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.     

Isolation and complete nucleotide sequence of the measles virus IMB-1 strain in China

The complete nucleotide sequence of the measles virus strain IMB-1, which was isolated in China, was determined. As in other measles viruses, its genome is 15,894 nucleotides in length and encodes six proteins. The full-length nucleotide sequence of the IMB-1 isolate differed from vaccine strains (including wild-type Edmonston strain) by 4%–5% at the nucleotide sequence level. This isolate has amino acid variations over the full genome, including in the hemagglutinin and fusion genes. This report is the first to describe the full-length genome of a genotype H1 strain and provide an overview of the diversity of genetic characteristics of a circulating measles virus.     

Rubella epidemics and genotypic distribution of the rubella virus in shandong province, china, in 1999-2010

Background

The rubella vaccine was introduced into the immunization program in 1995 in the Shandong province, China. A series of different rubella vaccination strategies were implemented at different stages of measles control in Shandong province.

Methodology/Principal Findings

The average reported incidence rate of rubella cases remained at a low level in Shandong province after 1999. However, rubella epidemics occurred repeatedly in 2001/2002, 2006, and 2008/2009. The age of the onset of rubella cases gradually increased during 1999–2010, which showed that most cases were found among the 10 years old in 1999 and among the 17 years old in 2010. Phylogenetic analysis was performed and a phylogenetic tree was constructed based on the World Health Organization standard sequence window for rubella virus isolates. All rubella viruses isolated in Shandong province were divided into 4 genotypes: 1E, 1F, 2A, and 2B. Genotype 1E viruses accounted for the majority (79%) of all these viruses. The similarity of nucleotide and amino acid sequences among genotype 1E viruses was 98.2–100% and 99.1–100%, respectively. All Shandong genotype 1E strains, differed from international genotype 1E strains, belonged to cluster 1 and interdigitated with the viruses from other provinces in mainland China. The effective number of infections indicated by a Bayesian skyline plot remained constant from 2001 to 2009.

Conclusions/Significance

The gradual shift of disease burden to an older age group occurred after a rubella-containing vaccine was introduced into the childhood immunization schedule in 1995 in Shandong province. Four genotypes, including 1E, 1F, 2A, and 2B, were found in Shandong province during 2000–2009. Genotype 1E, rather than genotype 1F, became the predominant genotype circulating in Shandong province from 2001. All Shandong genotype 1E viruses belong to the genotype 1E/cluster 1; they have constantly circulated, and co-evolved and co-circulated, with those from other provinces.     

2003～2007年中国风疹病毒基因特征分析

本研究用Vero细胞或Vero/SLAM细胞从我国10个省(直辖市、自治区,下同)2003～2007年风疹暴发和散发病例的咽拭子标本中分离到57株风疹病毒,用RT-PCR方法扩增了57株风疹病毒E1基因1 107个核苷酸的片段,并对该PCR产物进行序列测定和分析.结果提示,在基于WHO基因定型靶序列739个核苷酸片段构建的基因亲缘关系树上,其中55株风疹病毒株属于1E基因型,相对于其他国家的1E基因型,形成一个独立分支;另外2株风疹病毒属于2B基因型.57株风疹病毒大部分核苷酸的突变为无义突变,氨基酸序列高度保守,除了2株风疹病毒在E1蛋白血凝抑制和中和位点区域第212位氨基酸由Thr变为Ser,其他病毒株均无重要抗原位点的改变;所有我国已分离到的1E基因型风疹病毒在E1蛋白第338位氨基酸共享突变位点(Leu338→Phe338),而其他基因型以及其他国家的1E基因型风疹病毒在该位点均未发生突变,提示该氨基酸(Phe338)可能是我国1E基因型风疹病毒所特有.2003～2007年在我国10个省均分离到1E基因型,而2B基因型只在2006年从四川省的越南输入病例中分离到,提示1E为绝对优势基因型,2B基因型为输入基因型.与1979～1984年和1999～2002年我国流行的风疹基因型不同,发生了基因型的更替,近年我国风疹的流行是由1E基因型为主的风疹野病毒的多个传播链引起.     

Implementation of a National Measles Elimination Program in Iran: Phylogenetic Analysis of Measles Virus Strains Isolated during 2010–2012 Outbreaks

Measles virus (MV) causes small and large outbreaks in Iran. Molecular assays allow identifying and the sources of measles imported from neighboring countries. We carried out a phylogenetic analysis of measles virus circulating in Iran over the period 2010–2012. Specimens from suspected cases of measles were collected from different regions of Iran. Virus isolation was performed on urine and throat swabs. Partial nucleoprotein gene segments of MV were amplified by RT-PCR. PCR products of 173 samples were sequenced and analyzed. The median age of confirmed cases was 2 years. Among all confirmed cases, 32% had unknown vaccination status, 20% had been vaccinated, and 48% had not been vaccinated. Genotypes B3 and D8 (for the first time), H1 and D4 were detected mainly in unvaccinated toddlers and young children. Genotype B3 became predominant in 2012 and was closely related to African strains. H1 strains were also found in small and large outbreaks during 2012 but were not identical to Iranian H1-2009 strains. A majority of the Iranian D4 strains during 2010–2012 outbreaks were linked to the D4 strain identified in the Pakistan in 2007. We identified a single case in 2010 belonging to D8 genotype with 99.7% identity to Indian isolates. Although the vaccination program is currently good enough to prevent nationwide epidemics and successfully decreased measles incidence in Iran, the fraction of protected individuals in the population was not high enough to prevent continuous introduction of cases from abroad. Due to increasing number of susceptible individuals in some areas, sustained transmission of the newly introduced viral genotype remains possible.     

New genotype of avian influenza H5N1 viruses isolated from tree sparrows in China

The 2004 outbreaks of highly pathogenic avian influenza H5N1 disease in China led to a great poultry loss and society attention. A survey of avian influenza viruses was conducted on tree sparrows (Passer montanus) collected in China in 2004. Four viruses were isolated from free-living tree sparrows. The results of the whole-genome analysis indicated that an H5N1 virus with a new genotype is circulating among tree sparrows. The hemagglutinin and neuraminidase genes of the new genotype were derived from Gs/Gd/96-like viruses and the nuclear protein gene descended from the 2001 genotype A H5N1 viruses, while the other inner genes originated from an unknown influenza virus. In experimental infection, all four viruses were highly pathogenic to chickens but not pathogenic to ducks or mice. The four tree sparrow viruses were different from the 2003 tree sparrow strain (genotype Z) in Hong Kong. The results suggested that H5N1 viruses might be distributed widely in tree sparrows.     

Novel evidence of HBV recombination in family cluster infections in western China

Two hepatitis B virus (HBV) C/D recombinants were isolated from western China. No direct evidence indicates that these new viruses arose as a result of recombination between genotype C and D or a result of convergence. In this study, we search for evidence of intra-individual recombination in the family cluster cases with co-circulation of genotype C, D and C/D recombinants. We studied 68 individuals from 15 families with HBV infections in 2006, identified individuals with mixed HBV genotype co-infections by restriction fragment length polymorphism and proceeded with cloning and DNA sequencing. Recombination signals were detected by RDP3 software and confirmed by split phylogenetic trees. Families with mixed HBV genotype co-infections were resampled in 2007. Three of 15 families had individuals with different HBV genotype co-infections in 2006. One individual (Y2) had a triple infection of HBV genotype C, D and C/D recombinant in 2006, but only genotype D in 2007. Further clonal analysis of this patient indicated that the C/D recombinant was not identical to previously isolated CD1 or CD2, but many novel recombinants with C2, D1 and CD1 were simultaneously found. All parental strains could recombine with each other to form new recombinant in this patient. This indicates that the detectable mixed infection and recombination have a limited time window. Also, as the recombinant nature of HBV precludes the possibility of a simple phylogenetic taxonomy, a new standard may be required for classifying HBV sequences.     

Molecular Characterization of the Measles Virus Genotypes in JiLin Province,China

Measles remains a severe global health threat, and nearly 30 million new cases are reported annually. Although many studies have analyzed measles viruses (MV) at the epidemiologic and phylogenetic levels, no study has yet to integrate these two types of data. To this end, we isolated 16 wild-type MV strains China''s Jilin province. The MV genotype H1 was the most prevalent strain. After sequencing the nucleoprotein (N) genes of these strains, a maximum clade credibility tree was constructed by the Bayesian Markov Chain Monte Carlo method using 450 MV strains from GenBank with epidemiological information. The MV N gene evolution rate was 1.127E-3. Analysis of the time of the most recent common ancestor (TMRCA) for genotypes A/B/C/G/H revealed that genotypes D and B had the largest and smallest TMRCA (45.86 and 26.63, respectively). The highest level of genetic diversity for the MV N gene occurred around the year 2000. Here in this study, we uncovered the MV genotypes circulating in China''s Jilin Province and estimated the epidemiologic and phylogenetic relationship for the six different genotypes of MV.     

Genetic structure of human A/H1N1 and A/H3N2 influenza virus on Corsica Island: phylogenetic analysis and vaccine strain match, 2006-2010

Background

The aim of this study was to analyse the genetic patterns of Hemagglutinin (HA) genes of influenza A strains circulating on Corsica Island during the 2006–2009 epidemic seasons and the 2009–2010 pandemic season.

Methods

Nasopharyngeal samples from 371 patients with influenza-like illness (ILI) were collected by General Practitioners (GPs) of the Sentinelles Network through a randomised selection routine.

Results

Phylogenetic analysis of HA revealed that A/H3N2 strains circulating on Corsica were closely related to the WHO recommended vaccine strains in each analyzed season (2006–2007 to 2008–2009). Seasonal Corsican influenza A/H1N1 isolated during the 2007–2008 season had drifted towards the A/Brisbane/59/2007 lineage, the A/H1N1 vaccine strain for the 2008–2009 season. The A/H1N1 2009 (A/H1N1pdm) strains isolated on Corsica Island were characterized by the S220T mutation specific to clade 7 isolates. It should be noted that Corsican isolates formed a separate sub-clade of clade 7 as a consequence of the presence of the fixed substitution D222E.The percentages of the perfect match vaccine efficacy, estimated by using the p _epitope model, against influenza viruses circulating on Corsica Island varied substantially across the four seasons analyzed, and tend to be highest for A/H1N1 compared with A/H3N2 vaccines, suggesting that cross-immunity seems to be stronger for the H1 HA gene.

Conclusion

The molecular analysis of the HA gene of influenza viruses that circulated on Corsica Island between 2006–2010 showed for each season the presence of a dominant lineage characterized by at least one fixed mutation. The A/H3N2 and A/H1N1pdm isolates were characterized by multiples fixation at antigenic sites. The fixation of specific mutations at each outbreak could be explained by the combination of a neutral phenomenon and a founder effect, favoring the presence of a dominant lineage in a closed environment such as Corsica Island.     

华东地区家鸭中N1亚型禽流感病毒NA基因的遗传进化分析

为了解华东地区家鸭内禽流感病毒的遗传进化情况,对2002~2006年分离自华东地区家鸭的3种主要N1亚型的禽流感病毒:2株H1N1、10株H3N1和14株H5N1,共26株病毒的NA基因进行了遗传进化分析。结果表明,华东地区家鸭中的N1亚型的禽流感病毒正处于不断进化状态中。14株H5N1禽流感病毒均在NA的茎部缺失20个氨基酸(49~68位),而其他N1亚型的禽流感病毒的NA都未见发生此缺失。H3N1病毒可能与H1N1病毒发生了NA基因的重排,但是目前还没有直接证据表明华东地区家鸭中H5N1禽流感参与了基因重排。     

Predominance of HA-222D/G Polymorphism in Influenza A(H1N1)pdm09 Viruses Associated with Fatal and Severe Outcomes Recently Circulating in Germany

Influenza A(H1N1)pdm09 viruses cause sporadically very severe disease including fatal clinical outcomes associated with pneumonia, viremia and myocarditis. A mutation characterized by the substitution of aspartic acid (wild-type) to glycine at position 222 within the haemagglutinin gene (HA-D222G) was recorded during the 2009 H1N1 pandemic in Germany and other countries with significant frequency in fatal and severe cases. Additionally, A(H1N1)pdm09 viruses exhibiting the polymorphism HA-222D/G/N were detected both in the respiratory tract and in blood. Specimens from mild, fatal and severe cases were collected to study the heterogeneity of HA-222 in A(H1N1)pdm09 viruses circulating in Germany between 2009 and 2011. In order to enable rapid and large scale analysis we designed a pyrosequencing (PSQ) assay. In 2009/2010, the 222D wild-type of A(H1N1)pdm09 viruses predominated in fatal and severe outcomes. Moreover, co-circulating virus mutants exhibiting a D222G or D222E substitution (8/6%) as well as HA-222 quasispecies were identified (10%). Both the 222D/G and the 222D/G/N/V/Y polymorphisms were confirmed by TA cloning. PSQ analyses of viruses associated with mild outcomes revealed mainly the wild-type 222D and no D222G change in both seasons. However, an increase of variants with 222D/G polymorphism (60%) was characteristic for A(H1N1)pdm09 viruses causing fatal and severe cases in the season 2010/2011. Pure 222G viruses were not observed. Our results support the hypothesis that the D222G change may result from adaptation of viral receptor specificity to the lower respiratory tract. This could explain why transmission of the 222G variant is less frequent among humans. Thus, amino acid changes at HA position 222 may be the result of viral intra-host evolution leading to the generation of variants with an altered viral tropism.     

宁夏2004—2007年麻疹监测分析

为了客观评价宁夏麻疹监测系统现状,探讨控制和消除麻疹的策略。对宁夏2004—2007年麻疹流行病学和实验室监测工作进行分析。结果显示,宁夏麻疹监测系统的灵敏性较高,疑似麻疹病例标本采集率和血清学诊断率等主要监测指标已达较高水平。同时,对监测到的36株麻疹野病毒基因型进行了探讨,全部为H1基因型H1a亚型,未发现其它基因亚型。因此,宁夏亟需加速控制麻疹,应继续做好麻疹监测和麻疹疫苗免疫接种。     

浙江省麻疹病毒的基因特征分析

目的分析浙江省流行的麻疹病毒(MV)的基因特征,为更好地防控麻疹提供科学参考。方法从GenBank检索并下载浙江省所有麻疹病毒株、中国麻疹疫苗株和WHO推荐参考株的血凝素蛋白(H)和核蛋白(N)的基因组序列,利用MEGA 6.0软件进行比对分析,构建种系进化树,确定浙江省麻疹病毒流行的基因型别,并进行同源性和基因变异分析。结果浙江省麻疹病毒以H1基因型为主(27株),其他基因型为辅(2株B3型)。H1a亚型(21/27)占绝对优势,其次为H1b亚型(6/27)。浙江省所有毒株间H蛋白的氨基酸同源性为96.9%~100.0%,与疫苗株Shanghai-191和Changchun-47的同源性均为95.0%~96.0%。浙江省所有毒株间N蛋白羧基末端的氨基酸同源性为82.7%~100.0%,与疫苗株Shanghai-191的同源性为85.8%~89.5%,与疫苗株Changchun-47的同源性为87.3%~91.0%。结论麻疹病毒H1基因型为浙江省麻疹流行的优势株,与现行参考疫苗株(A基因型)差异较大,因此针对麻疹病毒H1基因型疫苗的研制是今后浙江省麻疹病毒防控的关键。     

Evolution of highly pathogenic H5N1 avian influenza viruses in Vietnam between 2001 and 2007

Highly pathogenic avian influenza (HPAI) H5N1 viruses have caused dramatic economic losses to the poultry industry of Vietnam and continue to pose a serious threat to public health. As of June 2008, Vietnam had reported nearly one third of worldwide laboratory confirmed human H5N1 infections. To better understand the emergence, spread and evolution of H5N1 in Vietnam we studied over 300 H5N1 avian influenza viruses isolated from Vietnam since their first detection in 2001. Our phylogenetic analyses indicated that six genetically distinct H5N1 viruses were introduced into Vietnam during the past seven years. The H5N1 lineage that evolved following the introduction in 2003 of the A/duck/Hong Kong/821/2002-like viruses, with clade 1 hemagglutinin (HA), continued to predominate in southern Vietnam as of May 2007. A virus with a clade 2.3.4 HA newly introduced into northern Vietnam in 2007, reassorted with pre-existing clade 1 viruses, resulting in the emergence of novel genotypes with neuraminidase (NA) and/or internal gene segments from clade 1 viruses. A total of nine distinct genotypes have been present in Vietnam since 2001, including five that were circulating in 2007. At least four of these genotypes appear to have originated in Vietnam and represent novel H5N1 viruses not reported elsewhere. Geographic and temporal analyses of H5N1 infection dynamics in poultry suggest that the majority of viruses containing new genes were first detected in northern Vietnam and subsequently spread to southern Vietnam after reassorting with pre-existing local viruses in northern Vietnam. Although the routes of entry and spread of H5N1 in Vietnam remain speculative, enhanced poultry import controls and virologic surveillance efforts may help curb the entry and spread of new HPAI viral genes.     

Molecular Evolution of Human H1N1 and H3N2 Influenza A Virus in Thailand, 2006–2009

Background

Annual seasonal influenza outbreaks are associated with high morbidity and mortality.

Objective

To index and document evolutionary changes among influenza A H1N1 and H3N2 viruses isolated from Thailand during 2006–2009, using complete genome sequences.

Methods

Nasopharyngeal aspirates were collected from patients diagnosed with respiratory illness in Thailand during 2006–2009. All samples were screened for Influenza A virus. A total of 13 H1N1 and 21 H3N2 were confirmed and whole genome sequenced for the evolutionary analysis using standard phylogenetic approaches.

Results

Phylogenetic analysis of HA revealed a clear diversification of seasonal from vaccine strain lineages. H3N2 seasonal clusters were closely related to the WHO recommended vaccine strains in each season. Most H1N1 isolates could be differentiated into 3 lineages. The A/Brisbane/59/2007 lineage, a vaccine strain for H1N1 since 2008, is closely related with the H1N1 subtypes circulating in 2009. HA sequences were conserved at the receptor-binding site. Amino acid variations in the antigenic site resulted in a possible N-linked glycosylation motif. Recent H3N2 isolates had higher genetic variations compared to H1N1 isolates. Most substitutions in the NP protein were clustered in the T-cell recognition domains.

Conclusion

In this study we performed evolutionary genetic analysis of influenza A viruses in Thailand between 2006–2009. Although the current vaccine strain is efficient for controlling the circulating outbreak subtypes, surveillance is necessary to provide unambiguous information on emergent viruses. In summary, the findings of this study contribute the understanding of evolution in influenza A viruses in humans and is useful for routine surveillance and vaccine strain selection.     

Displacement of the predominant dengue virus from type 2 to type 1 with a subsequent genotype shift from IV to I in Surabaya, Indonesia 2008-2010

Indonesia has annually experienced approximately 100,000 reported cases of dengue fever (DF) and dengue hemorrhagic fever (DHF) in recent years. However, epidemiological surveys of dengue viruses (DENVs) have been limited in this country. In Surabaya, the second largest city, a single report indicated that dengue virus type 2 (DENV2) was the predominant circulating virus in 2003-2005. We conducted three surveys in Surabaya during: (i) April 2007, (ii) June 2008 to April 2009, and (iii) September 2009 to December 2010. A total of 231 isolates were obtained from dengue patients and examined by PCR typing. We found that the predominant DENV shifted from type 2 to type 1 between October and November 2008. Another survey using wild-caught mosquitoes in April 2009 confirmed that dengue type 1 virus (DENV1) was the predominant type in Surabaya. Phylogenetic analyses of the nucleotide sequences of the complete envelope gene of DENV1 indicated that all 22 selected isolates in the second survey belonged to genotype IV and all 17 selected isolates in the third survey belonged to genotype I, indicating a genotype shift between April and September 2009. Furthermore, in December 2010, isolates were grouped into a new clade of DENV1 genotype I, suggesting clade shift between September and December 2010. According to statistics reported by the Surabaya Health Office, the proportion of DHF cases among the total number of dengue cases increased about three times after the type shift in 2008. In addition, the subsequent genotype shift in 2009 was associated with the increased number of total dengue cases. This indicates the need for continuous surveillance of circulating viruses to predict the risk of DHF and DF.     

Molecular epidemiology of measles virus

Genetic analysis of viruses associated with recent outbreaks of measles in the United States indicated that at least four genotypes were present during 1994 and 1995. None of these more recent genotypes were related to the genotype responsible for the resurgence of measles cases in the United States between 1989 and 1992. The sequence data confirmed that the majority of measles cases that occurred in the United States between 1994 and 1995 were the result of international importation of virus. The data also suggested that transmission of the genotype associated with the resurgence had been interrupted by aggressive control measures. Therefore, molecular epidemiologic studies will provide a powerful means to measure the success of measles control strategies.     

Emergence of a C-Terminal Seven-Amino-Acid Elongation of NS1 in Around 1950 Conferred a Minor Growth Advantage to Former Seasonal Influenza A Viruses

Influenza A viruses circulating in humans from ∼1950 to ∼1987 featured a nonstructural (NS1) protein with a C-terminal extension of seven amino acids. The biological significance of this NS1 elongation remained elusive. We observed that replication kinetics of the wild-type virus A/Hong Kong/01/68 (H3N2) and a mutant encoding a truncated NS1 were indistinguishable in most experimental systems. However, wild-type virus outcompeted the mutant during mixed infections, suggesting that the NS1 extension conferred minor growth advantages.     

Subacute sclerosing panencephalitis in Croatia (1994-2004)

We studied five patients with SSPE during a 10-year period (1994-2004). The first clinical symptoms developed at the age of 5-11 years. All patients were vaccinated regularly against measles according to the official immunization schedule. One patient had measles at the age of 18 months. Two of them had a history of morbilliform rash (unrecognized measles) at the age of six and seven months, respectively. In two patients, with no history of measles before vaccination the disease started after varicella infection. Using complement-fixation (F) test and EIA, antibodies to measles virus (MV) were detected in the CSF and sera of all patients. The CF-antibody titers ranged from 1:1024 to 1:65536 in sera and from 1:16 to 1:128 in CSF samples. MV antigen was detected in brain imprints using IFA in two patients. Electron microscopic analysis revealed intranuclear viral inclusions (MV nucleocapsids). Using RT-PCR, viral RNA was found in both patients. Nucleotide sequence analysis showed that the viruses found in the brain tissue belonged to the wild-type MV D6 genotype [7].