Current knowledge of hepatitis B virus (HBV) sequence heterogeneity is based mainly on sequencing of amplified subgenomic HBV fragments. Here, we describe a method which allows sensitive amplification and simplified functional analysis of full-length HBV genomes with or without prior cloning. By this method, a large number of HBV genomes were cloned from sera of six immunosuppressed kidney transplant patients. Two size classes of HBV genomes, one 3.2 kb and another about 2.0 kb in size, were found in all patients. The genome population from one serum sample was studied in detail by size analysis of subgenomic PCR fragments and sequencing. Regions with deletions and insertions were mapped in the C gene and pre-S region. Up to 100% of HBV genomes in all other immunosuppressed patients also had deletions in the C gene. Our results demonstrate the potential of the established method for the structural and functional characterization of heterogeneous populations of complete virion-encapsidated HBV DNAs and suggest that HBV genomes with C gene deletions can have a selective advantage in immunosuppressed patients.

A novel method for efficient amplification of whole hepatitis B virus genomes permits rapid functional analysis and reveals deletion mutants in immunosuppressed patients.

https://aasldpubs.onlinelibrary.wiley.com/doi/pdf/10.1053/jhep.2003.50453

Exploring the biological basis of hepatitis B e antigen in hepatitis B virus infection

The emergence of HBe-minus hepatitis B virus (HBV) mutants, usually through a UAG nonsense mutation at codon 28 of the precore region, helps the virus to survive the anti-HBe immune response of the host. Host and viral factors that predispose to the emergence of such mutants are not well characterized. The fact that the precore region forms a hairpin structure essential for the packaging of viral pregenomic RNA may explain the extremely high prevalence of the UAG mutation at codon 28. It converts a wobble U-G pair in the packaging signal between nucleotide 3 of codon 15 (CCU) and nucleotide 2 of codon 28 (UGG) into a U-A pair. Since genotype A of HBV has a CCC sequence at codon 15, the UAG mutation would, instead, disrupt a C-G pair present in the wild-type virus. This alteration was shown by transfection experiments to greatly compromise the packaging of pregenomic RNA. The implication of this finding was elucidated by molecular epidemiological studies. Genotype A was found to be the most prevalent genotype in the wild-type virus populations in France but was found in only 1 of the 46 isolates of HBe-minus mutants found there. These mutants were contributed chiefly by genotype D, the second most prevalent genotype in France, which is characterized by a CCU sequence at codon 15. The role of the single nucleotide at codon 15 was confirmed by the finding of the single genotype A isolate in which both wild-type and mutant viruses were present. Interestingly, nearly all of the mutants had a codon 15 sequence of CCU instead of the CCC present in the wild-type viruses. Our results suggest that genotype A of HBV rarely circulates as HBe-minus mutants, probably because of a requirement for a simultaneous sequence change at codon 15. These data, together with the virtual absence of genotype A in the Chinese samples examined, may provide some insights into the uneven prevalence of HBe-minus mutants in the world.

Hepatitis B virus genotype A rarely circulates as an HBe-minus mutant: possible contribution of a single nucleotide in the precore region.

Publisher Summary This chapter provides an overview of the epidemiology of hantavirus infections in Europe and the genetics of European hantaviruses. Hantaviruses form a separate genus within the family Bunyaviridae . Unlike other members of the family, hantaviruses are not arthropode-borne agents. They are maintained in persistently infected rodent hosts but are not transmitted by any insect vectors and seem to coevolve with their natural hosts; each hantavirus species being predominantly associated with one specific rodent species. This results in the circulation of distinct hantaviruses on different continents and in the geographical clustering of hantavirus genetic variants. When transmitted to humans, pathogenic hantaviruses from Eurasia, which are carried by Murinae or Arvicolinae rodents of the Muridae family, can cause hemorrhagic fever with renal syndrome while most of their relatives from the Americas, which are maintained in indigenous Sigmodontinae rodents of the Muridae family, can cause hantavirus pulmonary syndrome. During recent years, knowledge of the distribution and significance of hantaviruses in Europe has been increasing rapidly. Several outbreaks caused by Puumala hantavirus, known previously as a pathogen almost exclusively in Scandinavia, Russia, and the Balkans, have been registered in Germany and Belgium.

https://www.thelancet.com/pdfs/journals/laninf/PIIS1473-3099(03)00774-6.pdf

Hantavirus infections in Europe

Publisher Summary Although most living biological systems have developed intricate mechanisms to keep the genetic information stable, many viruses, including hepatitis B virus (HBV) can undergo rapid and drastic sequence changes. This discusses the data from extensive research on the genetic variability of HBV done during the past decade and the implications for the life cycle of the virus, immune recognition, and pathogenesis. It also discusses currently available data on the epidemiology of mutants and their functional significance. It is still largely unknown whether mutants, particularly those with combinations of different mutations, have an impact on liver disease, virus persistence, and course and outcome, especially of chronic infection. Therefore, more long-term follow-up studies, analyses of complete genomes, and functional studies of variants in appropriate animal models are needed. Important questions to be answered in the future concern the driving forces for the selection of HBV variants and the influence of mutations on immune recognition. Answering these questions will deepen the understanding of the virus–host interplay and eventually help to develop new strategies for the successful therapy and eradication of HBV infection.

Naturally occurring variants of hepatitis B virus.

Frequent and rapid emergence of mutated pre-C sequences in HBV from e-antigen positive carriers who seroconvert to anti-HBe during interferon treatment

The hantavirus strain Vranica was previously reported to have been isolated from a bank vole in Bosnia-Hercegovina and associated with the occurrence of hemorrhagic fever with renal syndrome (HRFS) in humans. The complete cDNA nucleotide sequences of the small (S) and medium (M) genomic RNA segments of this virus were determined. Major open reading frames were found in the S and M segment between nucleotide positions 43 and 1341 coding for a polypeptide of 433 amino acid residues and between nucleotide positions 41 and 3 484 coding for 1 148 amino acid residues, respectively. The analysis and the alignment of the nucleotide and the derived amino acid sequences with known sequences of other hantavirus strains demonstrate that Vranica resembles Swedish strains and represents a new virus subtype of the Puumala serotype distinct from the subtypes represented by virus strains CG18–20 and Sotkamo.

Coding strategy of the S and M genomic segments of a hantavirus representing a new subtype of the Puumala serotype.

Human cytomegalovirus (HCMV) encephalitis in adult nonimmunosuppressed patients has rarely been reported. We have diagnosed HCMV encephalitis in an anti-HCMV immunoglobulin G-negative, nonimmunosuppressed young woman by HCMV DNA PCR and virus isolation from cerebrospinal fluid (CSF). At the same time, HCMV antigen and HCMV DNA could be demonstrated in peripheral blood leukocytes, and the virus was isolated in fibroblast cultures. After 22 days of acute illness, the virus disappeared from the CSF. Remarkably, the patient did not generate detectable anti-HCMV antibodies within 5 months after the beginning of illness. To investigate the significance of HCMV DNA detection in CSF, samples of CSF, blood cells, and serum from 35 nonimmunosuppressed patients with various neurological disorders (but no herpes simplex virus central nervous system [CNS] disease) were tested for HCMV DNA, antigen, and antibodies. Eleven of these patients were found to be positive for virus DNA and/or antigen in peripheral blood leukocytes. Additionally, HCMV DNA was detected in the CSF of two patients with noninflammatory CNS diseases. A causative role of HCMV in the CNS diseases of these two patients was not evident. In summary, HCMV DNA amplification from CSF samples is a very suitable method to verify HCMV-associated encephalitis, but it should be taken into consideration that there are few cases of positive PCR with DNA from CSF without any known clinical correlative.

Human Cytomegalovirus (HCMV) Encephalitis in an Immunocompetent Young Person and Diagnostic Reliability of HCMV DNA PCR Using Cerebrospinal Fluid of Nonimmunosuppressed Patients

Angela Reip

Papers

Frequent and rapid emergence of mutated pre-C sequences in HBV from e-antigen positive carriers who seroconvert to anti-HBe during interferon treatment

Coding strategy of the S and M genomic segments of a hantavirus representing a new subtype of the Puumala serotype.

Human Cytomegalovirus (HCMV) Encephalitis in an Immunocompetent Young Person and Diagnostic Reliability of HCMV DNA PCR Using Cerebrospinal Fluid of Nonimmunosuppressed Patients