Genotype D4 Research Articles

Molecular Epidemiology of Measles Viruses in the United States, 1997–2001

From 1997 to 2001, sequence data from 55 clinical specimens were obtained from confirmed measles cases in the United States, representing 21 outbreaks and 34 sporadic cases. Sequence analysis indicated the presence of 11 of the recognized genotypes. The most common genotypes detected were genotype D6, usually identified from imported cases from Europe, and genotype D5, associated with importations from Japan. A number of viruses belonging to genotype D4 were imported from India and Pakistan. Overall, viral genotypes were determined for 13 chains of transmission with an unknown source of virus, and seven different genotypes were identified. Therefore, the diversity of Measles virus genotypes observed in the United States from 1997 to 2001 reflected multiple imported sources of virus and indicated that no strain of measles is endemic in the United States.

Genetic homogeneity of measles viruses associated with a measles outbreak, São Paulo, Brazil, 1997.

During a resurgence of measles in São Paulo, Brazil, in 1997, >40,000 cases (peak incidence rate of 246/100,000 inhabitants) and 42 measles-related deaths were reported. Reverse transcriptase-polymerase chain reaction and nucleotide sequencing were used to analyze specimens from patients who had typical clinical measles infection during this outbreak and from six patients who had had measles in 1995 and 1996. Although wild-type measles viruses (genotypes D5 and D6) were present in São Paulo before this resurgence, we detected only D6 viruses. The genotype D6 viruses isolated during this outbreak had identical sequences to genotype D6 viruses isolated in other parts of Brazil and South America in 1997 and 1998, suggesting that a single chain of transmission was responsible. We also identified genotype A viruses in two vaccine-associated cases from 1995 and 1996. Our findings extend the knowledge of the circulation patterns of measles virus in South America, contributing to measles control efforts in the Americas.

Molecular analysis of measles virus genome derived from SSPE and acute measles patients in Papua, New Guinea

A very high annual incidence of 56 per million population below the age of 20 years for subacute sclerosing panencephalitis (SSPE) has been reported from Papua New Guinea (PNG). In a more recent study, we have confirmed this unusual high incidence for Eastern Highlands Province (EHP) of PNG. In the study, it was observed that the vaccination rate among SSPE patients registered at Goroka Base General Hospital (GBGH) in EHP was higher than that of other infants in the province in recent years. To identify the measles virus (MV) responsible for SSPE in EHP, sequence analysis of hypervariable region of the N gene was performed from 13 MV genomes: 2 amplified from clinical specimens of SSPE patients and 11 from acute measles patients. In 2 cases among the 11 with acute measles, nucleotide sequence of the entire H gene derived from isolated viruses was determined. Both nucleotide sequence and phylogenetic tree analyses showed that the amplified MV cDNAs were closely related to one another and belonged to the D3 genotype though they were different from any previously reported MV sequences. No genome sequences of vaccine strains were detected. These findings suggest that the MV strains prevailing in the highlands of PNG belong to genotype D3 of the MV and this wild-type MV rather than the vaccine strains was likely to be responsible for SSPE in these patients.

Suboptimal measles-mumps-rubella vaccination coverage facilitates an imported measles outbreak in ireland.

The year 2000 saw a dramatic increase in the incidence of measles infections in Ireland, with >1500 cases documented. Initial cases were reported from an area of Dublin with low vaccine uptake and a large immigrant population. Molecular epidemiologic findings revealed that the strain of measles virus responsible for this outbreak was the genotype D2 strain, which is closely related to strains initially identified in South Africa. It is suggested that suboptimal vaccine uptake facilitated the spread of imported measles infection.

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The incidence of acute hepatitis C (AHC) among HIV-infected men who have sex with men (MSM) has increased significantly in the last 10 years. Several studies point to a social and sexual network of HIV-positive MSM that extends internationally.The aim of our study was to investigate the dynamics of HCV transmission in an outbreak of AHC in HIV-infected MSM in Barcelona by ultra-deep pyrosequencing.Between 2008 and 2013, 113 cases of AHC in HIV-infected MSM were diagnosed in the Infectious Diseases Unit, Hospital Clínic, Barcelona. Massive sequencing was performed using the Roche 454 GS Junior platform. To define possible transmission networks, maximum likelihood phylogenetic trees were constructed, and levels of genetic diversity within and among patients were compared.Among the 70 cases analyzed, we have identified 16 potential clusters of transmission: 8 for genotype 1a (23 cases involved), 1 for genotype 1b (3 cases) and 7 for genotype 4d (27 cases). Although the initial phylogenetic reconstruction suggested a local transmission cluster of HCV gt4d, our approach based on low genetic differentiation did not corroborate it. Indeed, gt4d strains formed 4 independent groups related to patients from other countries.Frequent clustering of HIV-positive MSM shows that HCV infection has spread through a local network in Barcelona. This outbreak is related to a large international HCV transmission network among MSM. Public health efforts are needed to reduce HCV transmission among this high-risk group.

Characterization of measles virus strains causing SSPE: a study of 11 cases.

Eleven subacute sclerosing panencephalitis (SSPE) cases diagnosed in the UK between 1965 and 2000 were investigated. The entire or partial matrix (M), hemagglutinin (H), and nucleoprotein (N) genes of measles virus (MV) were sequenced following direct RT-PCR amplification from brain tissues. All the M genes showed the characteristic biased hypermutations and a premature termination codon was detected in 5/11 M sequences. Based on the more highly conserved H and N genes observed in persistent MV studies, phylogenetic analysis showed that two of three strains from patients likely to have acquired infection in the 1950s were related to clade C (WHO designation) and one appears to be a novel genotype. Three strains from patients infected in the 1960s and 1970s were clearly related to a MV strain isolated in 1974 belonging to genotype D1. Four strains from patients infected in the 1980s clustered with genotype D7 strains. One sequence from a patient infected in 1990s was identified as genotype D6. No vaccine strains were detected although five of these patients had been previously immunized. The sequence data obtained from these historic strains do not support the view that vaccine strains are associated with SSPE and provide valuable information for further studies of MV epidemiology, evolution, and pathogenesis in SSPE.

CUMULATIVE AUTHOR INDEX FOR MUTREV 2001

A nationwide measles outbreak occurred in Vietnam between 2013 and 2014.To provide an overview on the 2013–2014 measles outbreak in northern Vietnam using epidemiological and molecular analysis of the measles virus (MeV).Epidemiological information was collected from all suspected cases of measles/rubella. Serum and/or throat swabs were collected for laboratory confirmation of measles. MeV genomes were detected and sequenced for phylogenetic analysis.A total of 9577 confirmed measles cases were reported in northern Vietnam with an incidence rate of 116.4/1,000,000 population. Of these, approximately 76.3% had unvaccinated or unknown vaccination history and 55.7% were under five years old. The outbreak started in a minority population from the mountainous area bordering Lao PDR and China and exploded in high-density population areas. Molecular analysis of MeV revealed co-circulation of genotypes H1 and D8, with H1 as the predominant strain, and divided into two clusters: cluster 1, sharing high similarity with those detected in China and Lao PDR, and cluster 2, circulating locally with unidentified origin. The minor D8 strains belonged to the D8-Frankfurt cluster.The outbreak originated in and spread from a population with limited access to vaccines. Molecular analysis revealed co-circulation of MeVs with three different origins during the outbreak. This is the first report to provide an overview of the 2013–2014 measles outbreak in northern Vietnam, demonstrating the need for vaccination strategies against measles that are tailored to local conditions with enhanced molecular surveillance to achieve measles elimination.

Genomic characterization of wild-type measles viruses that circulated in different states in Brazil during the 1997 measles epidemic.

Despite the marked reduction in the incidence of measles in Brazil, a measles epidemic occurred in this country in 1997. The measles cases observed during this epidemic began to reappear in large numbers in São Paulo, and spread to Rio de Janeiro and other Brazilian states. In the present study molecular biology techniques were used for the detection and genomic characterization of measles viruses from clinical samples such as urine and nasopharyngeal secretions collected in the states of Rio de Janeiro, Minas Gerais and Paraná, during the 1997 epidemic. RT-PCR and nucleotide sequence analysis of part of the carboxyl-terminal region of the nucleoprotein gene of measles viruses obtained directly from clinical samples or from infected cell cultures during this epidemic classified all as wild-type of genotype D6. As the genotype D6 was identified in different Brazilian states, this study demonstrated that this genotype was circulating in Brazil during the 1997 epidemic.

Nosocomial spread of hepatitis B virus in two hemodialysis units, investigated by restriction fragment length polymorphism analysis.

A method for genotyping hepatitis B virus (HBV) strains, based on restriction fragment length polymorphism (RFLP) analysis of four different amplified fragments of the HBV genome, was used to investigate nosocomial infections that occurred in two Brazilian hemodialysis centers. Viral isolates from hepatitis B surface antigen (HBsAg)-positive serum samples from 27 hemodialysis patients and 39 HBV-positive unrelated control patients were grouped according to their RFLP patterns. Strains isolated from the control patients were divided into nine RFLP patterns: A1, A2, A3 (genotype A), D1, D2, D3, D4 (genotype D), F1, and F2 (genotype F). In hemodialysis unit A (Rio de Janeiro), 14 HBV isolates were grouped into five different RFLP patterns: A1, A2, A3, D3, and D4. Pattern A2, present at a relatively low prevalence (18%) in the control group, was observed in the majority (53%) of the hemodialysis patients. Notably, all five patients who seroconverted to HBsAg positivity in 1995 carried the strain A2. In hemodialysis unit B (state of São Paulo), where an outbreak of HBV infection occurred in 1996-1997, RFLP analysis showed that all 13 patients who seroconverted were infected with HBV isolates of genotype D. Coinfection with strain A1 was detected in seven of them. The results demonstrate the value of RFLP analysis in establishing common sources of infection in hemodialysis centers.

Genotyping of measles virus isolates from Central Europe and Russia

Sequence analysis of 285 nucleotides located on the variable part of the N gene was undertaken on measles virus (MV) samples collected from acutely infected patients in Germany, the Czech Republic, Denmark, Poland, and Russia. Two distinct genotypes (C2 and D6) have circulated in Germany between 1993 and 1996. Isolates of genotype C2 were related to strains reported in Germany before 1993. This genotype was also found in the Czech Republic in 1992 and in Denmark in 1997. The occurrence of genotype D6 in Germany is described below for the first time. In 1998, this genotype was identified in Poland. Genotypes C2 and D6 were also reported in Spain and in the United Kingdom between 1992 and 1996. Therefore, it is concluded that these genotypes are widely distributed over Europe. The analysis of the isolates from Russia revealed that genotype A was present in 1988 in the European part of the country and in 1996 in Siberia. An isolate identified in 1997 in Siberia belonged to genotype D6, which had never been found previously in Russia. We also analysed MV obtained from a case of subacute sclerosing panencephalitis (SSPE) in 1995 in Turkey. A comparison of this sequence with published sequences implied that this SSPE case was associated with a new genetic lineage of MV.

Genotyping of measles virus isolates from Central Europe and Russia

Sequence analysis of 285 nucleotides located on the variable part of the N gene was undertaken on measles virus (MV) samples collected from acutely infected patients in Germany, the Czech Republic, Denmark, Poland, and Russia. Two distinct genotypes (C2 and D6) have circulated in Germany between 1993 and 1996. Isolates of genotype C2 were related to strains reported in Germany before 1993. This genotype was also found in the Czech Republic in 1992 and in Denmark in 1997. The occurrence of genotype D6 in Germany is described below for the first time. In 1998, this genotype was identified in Poland. Genotypes C2 and D6 were also reported in Spain and in the United Kingdom between 1992 and 1996. Therefore, it is concluded that these genotypes are widely distributed over Europe. The analysis of the isolates from Russia revealed that genotype A was present in 1988 in the European part of the country and in 1996 in Siberia. An isolate identified in 1997 in Siberia belonged to genotype D6, which had never been found previously in Russia. We also analysed MV obtained from a case of subacute sclerosing panencephalitis (SSPE) in 1995 in Turkey. A comparison of this sequence with published sequences implied that this SSPE case was associated with a new genetic lineage of MV. J. Med. Virol. 58:313–320, 1999. © 1999 Wiley-Liss, Inc.

Genotypic and antigenic characterization of hemagglutinin proteins of African measles virus isolates

A comprehensive phylogenetic study based on the hemagglutinin (H) protein of all known African measles virus (MV) isolates is presented. The study includes 64 new H gene sequences from Ghana, Nigeria and South Africa as well as viruses from Zambia and The Gambia for which only incomplete sequencing data were available and that have previously not been genotyped. The results provide further support to the tentative assignment of the Nigerian and Ghanaian viruses to a new genotype B3 within clade B. A distinct geographic distribution pattern emerged with clade B viruses circulating exclusively in African countries north of the equator. All MV strains from southern Africa grouped in clades A and D with the majority of viruses belonging to genotype D4. The viruses considerably differed by their sensitivity to neutralization by monoclonal antibodies (mAb), but three selected antibodies were sufficient to distinguish between African MVs representing four different genotypes.

Sequence divergence of measles virus haemagglutinin during natural evolution and adaptation to cell culture.

Phylogenetic analysis of the sequence of the H gene of 75 measles virus (MV) strains (32 published and 43 new sequences) was carried out. The lineage groups described from comparison of the nucleotide sequences encoding the C-terminal regions of the N protein of MV were the same as those derived from the H gene sequences in almost all cases. The databases document a number of distinct genotype switches that have occurred in Madrid (Spain). Well-documented is the complete replacement of lineage group C2, the common European genotype at that time, with that of group D3 around the autumn of 1993. No further isolations of group C2 took place in Madrid after this time. The rate of mutation of the H gene sequences of MV genotype D3 circulating in Madrid from 1993 to 1996 was very low (5 x 10(-4) per annum for a given nucleotide position). This is an order of magnitude lower than the rates of mutation observed in the HN genes of human influenza A viruses. The ratio of expressed over silent mutations indicated that the divergence was not driven by immune selection in this gene. Variations in amino acid 117 of the H protein (F or L) may be related to the ability of some strains to haemagglutinate only in the presence of salt. Adaptation of MV to different primate cell types was associated with very small numbers of mutations in the H gene. The changes could not be predicted when virus previously grown in human B cell lines was adapted to monkey Vero cells. In contrast, rodent brain-adapted viruses displayed a lot of amino acid sequence variation from normal MV strains. There was no convincing evidence for recombination between MV genotypes.

Genotyping hepatitis C virus isolates from Spain, Brazil, China, and Macau by a simplified PCR method.

An improved and simplified method of genotyping was developed for classifying hepatitis C virus (HCV) isolates into the five common genotypes, i.e., I/1a, II/1b, III/2a, IV/2b, and V/3a, by PCR with genotype-specific primers deduced from the core gene. Sense and antisense primers, specific for each of the five common genotypes, were designed by comparison of 319 core gene sequences from HCV isolates of various genotypes from genetic groups 1 to 9. In the first round of PCR, a sequence of 433 bp representing nucleotides 319 to 751 was amplified with universal primers. The second round of PCR was performed with respective sense and antisense primers in two separate reactions, one for the amplification of genotypes I/1a and II/1b and the other for the amplification of genotypes III/2a, IV/2b, and V/3a. The specificity of genotyping was confirmed with a panel of 191 serum samples containing HCV isolates whose core gene sequences were known: 110 serum samples infected with HCV of the five common genotypes and 81 serum samples infected with HCV of other genotypes. The use of sense and antisense primers for genotype II/1b (primers 389 and 492) abolished the cross-reaction of the antisense primer for genotype II/1b (primer 133) with some HCV isolates of genotype I/1a found by our original method. The new method was used for genotyping 130 HCV isolates from Spain, 53 from Brazil, 106 from China, and 30 from Macau. A total of 329 bp of the NS5b region (nucleotides 8279 to 8607) of five isolates from Spain and five isolates from Macau which could not be classified as any of the five common HCV genotypes or genotype 2c were sequenced, and the sequences were compared with those of HCV isolates of known genotypes; two isolates from Spain were deduced to be of genotype 4d and one was deduced to be of genotype 1d, while the remaining two isolates from Spain had novel genotypes in genetic group 2; however, all five isolates from Macau were of genotype 6a.

Polymorphisms of the D4 Dopamine Receptor Alleles in Chronic Alcoholism

We have screened genomic DNA for the identification of D4 dopamine receptor polymorphisms. We show that the D4 dopamine receptor genotype in 72 severely affected chronic alcoholics is heterogenous, with individuals homozygous and heterozygous for the various D4 receptor alleles. Alcoholics demonstrated a greater prevalence of the D4(3) (p<0.005) and D4(6) (p<0.005) alleles than has been reported in normals. There was a high prevalence of nicotine abuse among all D4 genotypes. The frequency of other drug abuse was higher in the D4(3,3) and the D4(4,7) groups, and the family history was strongly positive in the D4(2,4) group. The distribution of the D2 alleles showed equivalence in all D4 genotypes, except in D4(4,6) and D4(4,7) in whom the prevalence of the D2 A1A2 allele was 2-fold higher. The polymorphic variations of the D4 receptor genes should be among the factors considered in the assessment of individual differences in susceptibility to disorders such as alcohol abuse or drug addiction that may be mediated through central dopaminergic systems.