Youngmee Jee

Improving molecular tools for global surveillance of measles virus

BACKGROUND The genetic characterization of wild-type measles viruses plays an important role in the description of viral transmission pathways and the verification of measles elimination. The 450 nucleotides that encode the carboxyl-terminus of the nucleoprotein (N-450) are routinely sequenced for genotype analysis. OBJECTIVES The objectives of this study were to develop improved primers and controls for RT-PCR reactions used for genotyping of measles samples and to develop a method to provide a convenient, safe, and inexpensive means to distribute measles RNA for RT-PCR assays and practice panels. STUDY DESIGN A newly designed, genetically defined synthetic RNA and RNA isolated from cells infected with currently circulating genotypes were used to compare the sensitivity of primer pairs in RT-PCR and nested PCR. FTA® cards loaded with lysates of measles infected cells were tested for their ability to preserve viral RNA and destroy virus infectivity. RESULTS A new primer pair, MeV216/MeV214, was able to amplify N-450 from viruses representing 10 currently circulating genotypes and a genotype A vaccine strain and demonstrated 100-fold increased sensitivity compared to the previously used primer set. A nested PCR assay further increased the sensitivity of detection from patient samples. A synthetic positive control RNA was developed that produced PCR products that are distinguishable by size from PCR products amplified from clinical samples. FTA® cards completely inactivated measles virus and stabilized RNA for at least six months. CONCLUSIONS These improved molecular tools will advance molecular characterization of circulating measles viruses globally and provide enhanced quality control measures.

Single endemic genotype of measles virus continuously circulating in China for at least 16 years.

The incidence of measles in China from 1991 to 2008 was reviewed, and the nucleotide sequences from 1507 measles viruses (MeV) isolated during 1993 to 2008 were phylogenetically analyzed. The results showed that measles epidemics peaked approximately every 3 to 5 years with the range of measles cases detected between 56,850 and 140,048 per year. The Chinese MeV strains represented three genotypes; 1501 H1, 1 H2 and 5 A. Genotype H1 was the predominant genotype throughout China continuously circulating for at least 16 years. Genotype H1 sequences could be divided into two distinct clusters, H1a and H1b. A 4.2% average nucleotide divergence was found between the H1a and H1b clusters, and the nucleotide sequence and predicted amino acid homologies of H1a viruses were 92.3%–100% and 84.7%–100%, H1b were 97.1%–100% and 95.3%–100%, respectively. Viruses from both clusters were distributed throughout China with no apparent geographic restriction and multiple co-circulating lineages were present in many provinces. Cluster H1a and H1b viruses were co-circulating during 1993 to 2005, while no H1b viruses were detected after 2005 and the transmission of that cluster has presumably been interrupted. Analysis of the nucleotide and predicted amino acid changes in the N proteins of H1a and H1b viruses showed no evidence of selective pressure. This study investigated the genotype and cluster distribution of MeV in China over a 16-year period to establish a genetic baseline before MeV elimination in Western Pacific Region (WPR). Continuous and extensive MeV surveillance and the ability to quickly identify imported cases of measles will become more critical as measles elimination goals are achieved in China in the near future. This is the first report that a single endemic genotype of measles virus has been found to be continuously circulating in one country for at least 16 years.

The complete genome sequence and molecular analysis of human hepatitis E virus genotype IV identified from a Korean patient

Hepatitis E virus (HEV) was originally identified as the etiological agent of non-HAV enterically transmitted hepatitis. One HEV strain (FJ763142) was identified from an acute viral hepatitis E patient with IgM anti-HEV in Korea. The complete genome sequence consisted of 7,238 nucleotides (nt) plus a 22-nt poly(A) tail. The strain belongs to genotype IV, with 91% homology compared with AB197674, which was found in a Japanese patient who had traveled to China. This finding suggests that HEV genotype IV already circulates in Korea, and this HEV might be the first example of an indigenous strain.

Monitoring progress toward measles elimination by genetic diversity analysis of measles viruses in China 2009–2010

With the achievement of high coverage for routine immunization and supplementary immunization activities (SIAs), measles incidence in mainland China reached its lowest level in 2010. The proportion of measles cases in the vaccination-targeted population decreased during 2007-2010 after the SIAs. More than 60% of measles cases were in adults or infants, especially in the coastal and eastern provinces during 2009 and 2010. A total 567 isolates of measles virus were obtained from clinical specimens from 27 of 31 provinces in mainland China during 2009 and 2010. Except for two vaccine-associated cases, one genotype D4 strain, two genotype D9 strains, and four genotype D11 strains, the other 558 strains were genotype H1 cluster H1a. Genotype H1 has been the only endemic genotype detected in China since surveillance began in 1993. Only genotype H1 was found in mainland China during 1993-2008, except for one detection of genotype H2. More recently, multiple genotypes of imported measles were detected even with the background of endemic genetotype H1 viruses. Analysis of the 450-nucleotide sequencing window of the measles virus N gene showed that the overall genetic diversity of the recent geneotype H1 strains decreased between 2008 and 2010. The lower genetic diversity of H1 strains suggested that enhanced vaccination may have reduced the co-circulating lineages of endemic genotype H1 strains in mainland China.

Epidemiology of Japanese Encephalitis in the Philippines: A Systematic Review

Background Japanese encephalitis virus (JEV) is an important cause of encephalitis in most of Asia, with high case fatality rates and often significant neurologic sequelae among survivors. The epidemiology of JE in the Philippines is not well defined. To support consideration of JE vaccine for introduction into the national schedule in the Philippines, we conducted a systematic literature review and summarized JE surveillance data from 2011 to 2014. Methods We conducted searches on Japanese encephalitis and the Philippines in four databases and one library. Data from acute encephalitis syndrome (AES) and JE surveillance and from the national reference laboratory from January 2011 to March 2014 were tabulated and mapped. Results We identified 29 published reports and presentations on JE in the Philippines, including 5 serologic surveys, 18 reports of clinical cases, and 8 animal studies (including two with both clinical cases and animal data). The 18 clinical studies reported 257 cases of laboratory-confirmed JE from 1972 to 2013. JE virus (JEV) was the causative agent in 7% to 18% of cases of clinical meningitis and encephalitis combined, and 16% to 40% of clinical encephalitis cases. JE predominantly affected children under 15 years of age and 6% to 7% of cases resulted in death. Surveillance data from January 2011 to March 2014 identified 73 (15%) laboratory-confirmed JE cases out of 497 cases tested. Summary This comprehensive review demonstrates the endemicity and extensive geographic range of JE in the Philippines, and supports the use of JE vaccine in the country. Continued and improved surveillance with laboratory confirmation is needed to systematically quantify the burden of JE, to provide information that can guide prioritization of high risk areas in the country and determination of appropriate age and schedule of vaccine introduction, and to measure the impact of preventive measures including immunization against this important public health threat.

Genetic characterization of the hemagglutinin genes of wild-type measles virus circulating in china, 1993-2009.

Background China experienced several large measles outbreaks in the past two decades, and a series of enhanced control measures were implemented to achieve the goal of measles elimination. Molecular epidemiologic surveillance of wild-type measles viruses (MeV) provides valuable information about the viral transmission patterns. Since 1993, virologic surveillnace has confirmed that a single endemic genotype H1 viruses have been predominantly circulating in China. A component of molecular surveillance is to monitor the genetic characteristics of the hemagglutinin (H) gene of MeV, the major target for virus neutralizing antibodies. Principal Findings Analysis of the sequences of the complete H gene from 56 representative wild-type MeV strains circulating in China during 1993–2009 showed that the H gene sequences were clustered into 2 groups, cluster 1 and cluster 2. Cluster1 strains were the most frequently detected cluster and had a widespread distribution in China after 2000. The predicted amino acid sequences of the H protein were relatively conserved at most of the functionally significant amino acid positions. However, most of the genotype H1 cluster1 viruses had an amino acid substitution (Ser240Asn), which removed a predicted N-linked glycosylation site. In addition, the substitution of Pro397Leu in the hemagglutinin noose epitope (HNE) was identified in 23 of 56 strains. The evolutionary rate of the H gene of the genotype H1 viruses was estimated to be approximately 0.76×10−3 substitutions per site per year, and the ratio of dN to dS (dN/dS) was <1 indicating the absence of selective pressure. Conclusions Although H genes of the genotype H1 strains were conserved and not subjected to selective pressure, several amino acid substitutions were observed in functionally important positions. Therefore the antigenic and genetic properties of H genes of wild-type MeVs should be monitored as part of routine molecular surveillance for measles in China.

Are we there yet? Assessing achievement of vaccine-preventable disease goals in WHO's Western Pacific Region.

Accelerated disease control goals have long been appreciated for their role in galvanizing commitment and bringing a sense of urgency for disease prevention. WHOs Western Pacific Region has 14 on-going communicable disease reduction goals including 1 targeting eradication, 10 targeting elimination, and 3 control initiatives. These goals cover mother-to-child transmission of HIV, congenital syphilis, tuberculosis, leprosy, five parasitic diseases and four vaccine-preventable diseases (VPD). The initiatives have distinct objectives, approaches, and means in which to measure achievement of the goals. Given the long history and experience with VPD initiatives in the Western Pacific Region, this manuscript focuses on the Regions following initiatives: (1) smallpox eradication, (2) polio eradication, (3) measles elimination, (4) maternal and neonatal tetanus elimination (MNTE), and (5) hepatitis B control. There is good consistency across the Regions VPD initiatives yet a pattern of more robust and representative data requirements, stricter evaluation criteria, and more formal evaluation bodies are linked to the intensity of the goal - with eradication being the peak. On the other end of this spectrum, the Regional hepatitis B control initiative has established efficient and low-cost approaches for measuring impact and evaluating if the goals have been met. Even within the confines of VPD initiatives there are some deviations in use of terminology and comparisons across other disease control initiatives in the Region are provided.

Maintaining Polio-Free Certification in the World Health Organization Western Pacific Region for Over a Decade

On 29 October 2000, the World Health Organization (WHO) Regional Commission for the Certification of Poliomyelitis Eradication in the Western Pacific certified the WHO Western Pacific Region as free of indigenous wild poliovirus. This status has been maintained to date: wild poliovirus importations into Singapore (in 2006) and Australia (in 2007) did not lead to secondary cases, and an outbreak in China (in 2011) was rapidly controlled. Circulation of vaccine derived polioviruses in Cambodia, China and the Philippines was quickly interrupted. A robust acute flaccid paralysis surveillance system, including a multitiered polio laboratory network, has been maintained, forming the platform for integrating measles, neonatal tetanus, and other vaccine-preventable disease surveillance and their respective control goals. While polio elimination remains one of the most important achievements in public health in the Western Pacific Region, extended delays in global eradication have, however, led to shifting and competing public health priorities among member states and partners and have made the region increasingly vulnerable.

Seroprevalence of Hepatitis A and E Viruses Based on the Third Korea National Health and Nutrition Survey in Korea

Objectives The purpose of this study was to investigate the seroprevalence of hepatitis A virus (HAV) and hepatitis E virus (HEV) in Korea during 2005. Methods Study subjects were selected from across Korea using a stratified multistage probability sampling design, and HAV and HEV seroprevalence was compared on the basis of sex, age, and residency. A total of 497 rural and urban people aged 10–99 years of age (mean ± SD age = 28.87 ± 17.63 years) were selected by two-stage cluster sampling and tested serologically for anti-HAV and anti-HEV IgG using an enzyme-linked immunosorbent assay. Results Among this population, the overall seroprevalence of HAV was 63.80% (55.21% aged in their 20s and 95.92% in their 30s, p < 0.01) and that of HEV was 9.40% (5.21% aged in their 20s and 7.14% in their 30s, p < 0.01). Seroprevalence also varied according to area of residence. HEV prevalence in rural areas was higher than that of urban regions based on the anti-HEV antibody, odds ratio 3.22 (95% confidence interval: 1.46–7.10, p < 0.01). There were no significant differences between male and female against anti-HAV/HEV antibodies. Conclusion Our study suggested that the seropositive rates of HAV and HEV might be related to age and environmental conditions.

WHO's Initiatives to Control Vaccine Preventable Diseases (VPD) and Labnet for Targeted VPDs in the Western Pacific Region

Immunization is a highly cost effective public health intervention that has achieved dramatic reductions in disease, disability and death. By the end of 2010, the Western Pacific Region remained polio-free for over 13 years, regional measles incidence decreased to 30 cases per million population, over 87% of the Region’s population lived in countries and areas with <2% prevalence of chronic hepatitis infection among children, and 31 countries and areas have eliminated maternal and neonatal tetanus (MNT) as a public health problem. Demand is increasing for new and underutilized vaccines that prevent major causes of diarrhoea, pneumonia, meningitis, and encephalitis, as well as cervical cancer, and with it an increased need to assure vaccine safety, quality and adequate supply chain management. Programme monitoring and high quality surveillance supported by an accredited laboratory network are increasingly needed to demonstrate progress or achievement of disease eradication, elimination and control initiatives, and systematic expansion of surveillance for diseases targeted by new or underutilized vaccines is needed to guide decision making and monitor impact. Since the Western Pacific Region established the twin goals of measles elimination and hepatitis B control by 2012 in 2005, the region has made good progress towards measles elimination and hepatitis B control. Despite measles outbreaks in number of countries (Vietnam and Philippines, the overall number of reported measles cases has been dramatically decreased by 2011. Most countries in this region conducted measles supplementary immunization activities (SIA) or catch-up campaigns. China performed a historic measles SIA in 2010 vaccinating more than 100 million population. Vietnam, Cambodia and Philippines also conducted measles campaigns in 2010-2011 to provide an opportunity for second dose measles vaccine. The measles laboratory network consisting of 382 laboratories has played a crucial role by providing timely and reliable laboratory confirmation and virus identification, while maintaining high standards of quality. Genotype and sequence data on circulating measles virus strains are critical to track virus transmission and verify measles elimination in each country. The Western Pacific Region celebrated the 10th anniversary of its certification as a polio-free region in October 2010. However, as many of you know, on 26 August, China’s Ministry of Health reported four laboratory-verified cases of wild polio virus in the Xinjiang Uygur Autonomous Region. Since then 18 wild polio cases were reported in Xinjiang province in China and as of 20 October 2011, 47 wild poliovirus strains were detected from 18 AFP, 16 contacts and 12 healthy people in China. Sequence comparison indicated 99% homology with type 1 wild poliovirus strains isolated from Pakistan in 2010 implicating the virus was imported from Pakistan. The WHO polio laboratory network including China CDC and US CDC was able to identity the source of virus within just a few hours to provide critical information to the national EPI programme. An emergency response involving oral poliovirus vaccine immunization has been initiated, and two rounds of OPV vaccination were conducted in September and October in Xinjiang province in China and third round using monovalent OPV is planned in November. This event highlights the important role of the laboratory networks and how quickly they can provide critical information to the national programmes and WHO. Inequities in routine immunization coverage were addressed through WHO-supported training on district level immunization strengthening and development of national multi-year immunization plans that included special efforts to vaccinate hard to reach children. WHO also organized a Regional Vaccination Week for the Western Pacific involving more than 30 countries and areas. The first Regional Vaccination Week last April 2011 celebrated the achievements of immunization programmes in promoting healthy communities throughout our Region. With the achievements made to date we have established the foundation for sustained change to improve the efficiency, equity, and effectiveness of immunization – and ultimately of health systems. However, to move into the next decade, we need to understand what our remaining challenges are. We have to do our analysis in an evidence-based way and work with Member States in a realistic and consensus-building fashion. Strengthening immunization systems remains at the core of EPI disease control efforts. Despite the many successes of EPI, disparities in immunization coverage remain between and within countries, threatening achievement of the Regional goals of measles elimination and hepatitis B control, placing at risk the Region’s polio free status and achievement of MNT elimination, and limiting the impact of new and underutilized vaccine introduction. To fully realize the benefits of immunization and help achieve Millennium Development Goals, the Regional Office EPI Unit has developed a strategic framework in line with the Global Immunization Vision and Strategy and that has five objectives: 1) ensure equitable access to vaccines of assured quality, including pandemic vaccines; 2) achieve targeted disease eradication, elimination or control; 3) promote the rational introduction of new vaccines; 4) .strengthen vaccine preventable disease (VPD) monitoring and surveillance systems, laboratory capacity, and data use; and 5) strengthen communication, partnerships and advocacy to support immunizations and promote integration of immunization with other health interventions. WHO support for programme monitoring and VPD surveillance included supportive assistance and corrective feedback on the WHO-UNICEF Joint EPI Reporting Form; development and training on data management tools for traditional and new vaccine surveillance; development of models for monitoring/assessing low performance (at national and subnational level) and epidemiological risk for diseases like Polio and Measles; and continuous supportive and corrective feedback on surveillance data quality. The WHO EPI Unit collaborated with other Regional Office divisions and units to improve various aspects of VPD surveillance. VPD surveillance sensitivity was enhanced by integrating VPD surveillance with event based surveillance training in collaboration with the Disease Surveillance and Response Unit. Laboratory networks for poliomyelitis, measles and rubella, and Japanese encephalitis continued to provide timely and reliable laboratory confirmation and virus identification. All poliomyelitis network laboratories and almost all measles and rubella network laboratories in the Region are fully accredited. The polio laboratory network introduced a new algorithm/protocol that will shorten the interval between specimen collection and virus isolation. Real time polymerase chain reaction (PCR) for the intratypic differentiation and the screening of vaccine derived polioviruses was successfully implemented by the laboratory network during 2010. Measles and AFP surveillance and laboratory performances are being strengthened through a series of training, feedback mechanisms and supplemental surveillance actitivities (e.g., environmental and enterovirus surveillance for poliovirus). The WHO measles regional reference laboratory (RRL) in Hong Kong provided genotyping results for countries including Cambodia, Lao People’s Democratic Republic, Malaysia, Mongolia, Philippines and Vietnam. Regional capacity to conduct measles genotyping was enhanced after conducting two hands on laboratory training for measles network laboratories in 2009 and 2010. A newly established Japanese encephalitis laboratory network began to provide laboratory confirmation and implement quality assurance measures, such as proficiency testing and confirmatory testing. The two hands-on training workshops were held in 2009 and 2010 to further improve laboratory performance and the quality of testing.