulmonary complications in Asian children with unknown mechanism. EV71 has one single serotype but can be phylogenetically classified into 3 main genogroups (A, B and C) and 11 genotypes (A, B1∼B5 and C1∼C5). In Taiwan, nationwide EV71 epidemics with different predominant genotypes occurred in 1998 (C2), 2000–2001 (B4), 2004–2005 (C4), and 2008 (B5). In this study, sera were collected to measure cross-reactive neutralizing antibody titers against different genotypes. We collected historical sera from children who developed an EV71 infection in 1998, 2000, 2005, 2008, or 2010 and measured cross-reactive neutralizing antibody titers against all 11 EV71 genotypes. In addition, we aligned and compared the amino acid sequences of P1 proteins of the tested viruses. Serology data showed that children infected with genogroups B and C consistently have lower neutralizing antibody titers against genogroup A (>4-fold difference). The sequence comparisons revealed that five amino acid signatures (N143D in VP2; K18R, H116Y, D167E, and S275A in VP1) are specific for genogroup A and may be related to the observed antigenic variations. This study documented antigenic variations among different EV71 genogroups and identified potential immunodominant amino acid positions. Enterovirus surveillance and vaccine development should monitor these positions. Recently, enterovirus 71 (EV71) has caused life-threatening outbreaks in tropical Asia. EV71 has one single serotype but can be phylogenetically classified into 3 main genogroups and 11 genotypes (A, B1∼B5 and C1∼C5). In Taiwan, nationwide EV71 epidemics with different predominant genotypes occurred in 1998(C2), 2000–2001(B4), 2004–2005(C4), and 2008(B5). In this study, historical sera from children infected with these 4 genotypes were collected to measure cross-reactive neutralizing antibody titers against 11 genotypes. In addition, amino acid sequences of P1 proteins of the tested viruses were compared. Serology data showed that children infected with genogroup B and C consistently have lower neutralizing antibody titers against genogroup A (>4-fold difference). Antigenic variations between genogroup B and C could be detected but did not have a clear pattern. Five amino acid signatures are specific for genogroup A and may be related to the observed antigenic variations. Vaccine development should monitor the antigenic and genetic variations to select vaccine strains.

Enterovirus 71 (EV71) causes life-threatening epidemics in Asia and can be phylogenetically classified into three major genogroups (A∼C) including 11 genotypes (A, B1∼B5, and C1∼C5). Recently, EV71 epidemics occurred cyclically in Taiwan with different genotypes. In recent years, human studies using post-infection sera obtained from children have detected antigenic variations among different EV71 strains. Therefore, surveillance of enterovirus 71 should include phylogenetic and antigenic analysis. Due to limitation of sera available from children with EV71 primary infection, suitable animal models should be developed to generate a panel of antisera for monitoring EV71 antigenic variations. Twelve reference strains representing the 11 EV71 genotypes were grown in rhabdomyosarcoma cells. Infectious EV71 particles were purified and collected to immunize rabbits. The rabbit antisera were then employed to measure neutralizing antibody titers against the 12 reference strains and 5 recent strains. Rabbits immunized with genogroup B and C viruses consistently have a lower neutralizing antibody titers against genogroup A (≧8-fold difference) and antigenic variations between genogroup B and C viruses can be detected but did not have a clear pattern, which are consistent with previous human studies. Comparison between human and rabbit neutralizing antibody profiles, the results showed that ≧8-fold difference in rabbit cross-reactive antibody ratios could be used to screen EV71 isolates for identifying potential antigenic variants. In conclusion, a rabbit model was developed to monitor antigenic variations of EV71, which are critical to select vaccine strains and predict epidemics. Enterovirus 71 (EV71) has caused several life-threatening epidemics in children in the Asia-Pacific region since 1997. EV71 has one single serotype as measured using hyper-immune animal antisera but can be phylogenetically classified into three major genogroups (A, B and C) and eleven genotypes (A, B1–B5, and C1–C5). Recently, epidemiological studies in the Asia-Pacific region have found that large-scale EV71 epidemics occurred cyclically with different genotypes. This observation of genotype replacement, in conjunction with the observed antigenic variations among different EV71 genogroups in human studies, has kindled the interest to establish animal models to monitor the antigenic variations of EV71. In this study, we developed a rabbit model to monitor antigenic variations of EV71, which could be further integrated into national enterovirus surveillance systems.

Current egg-based influenza vaccine production technology can't promptly meet the global demand during an influenza pandemic as shown in the 2009 H1N1 pandemic. Moreover, its manufacturing capacity would be vulnerable during pandemics caused by highly pathogenic avian influenza viruses. Therefore, vaccine production using mammalian cell technology is becoming attractive. Current influenza H5N1 vaccine strain (NIBRG-14), a reassortant virus between A/Vietnam/1194/2004 (H5N1) virus and egg-adapted high-growth A/PR/8/1934 virus, could grow efficiently in eggs and MDCK cells but not Vero cells which is the most popular cell line for manufacturing human vaccines. After serial passages and plaque purifications of the NIBRG-14 vaccine virus in Vero cells, one high-growth virus strain (Vero-15) was generated and can grow over 10⁸ TCID₅₀/ml. In conclusion, one high-growth H5N1 vaccine virus was generated in Vero cells, which can be used to manufacture influenza H5N1 vaccines and prepare reassortant vaccine viruses for other influenza A subtypes.

Enterovirus 71 (EV71) belongs to picornavirus family and could be classified phylogenetically into three major genogroups (A, B and C) including 11 genotypes (A, B1-B5 and C1-C5). Since 1997, EV71 has caused large-scale of epidemics with neurological complications in Asian children. In Taiwan, nationwide EV71 epidemics with different predominant genotypes have occurred cyclically since 1998. A nationwide EV71 epidemic occurred again in 2012. We conducted genetic and antigenic characterizations of the 2012 epidemic. Chang Gung Memorial Hospital (CGMH) is a medical center in northern Taiwan. In CGMH, specimens were collected from pediatric inpatients with suspected enterovirus infections for virus isolation. Enterovirus isolates were serotyped and genotyped and sera from EV71 inpatients were collected for measuring neutralizing antibody titers. There were 10, 16 and 99 EV71 inpatients identified in 2010, 2011 and 2012, respectively. There were 82 EV71 isolates genotyped, which identified 17 genotype C4a viruses and 65 genotype B5 viruses. The genotype B5 viruses were not detected until November 2011 and caused epidemics in 2012. Interestingly, the B5-2011 viruses were genetically distinguishable from the B5 viruses causing the 2008 epidemic and are likely introduced from China or Southeastern Asia. Based on antigenic analysis, minor antigenic variations were detected among the B5-2008, B5-2011, C4a-2008 and C4a-2012 viruses but these viruses antigenically differed from genotype A. Genotype B5 and C4a viruses antigenically differ from genotype A viruses which have disappeared globally for 30 years but have been detected in China since 2008. Enterovirus surveillance should monitor genetic and antigenic variations of EV71.