David Kiang

Reemergence of Enterovirus 71 in 2008 in Taiwan: Dynamics of Genetic and Antigenic Evolution from 1998 to 2008

ABSTRACT In recent years, enterovirus 71 (EV71) has been a cause of numerous outbreaks of hand-foot-and-mouth disease, with severe neurological complications in the Asia-Pacific region. The reemergence in Taiwan of EV71 genotype B5 in 2008 resulted in the largest outbreak of EV71 in Taiwan in the past 11 years. Phylogenetic analyses indicated that dominant genotype changes from B to C or C to B occurred at least three times between 1986 and 2008. Furthermore, antigenic cartography of EV71 by using neutralization tests revealed that the reemerging EV71 genotype B5 strains formed a separate cluster which was antigenically distinct from the B4 and C genotypes. Moreover, analyses of full-length genomic sequences of EV71 circulating in Taiwan during this period showed the occurrence of intra- and interserotypic recombination. Therefore, continuous surveillance of EV71 including the monitoring of genetic evolution and antigenic changes is recommended and may contribute to the development of a vaccine for EV71.

Rhinovirus Outbreak in a Long Term Care Facility for Elderly Persons Associated with Unusually High Mortality

Abstract During a 6-week period in 2003, 56 residents and 26 staff developed respiratory illness in a long-term facility; 12 residents died. Seven of 13 respiratory specimens were culture-positive for rhinovirus; 6 of the isolates were serotype 82. In elderly populations, severe illness may be associated with organisms typically considered to be “benign,” such as rhinovirus.

A Summer Outbreak of Human Metapneumovirus Infection in a Long-Term-Care Facility

Human metapneumovirus (hMPV), a recently discovered paramyxovirus, is thought to be primarily a winter-spring pathogen affecting young children with a clinical presentation similar to that of respiratory syncytial virus. In June-July 2006, a respiratory outbreak in a long-term-care facility was reported to the local health department and investigated. Surveillance identified 26 residents and 13 staff with acute respiratory illness; 8 residents (31%) developed radiographically confirmed pneumonia, and 2 (5%) were hospitalized. Five of 14 respiratory specimens were positive by polymerase chain reaction assay for hMPV; sequencing identified genotype A. In institutionalized elderly persons, hMPV may be an important cause of respiratory outbreaks year-round.

Assay for 5′ Noncoding Region Analysis of All Human Rhinovirus Prototype Strains

ABSTRACT Increasing recognition of the association of rhinovirus with severe lower respiratory tract illnesses has clarified the need to understand the relationship between specific serotypes of rhinovirus and their clinical consequences. To accomplish this, a specific and sensitive assay to detect and serotype rhinovirus directly from clinical specimens is needed. Traditional methods of serotyping using culture and serum neutralization are time-consuming, limited to certain reference laboratories, and complicated by the existence of over 100 serotypes of human rhinoviruses (HRVs). Accordingly, we have developed a sequence-based assay that targets a 390-bp fragment accounting for approximately two-thirds of the 5′ noncoding region (NCR). Our goal was to develop an assay permitting amplification of target sequences directly from clinical specimens and distinction among all 101 prototype strains of rhinoviruses. We determined the sequences of all 101 prototype strains of HRV in this region to enable differentiation of virus genotypes in both viral isolates and clinical specimens. We evaluated this assay in a total of 101 clinical viral isolates and 24 clinical specimens and compared our findings to genotyping results using a different region of the HRV genome (the VP4-VP2 region). Five specimens associated with severe respiratory disease in children did not correlate with any known serotype of rhinovirus and were found to belong to a novel genogroup of rhinovirus, genogroup C. Isolates were also found that corresponded to the genogroup A2 variant identified in New York and Australia and two other novel group A clusters (GAC1 and GAC2).

Laboratory Diagnosis and Genetic Analysis of an Echovirus 30-Associated Outbreak of Aseptic Meningitis in Taiwan in 2001

ABSTRACT A large outbreak of aseptic meningitis occurred from April to November 2001 in Taiwan. Of the 1,130 enterovirus-infected patients, echovirus 30 (E30) infection was diagnosed in 188 (16.6%), with the patients having various clinical manifestations including aseptic meningitis (73.9%), young infant fever (6.9%), respiratory symptoms or herpangina (13.3%), or others (5.9%). The majority of the E30-infected patients were between 3 and 10 years old. Of the 264 E30 strains identified, 94.3, 71, and 67.4% were isolated from RD, MRC-5, and A549 cells, respectively. Primary isolation of E30 required mean times of 3.7 days for RD cells and 4.1 days for MRC-5 and A549 cells. Among all E30-positive patients, virus was most frequently isolated from throat swab specimens (85.2%) and, to a lesser extent, stool (76.4%) or cerebrospinal fluid (70.1%) specimens. The virus isolates were initially identified as echovirus 4 (E4) on the basis of immunofluorescence staining with anti-E4 and anti-E30 (Bastianni prototype) monoclonal antibodies. However, upon performance of the neutralization test, E30-specific reverse transcription-PCR, and sequencing of the VP1 gene, the results identified these isolates as E30, not E4, indicating that the reagent used to type E30, which is produced with the Bastianni strain as the immunogen, is inadequate for the identification of recent E30 isolates in Taiwan. Phylogenetic analyses of the VP1 genes of these isolates showed that their sequences differed from those of E30 isolates from the GenBank database by 9.1 to 25.2%, suggesting that this outbreak was caused by a new variant strain of E30 introduced into Taiwan in 2000 that resulted in the widespread aseptic meningitis epidemic in 2001.

Evolution of re-emergent virus and its impact on enterovirus 71 epidemics:

Enterovirus 71 (EV71), a member of the Enterovirus genus in the Picornaviridae family, has become an emergent infectious disease worldwide, most notably in Asia. As a neurotropic virus, EV71 infection occasionally causes neurological diseases with pulmonary edema, which is fatal for children. In this review, we examine the epidemiology of EV71, with three waves of increased EV71 activity since the 1970s and discuss the genotypic changes in phylogeny between the outbreaks or epidemics. Genetic changes including mutations and recombinations as well as the diversity of antigenic properties among EV71 strains in various outbreaks are described. Furthermore, the impact of genetic changes on viral pathogenesis and vaccine candidate selection are addressed. In conclusion, these genetic and antigenic investigations of EV71 evolution have provided us with new insight into the trend of EV71 epidemiology, which may contribute to a better understanding of the viral pathogenesis and vaccine development.

Introduction of a strong temperature-sensitive phenotype into enterovirus 71 by altering an amino acid of virus 3D polymerase

In 1998, an enterovirus 71 (EV71) epidemic in Taiwan resulted in 78 deaths; however, the molecular basis of EV71 pathogenicity remains poorly understood. Comparison of the deduced amino acid sequences in 3D polymerases of EV71clinical isolates showed the T251V or T251I substitution from 1986 and 1998 outbreaks. An EV71 replicon system showed that introducing an I251T mutation did not affect luciferase activities at 35 degrees C when compared with wild type; however, lower luciferase activities were observed when they were incubated at 39.5 degrees C. In addition, the I251T mutation in the EV71 infectious clone not only reduced viral replication at 39.5 degrees C in vitro but also decreased the virulence of the mouse adaptive strain MP4 in neonatal mice in an i.p. infection model. Therefore, these results suggested that the threonine at position 251 results in a temperature sensitivity phenotype of EV71 which may contribute to the attenuation of circulating strains.

5' noncoding region alone does not unequivocally determine genetic type of human rhinovirus strains.

During the last couple of years there has been a delightful increase in interest in genetic typing of human rhinoviruses. This is to a large extent due to the discovery of a proposed novel clade, human rhinovirus C (HRV-C). As a consequence, new methods have been reported aiming at unequivocal distinction of traditional HRV prototype strains as well as the newly found uncultivable HRV-C strains. We read with interest the article by Kiang and coworkers (2) describing reverse transcription-PCR-sequencing applications for genetic typing of human rhinoviruses targeting the 5′noncoding region (5′ NCR). A similar approach with largely similar results was published earlier (6). Relatively conserved areas within this region enable broad-spectrum primer design for sensitive methods. However, there are several issues to consider when using the 5′ NCR for genetic typing of HRV. Current taxonomy and classification of picornaviruses are based on capsid region coding sequences. On the basis of this region, a group of previously characterized novel HRV strains form one distinct clade (5, 7) (Fig. ​(Fig.1A),1A), a fact that has also been the basis of the proposal of the Picornavirus Study Group to form a new species, HRV-C, within the Enterovirus genus (4). However, in the article by Kiang et al., on the basis of the partial 5′ NCR sequences, the designated HRV-C strains clustered within the HRV-A clade (2). In contrast, the strains labeled HRV-C in this article formed a clade of their own. As a consequence, because 5′ NCR sequences do not segregate the designated HRV-C from HRV-A (Fig. ​(Fig.1B),1B), they should not be used for typing of new strains. Nevertheless, this region is quite suitable for selected topics of molecular epidemiology, such as analysis of short-term transmission routes (1, 8) or tentative prediction of genetic type as in human enteroviruses (HEV) (9). The sequences nominated as HRV-C by Kiang et al. (2) and by Lee et al. (6) form a new clade in the 5′ NCR. The exact taxonomic position of this clade should be determined according to the clustering of these strains in the capsid region. Clearly, it is divergent from all known HRV and HEV clades in the 5′ NCR, but the decision on whether the strains represent HRV-C or some other picornavirus group cannot be made on the basis of the 5′ NCR alone. FIG. 1. Phylogenetic trees in the VP4/VP2 capsid coding region (A) and in the 5′ NCR (B) of different species of the enterovirus genus showing different clustering of the species in the two regions. Trees were constructed with MEGA4 using the neighbor-joining ... The area close to the beginning of the open reading frame in the 5′ NCR is known to be a recombination hot spot in HEV. Although frequent recombination has not yet been shown for HRV, the analysis of the complete genome sequence data of all HRV prototype strains has not yet been published. Furthermore, the number of completely sequenced genomes of circulating HRV strains has remained low and is too low to conclude that the evolution in the 5′ NCR is always congruent with that of the capsid. Therefore, we would see phylogenetic analysis of the 5′ NCR of HRV as a welcome addition to HRV research, but not a surrogate of capsid coding sequence-based typing.

Increasing Appearance of Reassortant Influenza B Virus in Taiwan from 2002 to 2005

ABSTRACT Genetic and antigenic analyses of influenza B virus field strains isolated in Taiwan from 1998 to 2005 were performed. To investigate the molecular evolution of influenza B viruses, sequence analysis of the hemagglutinin (HA1 subunit) and neuraminidase genes was performed. All influenza B viruses isolated between 1998 and 2000 belonged to the B/Yamagata/16/88 lineage. The B/Victoria/2/87 lineage, which was cocirculating with the Yamagata lineage, was identified in Taiwan in March 2001. Concurrently, there was an increasing prevalence of this lineage in many parts of the world, including North America and Europe, during the 2001-2002 season. Since 2002, genetic reassortants of influenza B virus with the Victoria lineage of hemagglutinin and the Yamagata lineage of neuraminidase have been found at a rate of 46%. Therefore, in 2002, at least three sublineages of influenza B virus strains, the B/Shanghai/361/2002-like strain (Yamagata lineage), the B/Hong Kong/330/01-like strain (Victoria lineage), and the B/Hong Kong/1351/02-like strain (B reassortant lineage), were identified in Taiwan. The results showed that genetically distinct lineages can cocirculate in the population and that the reassortment among these strains plays a role in generating the genetic diversity of influenza B viruses. Interestingly, from January to April 2005, B reassortant viruses became dominant (73%) in Taiwan, which indicated that a mismatch had occurred between the influenza B vaccine strain recommended for the 2004-2005 season in the Northern hemisphere by the World Health Organization and the epidemic strain.

Comparison of neutralizing and hemagglutination-inhibiting antibody responses for evaluating the seasonal influenza vaccine

Evaluation of the efficacy of influenza vaccines is essential for vaccine development. This study evaluated the neutralizing and hemagglutination-inhibition antibody response in subjects receiving the 2006-07 and 2007-08 seasonal influenza vaccines. ELISA-based microneutralization demonstrated a greater mean-fold increase and seroconversion rate than the hemagglutination-inhibition assay. The increase in the antibody titers against influenza H1 were higher than those against influenza H3 and influenza B, indicating that the H1 vaccine strain in the 2006-07 and 2007-08 seasons was more immunogenic. These data suggest that the neutralizing antibody response is a better measurement of influenza vaccine efficacy.