Complete coding regions of the prototypes enterovirus B93 and C95: phylogenetic analyses of the P1 and P3 regions of EV-B and EV-C strains.

Complete coding regions were sequenced for two new enterovirus genomes: EV-B93 previously identified by VP1 sequencing, derived from a child with acute flaccid paralysis in the Democratic Republic of Congo; and EV-C95 from a French soldier with acute gastroenteritis in Djibouti. The EV-B93 P1 had more than 30% nucleotide divergence from other EV-B types, with highest similarity to E-15 and EV-B80. The P1 nucleotide sequence of EV-C95 was most similar, 71%, to CV-A21. Complete coding regions for the new enteroviruses were compared with those of 135 EV-B and 176 EV-C strains representing all types available in GenBank. When strains from the same outbreak or strains isolated during the same year in the same geographical region were excluded, 27 of the 58 EV-B, and 16 of the 23 EV-C types were represented by more than one sequence. However, for EV-B the P3 sequences formed three clades mainly according to origin or time of isolation, irrespective of type, while for EV-C the P3 sequences segregated mainly according to disease manifestation, with most strains causing paralysis, including polioviruses, forming one clade, and strains causing respiratory illness forming another. There was no intermixing of types between these two clades, apart from two EV-C96 strains. The EV-B P3 sequences had lower inter-clade and higher intra-clade variability as compared to the EV-C sequences, which may explain why inter-clade recombinations are more frequent in EV-B. Further analysis of more isolates may shed light on the role of recombinations in the evolution of EV-B in geographical context.

Endemic hepatitis E in two Nordic countries.

Antibodies against hepatitis E virus (anti-HEV) were found in 248 Swedish and Danish patients between 1993 and 2007. Most patients were symptomatic and tested for anti-HEV due to travel abroad. Among patients with known country of infection, most were infected in Asia, mainly on the Indian subcontinent. However, 29 patients were infected in Europe, nine of these had HEV IgM and/or HEV RNA in serum. In sera from 65 of 141 tested patients HEV RNA could be detected, and 63 strains could be typed by limited sequencing within ORF2. HEV RNA was found in sera from 71% of the patients with HEV IgM and IgG and in 18% of the patients with only detectable HEV IgG. It was also found up to three weeks after the onset of disease in 67% of the patients with known date of onset. Patients infected in Europe were infected by genotype 3, and were older than those infected by genotype 1 (mean age 55.3 vs 30 years, p<0.001). Since it is known that genotype 3 can infect domestic pigs, HEV strains from 18 piglets in 17 herds in Sweden and Denmark were sequenced. Phylogenetic analyses of the genotype 3 strains showed geographical clades and high similarity between strains from patients and pigs from the same area. There are thus autochthonous hepatitis E cases in Scandinavia, and there are probably many undiagnosed ones. Patients with hepatitis of unknown etiology should therefore be investigated for anti-HEV even if they have not been outside Europe, since infections acquired from pigs or other animals should be taken into consideration.

A new core promoter mutation and premature stop codon in the S gene in HBV strains from Iranian patients with cirrhosis.

In order to define hepatitis B virus (HBV) mutational patterns in Iran, nucleotide sequences obtained from 91 patients and encompassing the precore, basal core promoter (BCP) and surface (S) regions, were compared. The patients were grouped as asymptomatic carriers, chronic active hepatitis or cirrhotic patients. Genotypes and mutations were determined by sequencing and phylogenetic analysis. All strains belonged to genotype D, and most of them to subgenotype D1. All but two strains specified ayw2, one ayw3 and one adw2 determinants. Two deletions of 8- or 20-bp were found in the X region in eight strains, six from patients with chronic active hepatitis. Eight of 21 strains from patients with cirrhosis harboured unusual mutations such as a stop codon at position 69 in the S region or a previously not described mutation in the BCP region ((1761)TC/ATTTG(1766)). All patients infected by strains with the stop codon mutation had detectable HBsAg and high viral load. The accumulation of mutations found in the BCP and S regions in HBV strains from patients with chronic active hepatitis and cirrhosis may predict disease progression in Iranian HBsAg carriers.

Presumed common source outbreaks of hepatitis A in an endemic area confirmed by limited sequencing within the VP1 region.

Hepatitis A virus isolates from anti-HAV IgM positive sera of 70 hepatitis cases in two outbreaks and 216 other cases in Central America, 136 sporadic cases and 53 cases from an hyper-endemic region in Costa Rica, were compared by phylogenetic analyses within the VP1 region. The outbreaks in all 531 cases, in 1992 and 1999, respectively, were presumed water borne. In the first outbreak, HAV RNA could be detected in 70% of the cases sampled during 6 weeks after onset of jaundice. In the hyper-endemic region of San Ramón in Costa Rica, 1,932 cases were registered between 1972 and 1985. All isolates belonged to subtype 1A. Background isolates from Costa Rica and El Salvador tended to form separate subclusters in the phylogenetic tree construction and were mostly unrelated to subtype 1A strains from other parts of the world. Based on their amino acid sequences, four HAV strains, all related to CR326 sampled in Costa Rica in 1960, were found to have circulated in the area during the last three decades. However, on the basis of nucleotide variability the isolates from the outbreaks could be distinguished from the strains from sporadic cases and sequence analysis could confirm the epidemiological homogeneity of both outbreaks. In the hyper-endemic region, 16 different sequences were encountered forming one single subcluster. Thus, limited sequencing within the VP1 region proved useful to identify outbreaks of hepatitis A in a highly endemic area, where most strains were local and only one subtype was prevalent.

Shift in predominating subtype of HCV from 1b to 3a in St. Petersburg mediated by increase in injecting drug use.

The genotypes of 149 HCV strains from St. Petersburg were determined by limited sequencing and phylogenetic analysis within the NS5B region. One hundred two strains derived from patients that attended infectious disease clinics, of whom 48 admitted injecting drug use, and 47 derived from dialysis patients. Subtype 3a was predominant in the patients from infectious disease clinics, both in patients that admitted injecting drug use (56%) and in those with unknown source of infection (46%). However, 89% of the strains from dialysis patients belonged to subtype 1b. Eleven of twelve characterised strains from recent cases of hepatitis C at these units were at phylogenetic analysis shown to be related to strains already circulating there, demonstrating that within the dialysis units nosocomial transmission is the most important route of HCV infection. The predominance of subtype 1b strains in dialysis patients indicates that these strains have been circulating for a long time in dialysis units. The predominance of subtype 3a also among patients who did not admit drug use and that their strains were intermixed with the strains from injecting drug users in the phylogenetic analysis shows that the increase in injecting drug use is the major factor that explains the recent spread of HCV in the St. Petersburg population. This supports the concept that injecting drug use remains the major route for HCV infection in developed countries and that the control of drug abuse is the most important measure to prevent its spread.

Hepatitis B virus genotypes and HBsAg subtypes in refugees and injection drug users in the United States determined by LiPA and monoclonal EIA.

Hepatitis B virus (HBV) genotyping and hepatitis B surface antigen (HBsAg) subtyping were carried out on sera from 196 HBsAg-positive patients, including 151 refugees entering the United States and 45 injection drug users in Seattle. HBsAg subtyping was performed by enzyme immunoassay (EIA) using a panel of monoclonal antibodies and the HBV genotype was determined by polymerase chain reaction (PCR) followed by detection of amplified HBV DNA by a reverse-phase hybridization line probe assay (LiPA) using genotype-specific probes. HBV DNA was detected by PCR in 155 (79%) of the 196 sera and all 155 were genotyped by LiPA. Samples from Southeast Asia were predominantly genotype B/subtype ayw1 and genotype C/adr; samples from the former Soviet Union and eastern Europe were mostly genotype D/ayw2 and genotype D/ayw3; samples from east Africa were mainly genotype A/adw2 and genotype D/ayw2; and samples from injection drug users were mostly genotype D/ayw3 and genotype A/adw2. Some strains of ayw3 gave atypical monoclonal antibody reactivity patterns in the subtyping assay due to a Val/Ala instead of a Thr at amino acid residue 118 and a Thr instead of a Met at residue 125. A strain of ayw2 also gave an atypical monoclonal antibody reactivity pattern due to an Ala instead of a Thr at amino acid residue 123. LiPA genotyping and monoclonal EIA subtyping can provide useful information for epidemiological studies.

[Hepatitis B outbreak in a day care center affected several families. It could have been prevented by vaccination of all children]. / Hepatit B-utbrott på daghem spred smitta till flera familjer. Med vaccinering av alla barn kunde detta ha förhindrats.

An outbreak of hepatitis B originating in a family day nursery affected several children with Somali background. The transmission chain was confirmed by sequence analysis of the S gene. In Africa hepatitis B is often spread horizontally among children of pre-school age, a pattern of transmission that was retained in this outbreak. To limit the outbreak 126 children in the nursery and 50 members of staff had to be vaccinated. The total cost for this intervention was estimated to about 300,000 SEK. Considering the great number of immigrants in Sweden from areas highly endemic for hepatitis B the inclusion of vaccination in the general child immunisation program seems to be the most cost effective measure for long term prevention not only of hepatitis B transmission among children but also of venereal spread in early adulthood.

Homotypic echoviruses share aminoterminal VP1 sequence homology applicable for typing.

Molecular typing of enteroviruses should ideally focus on regions encoding determinants for neutralization. Mapping of monoclonal neutralizing antibodies has shown the VPI protein, in particular its aminoterminal part, encompassing the B-C loop, to be one major antigenic region. We therefore sequenced 570 nucleotides from the 5'-end of the VP1 region of the genome for all 28 echovirus prototypes, and for 61 clinical isolates representing all different echovirus types. An analysis of 133 sequences, including 39 sequences retrieved from GenBank, classified all echoviruses in enterovirus group B confirming results from sequencing within the VP2 region. The nucleotide and amino acid divergence of VP1 sequences of homotypic strains varied from 7.5-23.0% and from 0.0-5.3%, respectively, when compared to their corresponding prototypes, whereas strains belonging to different serotypes these divergences were 22.1-38.9 % and 4.9-16.4 %, respectively. Despite these minimal overlaps, the VP1 sequence was always more similar to that of the homotypic prototype than to that of any heterotypic strain. For 13 out of 14 echovirus types, where multiple isolates were available, the corresponding VP1 sequences diverged more from those of the prototype than from the other homotypic sequences as a reflection of genetic drift. Because there was a complete concordance between the sequences of the region encoding the VP1 aminoterminus and the serotype (P< 0.00001) sequence analysis of this region might complement typing by neutralization, and classify correctly echovirus isolates that may not be typed conveniently by the antisera in hand.

Preclinical evaluation of two human anti-hepatitis B virus (HBV) monoclonal antibodies in the HBV-trimera mouse model and in HBV chronic carrier chimpanzees.

Two human monoclonal antibodies (mAbs) against hepatitis B surface antigen (HBsAg) generated in the Trimera mouse system are described. Both mAbs 17.1.41 and 19.79.5 are of the IgG1 isotype and have high affinity constants for HBsAg binding in the range of 10(-10) mol/L. Monoclonal antibody 17.1.41 recognizes a conformational epitope on the a determinant of HBsAg whereas mAb 19.79.5 recognizes a linear one. The 2 mAbs bind to a panel of hepatitis B virus (HBV) subtypes with distinct patterns. The neutralizing activity of these antibodies was tested in 2 different animal model systems. Administration of each mAb to HBV-Trimera mice, a system that provides a mouse model for human hepatitis B infection, reduced the viral load and the percentage of HBV-DNA-positive mice in a dose-dependent manner. These 2 mAbs were more effective than a polyclonal antibody preparation (Hepatect; Biotest Pharma, Dreieich, Germany) in both inhibition of HBV liver infection and reduction of viral load. A single administration of a mixture of these mAbs into HBV chronic carrier chimpanzees resulted in immediate reduction in HBsAg levels followed by recurrence to initial levels within few days. Thus, these mAbs may be potential candidates for preventive therapy or in combination with other antiviral agents against HBV. Further studies in humans are needed to assess these mAbs in various clinical indications.

Confirmation of nosocomial transmission of hepatitis C virus by phylogenetic analysis of the NS5-B region.

Four hepatitis C virus transmission chains at three dialysis units were disclosed by limited sequencing; three of these were disclosed by analysis of the NS5-B region of the genome. Dialysis on the same shift as that during which infected patients were dialyzed was the common factor for seven patients in two chains. Two nurses exposed to needle sticks and their sources of infection constituted two other chains. The strains of three chains belonged to subtype 1a and formed clusters with an intrachain variability of 0 to 6 nucleotides compared to 8 to 37 nucleotides for unrelated strains within this subtype. The clusters were supported by bootstrap values ranging from 89 to 100%.

Antigenic diversity of hepatitis B virus strains of genotype F in Amerindians and other population groups from Venezuela.

The adw4 subtype of hepatitis B virus (HBV) belongs to a unique genomic group (genotype F) representing the original HBV strains from the New World. Data regarding the prevalence of this subtype among HBV carriers in South America are, however, scarce, and those concerning HBV genotype F are based on only a few samples from Latin America. In this study, serum samples were obtained from 141 hepatitis B surface antigen (HBsAg) carriers from Amerindians and urban populations from Venezuela. The HBsAg subtype was identified with monoclonal antibodies in 105 samples, and the HBV genotype was identified by reverse-phase hybridization with DNA fragments in 58 samples. The adw4 subtype was highly prevalent in the population studied (75%); among the Amerindians, the prevalence was 97%. The adw2 subtype was also present (10%), while other subtypes (ayw3 and ayw4) were only occasionally found. The HBV subtype was associated with the expected genotype in most cases (80%), and thus genotype F was highly prevalent. Sequencing of viral strains that gave genotypes unpredicted by the HBsAg subtyping confirmed seven of them as belonging to not previously described genotype-subtype associations: namely, adw2 and ayw4 within genotype F.

Molecular epidemiology of hepatitis B virus in Central America reflected in the genetic variability of the small S gene.

The S genes of 31 Central American hepatitis B virus (HBV) strains belonging to genotypes A, C, D, and F (4, 1, 4, and 22 strains, respectively) were compared with 104 published S genes. According to the deduced S gene product, 21 genotype F strains encoded adw4, while 1 encoded ayw4. Three clusters were revealed within genotype F, which correlated with substitutions at residue 45. In a cluster of 18 Central American and 1 Alaskan strain, all had Thr45. One cluster included 2 Central American strains and 6 strains from South America and Europe, which had Leu45. Two Nicaraguan strains differed by five substitutions, including a Pro45 in the S gene product from other F strains. In conclusion, the dominating HBV genotype was F, which might be the reason for a low prevalence of HBV in the area, despite high prevalence of hepatitis A. These infections otherwise vary in parallel and are considered to reflect socioeconomic conditions.

High prevalence of GB virus C strains genetically related to strains with Asian origin in Nicaraguan hemophiliacs.

The presence of hepatitis GB virus C (GBV-C), also known as hepatitis G virus (HGV), and hepatitis C virus (HCV) were investigated in sera from 45 hemophiliacs from nine locations in Nicaragua using a nested polymerase chain reaction (PCR). Primers used to detect GBV-C and HCV derived from the helicase region and 5'UTR, respectively. Seventeen (38%) patients were positive for GBV-C RNA in serum by PCR. Twelve (27%) patients were positive for HCV RNA by PCR. Six (13%) of these were coinfected with GBV-C. Anti-HCV was detected in all the 12 HCV RNA positive hemophiliacs and in another 14 (31%) individuals, in whom GBV-C RNA was found in 2. Ten patients (22%) lacked markers for both GBV-C and HCV. The mean age of the patients positive for GBV-C but negative for HCV by PCR was significantly lower than for those negative for GBV-C but positive for HCV by PCR (P < 0.05; Student's t-test), indicating that the risk for this group of hemophiliacs to acquire GBV-C infection is higher as compared to the risk of acquiring HCV infection. Eleven GBV-C strains were sequenced in the 5'UTR. Sequence comparison to previously published GBV-C strains revealed that all 11 strains were more similar to Asian strains than to strains of European and African origin. Sequences in the NS5-B region were available for 8 HCV strains, all of which were found to belong to genotype 1a. The similarity of the Nicaraguan GBV-C strains to strains from Asia indicates that the GBV-C strains in the region presumably have an Amerindian origin. It is also considered that the HTLV II strains in the New World aboriginal populations are ancient and brought there by the ancestral Amerindian populations from Asia. Further, the genotype F of hepatitis B virus, known to represent the strains in populations with Amerindian background, predominates in Central American populations with Hispanic background. It remains to be clarified why Amerindian strains of GBV-C as well as of HBV predominate also in populations with mixed ethnic background in Central America.

Genotype F prevails in HBV infected patients of hispanic origin in Central America and may carry the precore stop mutant.

The distribution of HBV genotypes and the presence of the precore stop mutation were investigated in HBV strains from Central America. 333 HBsAg positive sera from chronic HBsAg carriers and acute hepatitis B cases from five different countries (Costa Rica, Nicaragua, Honduras, EI Salvador and Guatemala) were tested for HBV DNA by nested PCR. Genotyping by limited sequencing within the S gene was performed on 90 strains, 66 from sera with a high level of HBV DNA, and another 24 from sera positive for HBV DNA only after nested PCR. 23 of the samples were anti-HBe positive. Genotype F was found in 71 (79%), A in 13 (14%), D in 5 (6%) and C in one of the 90 sera. 18 patients with genotype F infection had anti-HBe and HBV DNA in serum. Since the three published precore sequences of genotype F strains have a C1858, which is known to prevent the precore stop mutation from G to A at position 1896, the precore and part of the core genes were sequenced from 19 anti-HBe positive sera with HBV DNA, 17 with genotype F and 2 with genotype A. The A1896 mutation was found in 11 of the 17 genotype F strains. All these had a T1858, which was also present in 5 of the 6 genotype F strains with G1896. The precore region was therefore sequenced from genotype F strains from 5 HBeAg positive sera from the five different Central American countries. These also had a T1858, which thus is the wild type substitution in genotype F in Central America. A number of mutations were recorded between residues 57 and 68 in the core protein corresponding to a unique clustering region of the genotype F strains. The predominance of genotype F in Central American populations of Hispanic origin was not anticipated since this genotype is regarded as indigenous to the Amerindian populations of the New World.

Interferon-alpha 2b treatment in hepatitis B carriers. Effect on hepatitis B virus DNA levels in children infected with different genotypes.

Eleven hepatitis B virus (HBV) carrier children, infected with genotypes A-D, were treated with interferon-alpha. Two children had a sustained loss of hepatitis B e-antigen and HBV DNA. They were infected with the non-Asian genotypes A and D, and had low HBV DNA and high ALT levels in serum before treatment. However, HBV DNA titres decreased during treatment also in children infected with the East-Asian genotypes B and C.

Viral etiology of acute childhood encephalitis in Beijing diagnosed by analysis of single samples.

OBJECTIVE: To understand the viral etiology of acute childhood encephalitis in Beijing. METHODS: Ninety-seven Chinese children (between 7 months and 13 years of age) with acute encephalitis were retrospectively investigated. They were treated in Beijing Children's Hospital between June, 1991, and October, 1994. Different serologic methods (immunofluorescence assay, enzyme-linked immunosorbent assay, solid phase reverse immunosorbent test) were used for detection of IgM antibody to enteroviruses, herpesviruses, mumps, measles, rubella and Japanese encephalitis virus. The viral DNA of six herpesviruses was detected by polymerase chain reaction. RESULTS: Viral etiology was identified in 35 of 97 (36.0%) cases. The most frequently identified pathogens were enteroviruses (15; 15.4%), followed by mumps (7; 7.2%), rubella (6; 6.1%), Japanese encephalitis virus (5; 5.1%), human herpesvirus 6 (2; 2.0%), herpes simplex virus (2; 2.0%) and Epstein-Barr virus (1; 1.0%). IgM antibody in cerebrospinal fluid was detected for enterovirus, mumps and rubella viruses. CONCLUSIONS: Enteroviruses were the most frequent viral pathogens of acute childhood encephalitis in Beijing. Detection of IgM in cerebrospinal fluid may be useful for diagnosis in certain cases of viral encephalitis.

Genetic relatedness of Sindbis virus strains from Europe, Middle East, and Africa.

The relatedness of 40 strains of Sindbis virus (SIN) from Europe, the Middle East, and Africa was investigated by limited sequencing within the gene encoding the E2 glycoprotein corresponding to amino acid residues 117 to 229 and encompassing one of the major neutralization epitopes. Phylogenetic analyses using distance matrix and parsimonious methods identified two major genetic clusters of western SIN strains, although the variability was less than that of the corresponding region for Venezuelan equine encephalomyelitis virus with a maximum divergence of 12.4% versus 28.5%, respectively. One cluster comprising 19 strains included the HR derivate of the Egypt SIN prototype, AR339, and strains from Israel, Saudi-Arabia, Italy, Slovak Republic, Azerbaijan, as well as three Swedish strains. Another cluster of 17 strains included the Ockelbo virus (OCK) prototype, Edsbyn 5/82, and the majority of SIN strains from northern Europe including strains from Sweden, Norway, and Karelia, as well as two strains from South Africa. A third cluster, supported by the Neighbor joining method, was made up of four strains from South Africa, Uganda, and Cameroon. Residue 212, either Ser or Thr, previously appointed important for the differences in neutralization assays between SIN and Edsbyn 5/82, respectively, correlated with the two major genetic clusters, but was a Thr for two of the three Swedish strains in the SIN prototype cluster, and a Ser in one Swedish and one Karelian strain in the OCK cluster. The finding of strains similar to prototype SIN in Middle Sweden and of strains in South Africa relating to the northern cluster of SIN strains supports the notion of the dispersal of SIN by migrating birds as previously suggested for New World alphaviruses.

An increase in sudden unexpected cardiac deaths among young Swedish orienteers during 1979-1992.

BACKGROUND: Sixteen cases of sudden unexpected cardiac death, 15 males and one female, are known to have occurred among young Swedish orienteers from 1979 to 1992, of which seven cases occurred between 1989 and 1992. This is considered to be indicative of an increased death rate. RESULTS: Histopathological evaluation showed myocarditis in a higher than expected proportion of cases. In one such case, which we studied before the sudden unexpected death occurred, the victim had suffered a Chlamydia pneumoniae infection verified by serology, and a nucleotide sequence was found in the heart and lung by means of the polymerase chain reaction (PCR) that hybridized with a probe specific for that organism. Male Swedish orienteers do not, however, seem to have an increased rate of exposure to this agent. No further sudden unexpected deaths among young orienteers have occurred over the past 3.5 years. At the beginning of that period, attempts were made to modify training habits and attitudes.

Complete sequencing of a gibbon hepatitis B virus genome reveals a unique genotype distantly related to the chimpanzee hepatitis B virus.

We have sequenced the complete genome of a hepatitis B virus (HBV) strain that was transmitted from a gibbon with chronic hepatitis B to a chimpanzee that subsequently developed acute hepatitis B. The genome was 3,182 nucleotides long and had a genetic organization identical to and including the characteristics of other mammalian hepadnaviruses. Thus, the regulatory elements, the direct repeats, and the four open reading frames (ORFs) of this virus were all maintained, although there were amino acid substitutions affecting all the ORFs. Within the S gene encoding for the hepatitis B surface antigen (HBsAg), the subtype could be deduced as ayw3 in accordance with previous serological results. There were 25 amino acid substitutions affecting the P gene, 12 of which were within the spacer region. This region, which was the most divergent part of the genome compared to other HBV strains, also encodes for the pre-S proteins. A comparison with sequences of other hepadnaviruses revealed that the genome of gibbon HBV was unique as compared to previously described HBV genotypes. It was most similar to the chimpanzee HBV strain with which it shared 90.3% nucleic acid homology at the level of the complete genome and 96.3% homology at the level of the S-gene region corresponding to HBsAg, although being a distinct genotype as compared to the latter virus. Analyses performed using five different algorithms for phylogenetic tree construction showed more than 99% bootstrap support for the gibbon and the chimpanzee HBV to be grouped within the human HBV strains and that they represented later offshoots than the HBV strains of genotype F. However, in most of the dendrograms both the gibbon and the chimpanzee strains represented early lineages, indicating that these viruses are indigenous to their respective hosts and not recent acquisitions from man.